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Designed High-Redox Potential Laccases Exhibit High Functional Diversity

Cite this: ACS Catal. 2022, 12, 21, 13164–13173
Publication Date (Web):October 13, 2022
https://doi.org/10.1021/acscatal.2c03006

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Abstract

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White-rot fungi secrete an impressive repertoire of high-redox potential laccases (HRPLs) and peroxidases for efficient oxidation and utilization of lignin. Laccases are attractive enzymes for the chemical industry due to their broad substrate range and low environmental impact. Since expression of functional recombinant HRPLs is challenging, however, iterative-directed evolution protocols have been applied to improve their expression, activity, and stability. We implement a rational, stabilize-and-diversify strategy to two HRPLs that we could not functionally express. First, we use the PROSS stability-design algorithm to allow functional expression in yeast. Second, we use the stabilized enzymes as starting points for FuncLib active-site design to improve their activity and substrate diversity. Four of the FuncLib-designed HRPLs and their PROSS progenitor exhibit substantial diversity in reactivity profiles against high-redox potential substrates, including lignin monomers. Combinations of 3–4 subtle mutations that change the polarity, solvation, and sterics of the substrate-oxidation site result in orders of magnitude changes in reactivity profiles. These stable and versatile HRPLs are a step toward generating an effective lignin-degrading consortium of enzymes that can be secreted from yeast. The stabilize-and-diversify strategy can be applied to other challenging enzyme families to study and expand the utility of natural enzymes.

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Introduction

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Enzymatic conversion of biomass into useful materials and energy sources is critical for the drive toward a greener, more energy-efficient economy. Lignocellulose, comprising cellulose, hemicellulose, and lignin, is the main component of the plant cell wall and the most abundant source of biomass on earth. The cellulose and hemicellulose components can be effectively degraded by hydrolytic enzymes or using mild chemical conditions to their monosaccharides for biofuel production. (1) Lignin, however, is a cross-linked aromatic heteropolymer that is intertwined with cellulose and hemicellulose. It is, therefore, extremely recalcitrant to depolymerization, requiring harsh conditions, such as ionic liquids, extreme pH, or high temperatures. Economically viable and environmentally benign methods for lignin depolymerization may increase access to cellulose and hemicellulose for biofuel production and mobilize the high-value aromatic monomers stored in lignin. (2−4)
White-rot fungi secrete a broad repertoire of oxidoreductases that together with other auxiliary enzymes synergistically oxidize lignin, leading to its depolymerization. (5,6) These oxidoreductases are grouped into two major types: heme-containing peroxidases (manganese, lignin, and versatile peroxidases) and copper-dependent polyphenol oxidases named laccases (E.C. 1.10.3.2). Although peroxidases have a higher redox potential (up to 1.40 V vs normal hydrogen electrode, NHE), they depend on hydrogen peroxide, which leads to heme bleaching and enzyme deactivation even in moderate hydrogen peroxide concentrations. (7,8) By contrast, laccases are highly promiscuous, O2-dependent oxidoreductases in which paramagnetic Cu2+ (type 1 copper, T1Cu) oxidizes diverse substrates in a partially exposed recognition site. Following oxidation, the electrons are transferred to and stored in a buried trinuclear copper cluster (T2/T3Cu) which reduces molecular oxygen to two water molecules. Thus, laccases exhibit two key advantages for industrial application: they use molecular oxygen rather than the harsher hydrogen peroxide, and their sole byproduct is water, making them both longer lasting compared to peroxidases and milder in relation to chemical oxidation. (9,10)
Laccases from white-rot fungi are of special interest as they exhibit a higher redox potential (high-redox potential laccases, or HRPLs; 0.720–0.790 V vs NHE) compared to other fungal, bacterial, or plant laccases (0.400–0.700 V vs NHE). HRPLs do not oxidize lignin directly but through laccase mediators─small aromatic compounds that can diffuse into the laccase active site, undergo oxidation, and then diffuse into the lignin mesh and attack it. (9,11,12) Despite these advantages, however, HRPLs typically express very poorly in heterologous hosts impeding research and applications. This is, among other reasons, due to their high content of irregular structures (approximately 50% of the protein comprises loops), several disulfide bonds, and glycosylation sites. (13,14) Previous HRPL engineering campaigns mostly used directed evolution, including recent computation-guided evolution, HRPL chimeragenesis, or in vitro evolution starting from a reconstructed ancestral HRPL. (9,15−18) These are powerful approaches to generate stable and efficient enzymes. In all these campaigns, however, the engineering of a single enzyme demands substantial and iterative experimental effort, from improving its heterologous expression to engineering its activity, stability, and substrate specificity. Furthermore, HRPLs, peroxidases, and other auxiliary enzymes attack lignin synergistically, demanding several enzymes from each family for efficient attack. (5,19) For these reasons, a common and facile expression host, such as S. cerevisiae, is essential for research and industrial applications, and the abovementioned engineering difficulties present a serious bottleneck to produce a cost-effective enzymatic cocktail.
Computational design strategies can be used as effective alternatives to iterative engineering approaches or in combination with them. The PROSS (20) and FuncLib (21) algorithms previously developed in our lab have been used to improve diverse proteins in one-shot design; that is, without iterating modeling, mutagenesis, and screening. (20−25) Both algorithms use Rosetta atomistic design calculations and phylogenetic data to restrict design to structurally tolerated substitutions that are likely to occur in natural evolution. PROSS designs mutants that exhibit higher stability and expressibility; hence, design is allowed only outside the active sites. (20,22) By contrast, FuncLib aims to improve the catalytic activity or alter the substrate scope, and accordingly, the design process is limited to residues in the active site. (21) In a typical FuncLib design calculation, hundreds of thousands of different active-site mutants are ranked according to their energy, and the lowest-energy (most stable) ones are nominated for experimental screening. Thus, PROSS and FuncLib are compatible and complementary methods that rationalize and dramatically accelerate many of the iterative steps that are necessary for enzyme optimization. We recently used PROSS to enable the functional expression of enzymes as complex as versatile peroxidases (VPs) in yeast. (25) This encouraged us to extend these methods to other ligninolytic oxidoreductases, with the ultimate aim of constructing an artificial yeast secretome for efficient lignin depolymerization. (5)
Here, we focused our design effort on three fungal HRPLs whose structures were determined but are hardly expressed functionally in yeast. We first used PROSS stability-design calculations to enable functional yeast expression of HRPLs from Trametes hirsuta and Trametes versicolor by introducing dozens of mutations outside the active site. We then selected the most stable and active PROSS designs for further design of the T1Cu site using the FuncLib algorithm, implementing 3–7 mutations within the substrate-oxidation site. Four Trametes hirsuta FuncLib designs were further characterized together with their PROSS progenitor, showing high stability and distinct reactivity profiles against laccase substrates, including orders of magnitude differences in substrate-oxidation profiles. Structural analysis reveals that the combination of subtle mutations leads to these dramatic changes in activity, demonstrating that the stabilize-and-diversify strategy can illuminate structure–function relationships even in challenging enzymes.

Results

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Design of HRPLs for Functional Expression in Yeast

Since many HRPLs hardly express in yeast, we began by designing more stable HRPL variants using the PROSS stability-design algorithm. (20) Furthermore, activity-enhancing mutations often destabilize proteins, and a stable scaffold would be more permissive of function-altering mutations. (26) The crystal structures of Trametes versicolor, Trametes hirsuta, and basidiomycete PM1 HRPLs (PDB entries 1GYC, (27)3FPX (28) and 5ANH, (29) respectively) were used as the starting points for the PROSS design (20,22) (Figure 1A,B). For each HRPL, we selected the wildtype and four PROSS designs with different mutational loads for experimental characterization (20–84 mutations in each design, corresponding to 4–16% of the protein sequence, see Table S1 and the designed sequences in SI). In three of the four designs for each wild type HRPL, we disabled mutations in known glycosylation sites and eliminated mutations that might impact the native N-glycosylation patterns since glycosylation often enhances stability. As glycosylation sites are not entirely conserved in laccases, however, we further tested a single design for each HRPL which was allowed to impact the glycosylation sites (designs named 9nL, where nL stands for nonlimited N-glycosylation). We note that all atomistic modeling was done in the absence of glycans. The DNA encoding each protein was incorporated into the pJRoC30 plasmid and transformed into S. cerevisiae. Since the wild type enzymes could not be functionally expressed in this system, as a reference for stability and activity, we used the highly secreted, stable, and active basidiomycete PM1 variant OB-1, which was optimized by in vitro evolution. (30)

Figure 1

Figure 1. The stabilize-and-diversify strategy. (A) The crystal structures of three HRPLs from Trametes versicolor, Trametes hirsuta, and basidiomycete PM1 (PDB entries 1GYC, (27)3FPX (28) and 5ANH, (29) respectively) were selected as the starting points for design. (B) First, the three HRPLs were designed for expressibility and stability using PROSS. (20) For each wild type protein, four PROSS designs were selected for experimental testing. Copper atoms (cyan) and some PROSS-designed positions (yellow) are indicated in spheres. (C) Two designs with highest activity and stability, one based on the HRPL from Trametes versicolor and the other from Trametes hirsuta, were selected for FuncLib active-site design (green spheres). (21) For each PROSS-designed progenitor, 30 designs were selected for experimental screening. (D) FuncLib designs show diverse reactivity profiles. Four designs from Trametes hirsuta (light pink, light purple, light blue, and light green) show dramatic improvements in catalytic efficiency against various HRPL substrates compared to their PROSS progenitor (purple).

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We initially screened all PROSS designs against the general oxidoreductase substrate 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). In this activity assay, as in all the assays described below (except where stated otherwise), we measured the time-dependent colorimetric change due to substrate oxidation. The screen indicated that while the wild type progenitors of all three HRPLs exhibited no functional expression, three PROSS designs of both Trametes versicolor (Tv) and Trametes hirsuta (Th) could be functionally expressed in yeast (Figure S1A). Two designs based on the HRPL from Trametes versicolor (Tv2 and Tv9nL with 28 and 79 mutations, respectively) and three designs of the HRPL from Trametes hirsuta (Th3, Th7, and Th9 with 20, 35, and 60 mutations, respectively) were further characterized, revealing diverse stability and reactivity profiles (Figures 2 and S1). For instance, Tv9nL with 79 mutations (representing 16% of the protein)─to our knowledge, an unprecedented mutational load for stability design─is highly thermostable, even compared to the evolved OB-1 (Figure 2A). Furthermore, Tv9nL exhibits remarkable pH stability (Figures 2B and S1D), including a high stability under a very acidic pH (85% residual activity after one-week incubation at pH 2). Such acidic conditions typically destabilize HRPLs although, paradoxically, lignin degradation is favored by high acidity. (31,32) This design is remarkable both for its unusually high mutational load and because it modifies four putative N-linked glycosylation sites: substitution His55Pro, Asn141Asp, and Thr253Pro abolish three glycosylation motifs and Ile301Asn introduces an Asn-X-Thr putative glycosylation motif. In fact, the glycosylation sites seen in the crystallographic structure of Tv at positions Asn54 and Asn251 are likely to be eliminated by these mutations. Our observation that glycosylation sites can in some cases be mutated without loss in activity or stability (and in fact, exhibiting a gain in activity by 2–5-fold relative to more restricted designs) may have implications for optimizing other glycoproteins.

Figure 2

Figure 2. High thermal and pH stability in designs. (A) Kinetic thermostability (t1/2) profiles were determined by incubating yeast supernatants at 60 or 70 °C and measuring the residual activity at times 0–120 or 0–150 min, compared to the initial activity. The results are presented as the mean ± S.D. of three independent experiments. See more data and direct comparison between designs in Figure S1C. (B) pH stability profiles were determined by incubating the supernatants at 100 mM borate-citrate–phosphate buffer with pH values ranging from 2 to 9 and measuring the residual activity at times 0–168 h, compared to the initial activity at each pH. The results are presented as the mean of three independent experiments.

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Th3, originating from a different HRPL and bearing only 20 mutations, shows low stability when compared to Tv9nL and OB-1 but high stability when compared to some of the other Th variants (Figures 2 and S1C,D). Nevertheless, it exhibits high activity compared to all other designs, including against the HRPL high-redox potential synthetic mediator violuric acid (VLA, Figure S1A,B). Although VLA is synthetic, it is relevant for research and applications since it belongs to the class of the most efficient laccase mediators. (12) Owing to their superior characteristics, Tv9nL and Th3 were selected as the starting points for FuncLib active-site design. (21)

Dramatic Changes in Stability and Substrate Specificity in FuncLib Designs

The second step in our stabilize-and-diversify strategy was to apply FuncLib (21) to the T1Cu site (Figure 1C), which is responsible for substrate specificity and redox potential. (9,18,33) FuncLib design was applied to 13 positions that impact the HRPL activity (15,16,29,30,34−36) in the T1Cu site of the two starting PROSS-designed HRPLs (Tv9nL and Th3), and 30 designs per progenitor were selected for experimental screening (Tables S2 and S3). We first screened all the FuncLib designs expressed under restrictive growth conditions in a 96-well plate (37) against three laccase substrates─ABTS, guaiacol (GUA, a lignin monomer), and VLA. Screening indicated that some of the FuncLib designs exhibited different specificity profiles compared to their progenitors (Figure S2A–C). Of those, 13 Th3 designs and seven Tv9nL designs were selected for a second screen against two additional lignin monomers─2,6-dimethoxyphenol (DMP) and sinapic acid (SA). Here, several designs showed much improved activity compared to their progenitors (Figure S2D). For example, while the PROSS designs Tv9nL and Th3 exhibited no activity against SA, the FuncLib designs Tv9nL.21, Th3.7, Th3.10, and Th3.14 were active, and Tv9nL.26 could oxidize VLA while its PROSS-designed progenitor could not. These results demonstrate that FuncLib design can produce substrate preferences that are not detected in their parental proteins.
We next investigated how the active-site mutations in the most active FuncLib designs (Tv9nL designs 21 and 26, and Th3 designs 1, 7, 10, and 14) impact their stability. The mutations in the Th3 FuncLib designs only slightly reduce stability, mainly in acidic conditions (for Th3 designs 1, 7, and 14, see Figures 2B and S3B), and the kinetic thermostability at 60 °C is not affected for designs 1 and 10, slightly reduced for design 14 and improved for design 7 (Figure 2A). The Tv9nL FuncLib designs, however, showed much reduced thermal and pH stability compared to their progenitor (Figure S3) but higher activities than Tv9nL. Nevertheless, since they showed consistently lower activity than the Th3 designs and low stability, they were not characterized further.

Designs Express as a Heterogeneous Mix of Glycosylated Forms

Following the encouraging screening results, we purified Th3 and its top four FuncLib designs, Th3.1, Th3.7, Th3.10, and Th3.14, to measure their kinetic activity and substrate-specificity profiles. We purified the designs using a similar protocol as used in the in vitro evolution campaign of PM1 variants (15,30) (see SI Materials and Experimental Procedures) and compared their kinetics and ability to degrade the recalcitrant dye reactive black 5 (RB5) using the laccase mediator system (see below). The purified Th3 designs exhibit the expected size calculated from their sequence (approximately 56 kDa, Figure S4A). During purification, however, we found that a large fraction of the expressed designs was heavily and heterogeneously glycosylated (observed as a high molecular weight smear in an SDS-PAGE analysis; see SI Materials and Experimental Procedures and Figure S4B), indicating that the designed Th3 laccases were expressed as both nonglycosylated and several heavily glycosylated forms. Hyper and differential glycosylation patterns are often observed in secreted proteins and were observed previously in HRPLs. (14,38) In fact, hyperglycosylation may underlie the high stability observed in our designs. (13,14,39)
We next applied the N-glycosidase PNGaseF to probe the source of glycosylation. Treatment of the highly glycosylated fraction resulted in the appearance of three major bands in an SDS-PAGE (Figure S4B), and proteolytic mass spectrometry showed that all the Th3 bands contained Th3 peptides. Two bands were of a larger size compared to the calculated size of the designed Th3 laccases and one of a smaller size, suggesting the designs undergo both N- and O-glycosylation and are potentially subject to proteolytic degradation. Since further purification efforts did not yield a homogeneous fraction, and as the mass spectrometry results showed that the heterogeneous, heavily glycosylated Th3 fraction also contained some S. cerevisiae native contaminants, we could not precisely calculate the secretion levels of the designed Th3s. Furthermore, the data obtained on purified Th3 designs do not precisely represent the activity observed in the secreted yeast supernatant and should be interpreted as an estimate only.

FuncLib Designs Exhibit Substantial Reactivity Differences

We characterized the activity profiles of the PROSS-designed Th3 and its top four FuncLib designs, Th3.1, Th3.7, Th3.10, and Th3.14 (Figure 1D). In nature, lignin decomposes under acidic conditions, and HRPL activity is acid-dependent. (31,32) We first measured the colorimetric change immediately after adding the enzyme to reaction mixtures, which contain the substrate at different pHs. This assay indicates that the designs exhibit significantly different pH activity profiles for different substrates (Figures 3A and S5). While pH 4 is optimal for Th3 relative to all substrates except ABTS (for which the activity in pH 4 is similar to the optimum activity at pH 3), for the FuncLib designs, this is not always the case. For example, Th3.1 and Th3.7 are optimal at higher pH (pH 5 with GUA, and at this pH Th3.1 is also optimal with DMP and VLA).

Figure 3

Figure 3. FuncLib designs exhibit striking diversity in reactivity profiles. (A) Activities of OB-1 (gray), the Th3 PROSS progenitor (purple) and its four FuncLib designs measured against five substrates. The pH-dependent activity profiles are shown as individual graphs for each substrate where the activity for each design is normalized to the activity at optimal pH. Complete pH-dependent activity data are available in Figure S5. Three kinetic parameters: the inverse Michaelis constant (KM–1), the catalytic rate (kcat), or the catalytic efficiency (kcat/KM) are shown. Longer bars represent improved activities. The results are presented as the mean of three independent experiments. OB-1 exhibits superior kcat values relative to all the designs and is not shown in the graphs here to clarify the differences among the designs; the OB-1 values are given in gray font for each substrate and kinetic parameter. Complete kinetic data are available in Table S4. (B) The activity of OB-1, the Th3 PROSS progenitor, and its four FuncLib designs was measured against the same five substrates as in (A) but in the secreted yeast supernatant and at saturating substrate concentrations. Here, for each substrate the activity of all designs is normalized to the activity of the most active variant. In media, the designs and OB-1 exhibit similar activity levels. The results are presented as the mean ± S.D. of three independent biological replicates. (C) RB5 oxidation was determined by incubating the purified OB-1; the Th3 PROSS progenitor and its four FuncLib designs for 6 h with RB5 and with the laccase mediators VLA and 1-HBT or without mediator and measuring the residual dye absorption at times 0–6 h, compared to the initial absorption for each design. The results are presented as the mean ± S.D. of three independent experiments.

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We further tested the activities of Th3 designs against the five substrates at their optimal pHs. We used the purified enzymes to study their kinetics by measuring the reaction rate at various substrate concentrations. Since the purified enzymes represent only one fraction of the secreted enzymes, the kinetic values only estimate the differences between the activities. The activities of the FuncLib designs differ dramatically from those of their Th3 PROSS-designed progenitor (Figure 3A and Table S4). First, for all the substrates, Th3 is not the best enzyme in terms of the catalytic rate (kcat), substrate affinity (KM), or the catalytic efficiency (kcat/KM). Second, while Th3 shows very low activity against SA (slight color developed only after a few hours of incubation), three designs gained SA oxidation activity. Third, Th3.7 exhibits the highest catalytic rate against all substrates while exhibiting the lowest affinity among the FuncLib designs (except for DMP, where Th3.1 is the worst, and in SA where Th3.1 is inactive). This trend is expected as often high rate and substrate affinity tradeoff, (40) but the observed magnitude of the change for just a handful of mutations is striking. Of all the designs, Th3.1 has the lowest catalytic efficiency across the board, including when compared to Th3 (except for DMP, where its catalytic rate is slightly higher than Th3 but much lower than the other designs). Fourth, in terms of substrate affinities, the designs exhibit diversity without any of the designs standing out as the best: while Th3.14 has the greatest affinity to ABTS and VLA, Th3.10 has the greatest affinity to SA and DMP, which are chemically similar (sharing the same syringol head groups, see Figure 3), and Th3.1 has the greatest affinity to GUA. Last, all these differences reflect well in the catalytic efficiencies: Th3.10 shows remarkable improvement for DMP of approximately 600-fold compared to its progenitor, while Th3.14 and Th3.10 show approximately 10- and 15-fold improvements in catalytic efficiency for ABTS and GUA, respectively. Thus, the FuncLib designs improve activity relative to their PROSS-designed progenitor in all aspects we tested and against all substrates with each showing a unique reactivity profile.
Although Th3 and the laboratory-evolved OB-1 do not originate from the same wild type sequence, comparing their kinetics is intriguing (Table S4). OB-1 is clearly a much faster enzyme than the Th3 designed variants. The affinity of OB-1 for the different substrates, however, is typically in the same range of Th3 and its designs, with the FuncLib designs exhibiting superior affinities for some of the substrates (Th3.10 shows tenfold improvement for DMP, Th3.1 shows twofold improvement for GUA, and Th3.14 shows 3.5-fold improvement to the high-redox mediator VLA). Of note, we could not compare the activity profiles of the designs relative to the wild type enzyme as it is not expressible in yeast. For this reason, we cannot determine whether the observed differences between the designs and OB-1 are due to the design process or to the inherent potential of the two wild type enzymes to be optimized.
The different activity profiles observed in the kinetic data reflect the behavior of only the purified designs and do not necessarily represent the activity of the glycosylated forms secreted from yeast. Since the highly glycosylated fraction could not be effectively separated to its different constituents, we next probed the activity of the secreted yeast supernatant by measuring the oxidation rate of the five substrates at saturating substrate concentrations (Vmax), at the optimum pH exhibited by each design (Figure 3B). This is particularly relevant for our yeast-secretome motivation where all enzymes would be produced in one host and used directly from the media. (5) Here, the results differ considerably from the trends seen for the purified fractions, and the fold change in activity relative to the progenitor and between designs is much smaller. The activities in media are also in the same range as that of OB-1. The most prominent change is observed for Th3.1, which is highly active in media but shows poor activity in its purified form. As Vmax directly relates to both enzyme concentration and catalytic rate, this can be explained either by much higher expression levels for Th3.1 compared to the other designs or by differences in the activity of the highly glycosylated forms relative to other forms. Since the different glycosylation forms preclude calculating their expression levels, the source of this difference is elusive and requires further analysis.
Last, we assessed the potential of the designs to degrade complex high-redox potential compounds through the laccase mediator system. We tested the enzymes’ ability to oxidize the dye RB5 (E = 0.92 V vs NHE) through two high-redox potential mediators, VLA (E = 0.92 V vs NHE) and 1-hydroxybenzotriazole (1-HBT, E = 1.08 V vs NHE). (15) 1-HBT is a highly efficient laccase mediator, and laccase-1-HBT systems were shown to effectively degrade lignin and other recalcitrant compounds. (12) Here, we incubated the purified enzymes with either one of the mediators together with RB5 and measured decolorization over time. While neither Th3 nor its FuncLib designs oxidize RB5 effectively in the absence of a mediator, Th3, Th3.7, and Th3.10 show improved oxidation of RB5 through both VLA and 1-HBT, and here too, the FuncLib designs are better than their progenitor. Importantly, although with a lower rate than OB-1, Th3.14 efficiently oxidizes RB5 through both VLA and 1-HBT (Figure 3C) consistent with its observed high catalytic efficiency against VLA.

Structural Basis for Functional Diversity in Designs

Lacasse structure–function studies are typically based on expert-guided substitutions in the active site. (18) Such mutations usually lead to loss-of-function or are studied individually on a specific active-site background. Furthermore, strategies such as directed evolution only rarely mutate the active site. (41) Each FuncLib design carries 3–4 active-site mutations relative to its PROSS-designed progenitor. Therefore, the designs cannot directly report on the impact of individual mutations, but they provide a complementary view to previous single-mutation analyses. (15,16,29,30,34−36) As we discuss below, this analysis illuminates the potential of combinations of subtle mutations to dramatically alter the enzyme activity profile.
The T1Cu site contributes significantly to the laccase redox potential. (9,18,33) For example, T1Cu shielding and hydrophobicity increase the redox potential, (42) and therefore, vicinal polar-to-hydrophobic mutations often result in enhanced activity and vice versa. By contrast with these observations, Th3.7, which has the highest catalytic rate against all substrates, carries two unique deshielding and polar-to-charged mutations in the T1Cu binding site: Ile455Val and Asn206Asp. Position 455 is in the first shell of T1Cu, eliminating a methyl group and partly deshielding T1Cu. The vicinal mutations Asn206Asp and the additional Th3.7 Ser427Asp mutation polarize the T1Cu site (Figure 4A). Nonetheless, we observed a high catalytic rate for Th3.7, demonstrating that, surprisingly, in some contexts enhanced polarity does not reduce activity. Interestingly, only one additional FuncLib design introduces the Asn206Asp and Ile455Val mutations (design Th3.8 with low activity for most substrates, see Table S3 and Figure S2D). While Th3.7 bears the additional mutation Ser427Asp, Th3.8 has the native Ser427, suggesting that this mutation compensates for the increased polarity.

Figure 4

Figure 4. Subtle changes to active-site electrostatics, solvation, and packing underlie the dramatic reactivity differences in designs. Th3 backbone, based on PDB entry 3FPX, (28) is presented as a purple cartoon. Th3 is colored in purple, Th3.1 in light pink, Th3.7 in light purple, Th3.10 in light blue, and Th3.14 in light green in all panels. The four copper atoms are presented as teal spheres and T1Cu chelating residues from Th3 are presented in teal sticks. 2,5-xylidine from the Trametes versicolor laccase structure (PDB entry 1KYA (43)) located in the T1Cu binding site is shown in yellow sticks. (A) Changes in T1Cu hydrophobicity. Substitutions Asn206Asp, Ser427Asp, and Ile455Val in Th3.7 are presented as sticks (left). Electrostatic potential maps of Th3 (middle) and Th3.7 (right) T1Cu sites were calculated using the APBS suite (44) through the PyMOL Molecular Graphics System (Version 2.4.1, Schrödinger, LLC) and are colored in scale of [−5,5] kT/e. (B) Changes in substrate accessibility. Substitutions Phe265Met and Ala393Ser in Th3.1 are presented in sticks. (C) Changes in T1Cu-chelating His395 environment. Substitution Ser427Asp in Th3.10 and neighboring positions are presented in sticks. Hydrogen bonds within the 427–429 triad and with His395 are indicated in dashed lines and colored as the designs. (D) Dense packing near the T1Cu site and obstruction of water molecules. Substitution Phe457Trp in T3.14 is shown in sticks, and residues near Th3 Phe457 are presented as purple lines. Phe457 from Trametes hirsuta laccase crystal structure (PDB entry 3FPX (28)) is presented as gray sticks and proximal water molecules as red spheres.

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T1Cu is also the substrate recognition site hence mutations can also change substrate preferences. Asp206 is conserved in basidiomycetes (35) and forms critical hydrogen bonds with laccase substrates, reducing the electron-transfer distance between the substrate and T1Cu-chelating His458. (45) In Trametes hirsuta laccase, however, position 206 exhibits an Asn. Th3.10 has the highest affinity for DMP (KM = 17.1 and 1280 μM for Th3.7 and Th3, respectively) and SA (KM = 150 μM for Th3.7 and Th3’s KM could not be determined) which share the same 2,6-dimethoxyphenol (syringol) functional group. Here, we propose that the addition of the negative charge (Asn206Glu) to the binding pocket stabilizes the substrate (35) in Th as well. Furthermore, Th3.14, which shows the second-best affinity to these substrates (KM = 44 and 185 μM for DMP and SA, respectively) carries the Asn206His substitution that also extends into the binding cleft but has a different impact on electrostatics. Last, Th3.7 carries Asn206Asp, which changes binding-site electrostatics, while Th3 and Th3.1 bear the native Asn206 residue (Figure 4A). Th3 and Th3.1 have dramatically lower kinetic parameters toward DMP and SA (see Figure 3A and Table S4). Of the 30 FuncLib designs, only designs 1, 6, 25, and 26 maintain the native Asn206, and while all of them are active against ABTS, they exhibit poor activity against DMP and SA (Table S3 and Figure S2). Since 206 is the only polar position mutated in the characterized designs that faces directly into the oxidation site, our results suggest that Asn in this position reduces dramatically the activity against syringol-type substrates. This is in agreement with previous studies of fungal laccases where mutations from Asp or Glu to Asn changed dramatically the activity toward DMP. (9,34,35)
We further observe that mutations in surface-exposed positions in the substrate entry pathway impact activity. (29,36) Two unique mutations in surface-exposed positions in Th3.1, Phe265Met, and Ala393Ser, which may interfere with the substrate entry to the T1Cu site, may underlie the comparably low activity of this design (Figure 4B). Position 427 is a second-shell position, mutations in which can impact the rigidity and stability of the T1Cu-chelating His395. The native Ser427 hydrogen bonds with Gly429, but the Ser427Asn or Ser427Asp mutations in the designs can change the hydrogen-bond network with positions 428 and 429, the first of which hydrogen bonds to His395. By influencing His395’s conformation and adding polar moieties close to the T1Cu (<10 Å), such mutations can impact electron abstraction by the T1Cu site (30) (Figure 4C). Last, position Phe457, which is adjacent to the T1Cu-chelating position His458, is substituted for Tyr or Trp in designs Th3.1, Th3.10, and Th3.14. According to the design models, these mutations improve the packing with several active-site and distant loops, potentially enhancing the rigidity of the helix on which His458 resides. Furthermore, introducing a larger side chain into a hydrophobic region may contribute to the desolvation of the T1Cu site, thus decreasing its polarity (Figure 4D). Of all the Funclib designs, only Th3.7 maintains the native Phe457, and it exhibits a shift toward activity at alkaline pHs and the highest catalytic rates. Although Phe457 does not face T1Cu (T1Cu-Phe457Cβ distance is 8 Å), mutations to polar identities at this position increase activity at alkaline pH, (34) similar to our observation with the smaller native Phe. This indicates that space-filling mutations at this position as well as its ability to rigidify His458 may dramatically impact activity and pH sensitivity. To conclude, the structure–activity analysis demonstrates that even subtle mutations (e.g., eliminating a single methyl group near the copper site or adding a hydroxyl at a second-shell position) can lead to dramatic changes in activity when applied simultaneously in the active site.

Discussion

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Lignin is the second most abundant terrestrial biopolymer and the only large-volume, renewable feedstock based on aromatic compounds. Due to its recalcitrance, however, it is not effectively valorized, and at present it is mostly used as an energy source. (3,4,46) In nature, lignin is decomposed effectively by white-rot fungi, which secrete a large repertoire of oxidoreductases and auxiliary enzymes for synergistic lignin oxidation. (47−49) Accordingly, adapting the fungal secretome to industrial needs is a promising path for environmentally benign and economically attractive lignin utilization. Because of the high complexity of the lignin substrate, however, it is essential to include in such a secretome not only one enzyme of each type but several paralogues with different catalytic activities for each of the lignin monomers. (5,19,50) Thus, the functional expression of dozens of highly stable enzymes with diverse substrate specificities and with a wide range of affinities and catalytic rates in one host organism is imperative to enable efficient lignin degradation. Since the expression of the fungal enzymes in an industrially relevant organism is challenging, experimental protein engineering methods are impractical for screening and optimizing many starting points. Furthermore, due to experimental constraints, these methods usually implement only very few mutations in the active site and hence are limited in the number of variants they can generate with versatile reactivity profiles. (18,41) Recently, we demonstrated how PROSS could generate several stable and functionally diverse VPs from natural sequences that could not be expressed in yeast. (25) The current work adds a significant capability by showing that diverse reactivity profiles can be generated by a combined stabilize-and-diversity strategy. Thus, evolution-guided atomistic design methods (51) can be used to construct a repertoire of stable and diverse enzymes using a two-step design protocol, again with a very low experimental effort. This strategy dramatically accelerates the process for obtaining functional diversity relative to even the most advanced laboratory engineering and evolution strategies. We envision that, ultimately, the computational design steps may be used to generate stable and diverse starting points that can be further optimized by laboratory evolution strategies if needed.
Both the PROSS and FuncLib designs generated here possess attractive properties for synergistic lignin depolymerization, as well as for other industrial applications. First, the designs are stable at elevated temperatures and in diverse pH ranges. Of all the designs, the Tv9nL PROSS design stands out with high thermostability and stability at very acidic pHs, which are desirable conditions for efficient lignin oxidation. Second, the designs show different pH-dependent activity profiles. For example, Th3.7 shows an activity shift to more alkaline pHs and may find uses in the many possible applications for HRPLs under physiological conditions. (34) Third, the purified designs show significant substrate specificity, gain activity against one substrate (SA) and reach up to 600-fold improvement in catalytic efficiency compared to their progenitor with another substrate (DMP).
Whereas the purified designs exhibit lower efficiency than the laboratory-evolved OB-1, in the yeast supernatant, they all exhibit similar efficiencies to one another and to OB-1. Although the heterogeneity in glycosylation patterns in the supernatant precludes detailed kinetic analysis, the supernatant is of special interest because it is the ultimate goal of a lignolytic yeast secretome. The high efficiency in the supernatant suggests either that some of the glycoforms have high turnover or that their yeast functional secretion levels are high. Furthermore, assuming that substrate affinities are the same among the different glycoforms and given the substantial differences we measured on the purified forms, the designs may indeed comprise an effective repertoire of high-efficiency oxidases for different applications depending on the substrate and required reaction conditions. Moreover, the large diversity in substrate specificity profiles suggests that an enzyme cocktail exhibiting various substrate affinities and catalytic rates against each lignin monomer may act more effectively on native lignin.
The stabilize-and-diversify approach is general and can be applied essentially to any enzyme family. As we show here for HRPLs, this pipeline can also highlight beneficial substitutions and serve as a platform for structure–function studies. In this case, the combination of subtle active-site mutations yielded large changes in activity profiles. Using FuncLib to generate a set of diverse active-site designs can therefore be a powerful strategy to extend our understanding of the rules that govern activity and substrate scope. Furthermore, the ability to heterologously express the enzymes and produce them effectively in the lab sheds light on the importance of other unanticipated molecular features, such as the existence of multiple glycosylation forms and their impact on the activity. Thus, the PROSS-FuncLib combination can generate stable, diverse, and efficient enzymes to address basic research and biotechnological challenges.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acscatal.2c03006.

  • A detailed description of the computational methods, materials, and experimental procedures; amino acid sequences of the wild type, PROSS designs and characterized FuncLib designs; Tables S1–S3 include the selected sequences’ origins, lengths, PDB entries and their PROSS-designed mutational loads, and the FuncLib mutations in all screened designs; Table S4 reports the full kinetic data; Figures S1–S5 show the full stability and activity profiles of PROSS and FuncLib designs and purification analysis of the Th3 designs. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (52) partner repository with the data set identifier PXD034630 and 10.6019/PXD034630. Plasmids encoding Th3, Th3.1, Th3.7, Th3.10, Th314, and Tv9nL are available from AddGene (IDs 188064–188069) (PDF)

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Author Information

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  • Corresponding Author
  • Authors
    • Shiran Barber-Zucker - Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot 7600001, Israel;  Present Address: Scala Biodesign LTD, 50 Dizengoff St., Tel Aviv 6433222, Israel;  Orcidhttps://orcid.org/0000-0003-1835-730X
    • Ivan Mateljak - Department of Biocatalysis, Institute of Catalysis, CSIC, Cantoblanco, Madrid 28049, Spain;  EvoEnzyme S.L., Parque Científico de Madrid, C/Faraday, 7, Campus de Cantoblanco, Madrid 28049, Spain
    • Moshe Goldsmith - Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot 7600001, Israel;  Orcidhttps://orcid.org/0000-0002-4257-0509
    • Meital Kupervaser - Nancy and Stephen Grand Israel National Center for Personalized Medicine, Weizmann Institute of Science, Rehovot 7600001, Israel
    • Miguel Alcalde - Department of Biocatalysis, Institute of Catalysis, CSIC, Cantoblanco, Madrid 28049, Spain;  Orcidhttps://orcid.org/0000-0001-6780-7616
  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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The authors thank Prof. Itzhak Hadar and Dr. Shira Albeck for fruitful discussion, Prof. Avraham A. Levy, Dr. Cathy Melamed-Bessudo, Dr. Ely Morag, and Dr. Rivka Elbaum for sharing their equipment and materials, and members of the Fleishman lab for comments and advice. The research in the Fleishman lab was supported by an Individual Grant from the Israel Science Foundation (1844/19), a Consolidator Award from the European Research Council (815379), the Weizmann Institute’s Sustainability and Energy Research Initiative, the Dr. Barry Sherman Institute for Medicinal Chemistry, and a donation in memory of Sam Switzer.

References

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This article references 52 other publications.

  1. 1
    Davidi, L.; Moraïs, S.; Artzi, L.; Knop, D.; Hadar, Y.; Arfi, Y.; Bayer, E. A. Toward Combined Delignification and Saccharification of Wheat Straw by a Laccase-Containing Designer Cellulosome. Proc. Natl. Acad. Sci. U. S. A. 2016, 113, 10854– 10859,  DOI: 10.1073/pnas.1608012113
    Google Scholar
    1
    Toward combined delignification and saccharification of wheat straw by a laccase-containing designer cellulosome
    Davidi, Lital; Morais, Sarah; Artzi, Lior; Knop, Doriv; Hadar, Yitzhak; Arfi, Yonathan; Bayer, Edward A.
    Proceedings of the National Academy of Sciences of the United States of America (2016), 113 (39), 10854-10859CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Efficient breakdown of lignocellulose polymers into simple mols. is a key technol. bottleneck limiting the prodn. of plant-derived biofuels and chems. In nature, plant biomass degrdn. is achieved by the action of a wide range of microbial enzymes. In aerobic microorganisms, these enzymes are secreted as discrete elements in contrast to certain anaerobic bacteria, where they are assembled into large multienzyme complexes termed cellulosomes. These complexes allow for very efficient hydrolysis of cellulose and hemicellulose due to the spatial proximity of synergistically acting enzymes and to the limited diffusion of the enzymes and their products. Recently, designer cellulosomes have been developed to incorporate foreign enzymic activities in cellulosomes so as to enhance lignocellulose hydrolysis further. In this study, we complemented a cellulosome active on cellulose and hemicellulose by addn. of an enzyme active on lignin. To do so, we designed a dockerin-fused variant of a recently characterized laccase from the aerobic bacterium Thermobifida fusca. The resultant chimera exhibited activity levels similar to the wild-type enzyme and properly integrated into the designer cellulosome. The resulting complex yielded a twofold increase in the amt. of reducing sugars released from wheat straw compared with the same system lacking the laccase. The unorthodox use of aerobic enzymes in designer cellulosome machinery effects simultaneous degrdn. of the three major components of the plant cell wall (cellulose, hemicellulose, and lignin), paving the way for more efficient lignocellulose conversion into sol. sugars en route to alternative fuels prodn.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhsV2kurrL&md5=dd7c20fdfdd3024eadad9b0bf9937402
  2. 2
    Ragauskas, A. J.; Williams, C. K.; Davison, B. H.; Britovsek, G.; Cairney, J.; Eckert, C. A.; Frederick, W. J., Jr.; Hallett, J. P.; Leak, D. J.; Liotta, C. L.; Mielenz, J. R.; Murphy, R.; Templer, R.; Tschaplinski, T. The Path Forward for Biofuels and Biomaterials. Science 2006, 311, 484– 489,  DOI: 10.1126/science.1114736
    Google Scholar
    2
    The path forward for biofuels and biomaterials
    Ragauskas, Arthur J.; Williams, Charlotte K.; Davison, Brian H.; Britovsek, George; Cairney, John; Eckert, Charles A.; Frederick, William J., Jr.; Hallett, Jason P.; Leak, David J.; Liotta, Charles L.; Mielenz, Jonathan R.; Murphy, Richard; Templer, Richard; Tschaplinski, Timothy
    Science (Washington, DC, United States) (2006), 311 (5760), 484-489CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    A review. Biomass represents an abundant carbon-neutral renewable resource for the prodn. of bioenergy and biomaterials, and its enhanced use would address several societal needs. Advances in genetics, biotechnol., process chem., and engineering are leading to a new manufg. concept for converting renewable biomass to valuable fuels and products, generally referred to as the biorefinery. The integration of agro-energy crops and biorefinery manufg. technologies offers the potential for the development of sustainable bio-power and biomaterials that will lead to a new manufg. paradigm.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XmvVylsw%253D%253D&md5=7640c8c07be990221399b3f9e2788022
  3. 3
    Tuck, C. O.; Pérez, E.; Horváth, I. T.; Sheldon, R. A.; Poliakoff, M. Valorization of Biomass: Deriving More Value from Waste. Science 2012, 337, 695– 699,  DOI: 10.1126/science.1218930
    Google Scholar
    3
    Valorization of Biomass: Deriving More Value from Waste
    Tuck, Christopher O.; Perez, Eduardo; Horvath, Istvan T.; Sheldon, Roger A.; Poliakoff, Martyn
    Science (Washington, DC, United States) (2012), 337 (6095), 695-699CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    A review is given. Most of the C-based compds. currently manufd. by the chem. industry are derived from petroleum. The rising cost and dwindling supply of oil have been focusing attention on possible routes to making chems., fuels, and solvents from biomass instead. In this context, many recent studies have assessed the relative merits of applying different dedicated crops to chem. prodn. We highlight the opportunities for diverting existing residual biomass, the byproducts of present agricultural and food-processing streams to this end.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhtFCktb%252FF&md5=325bb75a58d6e05aaf5a198268e91f28
  4. 4
    Ragauskas, A. J.; Beckham, G. T.; Biddy, M. J.; Chandra, R.; Chen, F.; Davis, M. F.; Davison, B. H.; Dixon, R. A.; Gilna, P.; Keller, M.; Langan, P.; Naskar, A. K.; Saddler, J. N.; Tschaplinski, T. J.; Tuskan, G. A.; Wyman, C. E. Lignin Valorization: Improving Lignin Processing in the Biorefinery. Science 2014, 344, 1246843  DOI: 10.1126/science.1246843
    Google Scholar
    4
    Lignin valorization: improving lignin processing in the biorefinery
    Ragauskas Arthur J; Beckham Gregg T; Biddy Mary J; Chandra Richard; Saddler Jack N; Chen Fang; Dixon Richard A; Davis Mark F; Davison Brian H; Gilna Paul; Tschaplinski Timothy J; Tuskan Gerald A; Keller Martin; Langan Paul; Naskar Amit K; Wyman Charles E
    Science (New York, N.Y.) (2014), 344 (6185), 1246843 ISSN:.
    Research and development activities directed toward commercial production of cellulosic ethanol have created the opportunity to dramatically increase the transformation of lignin to value-added products. Here, we highlight recent advances in this lignin valorization effort. Discovery of genetic variants in native populations of bioenergy crops and direct manipulation of biosynthesis pathways have produced lignin feedstocks with favorable properties for recovery and downstream conversion. Advances in analytical chemistry and computational modeling detail the structure of the modified lignin and direct bioengineering strategies for future targeted properties. Refinement of biomass pretreatment technologies has further facilitated lignin recovery, and this coupled with genetic engineering will enable new uses for this biopolymer, including low-cost carbon fibers, engineered plastics and thermoplastic elastomers, polymeric foams, fungible fuels, and commodity chemicals.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2cjjsVakug%253D%253D&md5=cecde205ef4a6be5b98d4b49334707b4
  5. 5
    Alcalde, M. Engineering the Ligninolytic Enzyme Consortium. Trends Biotechnol. 2015, 33, 155– 162,  DOI: 10.1016/j.tibtech.2014.12.007
    Google Scholar
    5
    Engineering the ligninolytic enzyme consortium
    Alcalde, Miguel
    Trends in Biotechnology (2015), 33 (3), 155-162CODEN: TRBIDM; ISSN:0167-7799. (Elsevier Ltd.)
    A review, with commentary. The ligninolytic enzyme consortium is one of the most-efficient oxidative systems found in nature, playing a pivotal role during wood decay and coal formation. Typically formed by high redox-potential oxidoreductases, this array of enzymes can be used within the emerging lignocellulose biorefineries in processes that range from the prodn. of bioenergy to that of biomaterials. To ensure that these versatile enzymes meet industry stds. and needs, they have been subjected to directed evolution and hybrid approaches that surpass the limits imposed by nature. This Opinion article analyzes recent achievements in this field, including the incipient groundbreaking research into the evolution of resurrected enzymes, and the engineering of ligninolytic secretomes to create consolidated bioprocessing microbes with synthetic biol. applications.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXmslenuw%253D%253D&md5=d7557e07dbc708ac4795c3e1f6ed0818
  6. 6
    Chen, C.-C.; Dai, L.; Ma, L.; Guo, R.-T. Enzymatic Degradation of Plant Biomass and Synthetic Polymers. Nat. Rev. Chem. 2020, 4, 114– 126,  DOI: 10.1038/s41570-020-0163-6
    Google Scholar
    There is no corresponding record for this reference.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=&md5=9874b665cc7a056b8e2f928dd3112440
  7. 7
    Ruiz-Dueñas, F. J.; Morales, M.; García, E.; Miki, Y.; Martínez, M. J.; Martínez, A. T. Substrate Oxidation Sites in Versatile Peroxidase and Other Basidiomycete Peroxidases. J. Exp. Bot. 2009, 60, 441– 452,  DOI: 10.1093/jxb/ern261
    Google Scholar
    7
    Substrate oxidation sites in versatile peroxidase and other basidiomycete peroxidases
    Ruiz-Duenas, Francisco J.; Morales, Maria; Garcia, Eva; Miki, Yuta; Martinez, Maria Jesus; Martinez, Angel T.
    Journal of Experimental Botany (2009), 60 (2), 441-452CODEN: JEBOA6; ISSN:0022-0957. (Oxford University Press)
    A review. Versatile peroxidase (VP) is defined by its capabilities to oxidize the typical substrates of other basidiomycete peroxidases: (i) Mn2+, the manganese peroxidase (MnP) substrate (Mn3+ being able to oxidize phenols and initiate lipid peroxidn. reactions); (ii) veratryl alc. (VA), the typical lignin peroxidase (LiP) substrate; and (iii) simple phenols, which are the substrates of Coprinopsis cinerea peroxidase (CIP). Crystallog., spectroscopic, directed mutagenesis, and kinetic studies showed that these hybrid properties are due to the coexistence in a single protein of different catalytic sites reminiscent of those present in the other basidiomycete peroxidase families. Crystal structures of wild and recombinant VP, and kinetics of mutated variants, revealed certain differences in its Mn-oxidn. site compared with MnP. These result in efficient Mn2+ oxidn. in the presence of only two of the three acidic residues forming its binding site. A solvent-exposed tryptophan is the catalytically-active residue in VA oxidn., initiating an electron transfer pathway to heme (two other putative pathways were discarded by mutagenesis). Formation of a tryptophanyl radical after VP activation by peroxide was detected using ESR. This was the first time that a protein radical was directly demonstrated in a ligninolytic peroxidase. In contrast with LiP, the VP catalytic tryptophan is not β-hydroxylated under hydrogen peroxide excess. It was also shown that the tryptophan environment affected catalysis, its modification introducing some LiP properties in VP. Moreover, some phenols and dyes are oxidized by VP at the edge of the main heme access channel, as found in CIP. Finally, the biotechnol. interest of VP is discussed.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXivFSmt78%253D&md5=f148e64ff9162c7f0a0db45d3bf7626e
  8. 8
    Valderrama, B.; Ayala, M.; Vazquez-Duhalt, R. Suicide Inactivation of Peroxidases and the Challenge of Engineering More Robust Enzymes. Chem. Biol. 2002, 9, 555– 565,  DOI: 10.1016/S1074-5521(02)00149-7
    Google Scholar
    8
    Suicide inactivation of peroxidases and the challenge of engineering more robust enzymes
    Valderrama, Brenda; Ayala, Marcela; Vazquez-Duhalt, Rafael
    Chemistry & Biology (2002), 9 (5), 555-565CODEN: CBOLE2; ISSN:1074-5521. (Cell Press)
    A review with 171 refs. As the no. of industrial applications for proteins continues to expand, the exploitation of protein engineering becomes crit. It is predicted that protein engineering can generate enzymes with new catalytic properties and create desirable, high-value, products at lower prodn. costs. Peroxidases are ubiquitous enzymes that catalyze a variety of O2-transfer reactions and are thus potentially useful for industrial and biomedical applications. However, peroxidases are unstable and are readily inactivated by their substrate, H2O2. Researchers rely on the powerful tools of mol. biol. to improve the stability of these enzymes, either by protecting residues sensitive to oxidn. or by devising more efficient intramol. pathways for free-radical allocation. Here, the authors discuss the catalytic cycle of peroxidases and the mechanism of the suicide inactivation process to establish a broad knowledge base for future rational protein engineering.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XjvVWisr0%253D&md5=30a1aeb64472d09878bbd32a41d7ecc7
  9. 9
    Mate, D. M.; Alcalde, M. Laccase Engineering: From Rational Design to Directed Evolution. Biotechnol. Adv. 2015, 33, 25– 40,  DOI: 10.1016/j.biotechadv.2014.12.007
    Google Scholar
    9
    Laccase engineering: From rational design to directed evolution
    Mate, Diana M.; Alcalde, Miguel
    Biotechnology Advances (2015), 33 (1), 25-40CODEN: BIADDD; ISSN:0734-9750. (Elsevier)
    A review. Laccases are multicopper oxidoreductases considered by many in the biotechnol. field as the ultimate "green catalysts". This is mainly due to their broad substrate specificity and relative autonomy (they use mol. oxygen from air as an electron acceptor and they only produce water as byproduct), making them suitable for a wide array of applications: biofuel prodn., bioremediation, org. synthesis, pulp biobleaching, textiles, the beverage and food industries, biosensor and biofuel cell development. Since the beginning of the 21st century, specific features of bacterial and fungal laccases have been exhaustively adapted in order to reach the industrial demands for high catalytic activity and stability in conjunction with reduced prodn. cost. Among the goals established for laccase engineering, heterologous functional expression, improved activity and thermostability, tolerance to non-natural media (org. solvents, ionic liqs., physiol. fluids) and resistance to different types of inhibitors are all challenges that have been met, while obtaining a more comprehensive understanding of laccase structure-function relationships. In this review we examine the most significant advances in this exciting research area in which rational, semi-rational and directed evolution approaches have been employed to ultimately convert laccases into high value-added biocatalysts.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtVCqtb8%253D&md5=dc273d4fd3c8036fa8de8f4116795b78
  10. 10
    Solomon, E. I.; Sundaram, U. M.; Machonkin, T. E. Multicopper Oxidases and Oxygenases. Chem. Rev. 1996, 96, 2563– 2606,  DOI: 10.1021/cr950046o
    Google Scholar
    10
    Multicopper Oxidases and Oxygenases
    Solomon, Edward I.; Sundaram, Uma M.; Machonkin, Timothy E.
    Chemical Reviews (Washington, D. C.) (1996), 96 (7), 2563-2605CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
    A review with 428 refs. on copper binding site and other characteristics of multicopper oxidases and oxygenases such as; tyrosinase, laccase, ascorbate oxidase, ceruloplasmin.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28Xmt1Gnu7k%253D&md5=d063642db6b6652a7611b11cb1266357
  11. 11
    Singh, G.; Kaur, K.; Puri, S.; Sharma, P. Critical Factors Affecting Laccase-Mediated Biobleaching of Pulp in Paper Industry. Appl. Microbiol. Biotechnol. 2015, 99, 155– 164,  DOI: 10.1007/s00253-014-6219-0
    Google Scholar
    11
    Critical factors affecting laccase-mediated biobleaching of pulp in paper industry
    Singh, Gursharan; Kaur, Kavleen; Puri, Sanjeev; Sharma, Prince
    Applied Microbiology and Biotechnology (2015), 99 (1), 155-164CODEN: AMBIDG; ISSN:0175-7598. (Springer)
    Next to xylanases, laccases from fungi and alkali-tolerant bacteria are the most important biocatalysts that can be employed for eco-friendly biobleaching of hard and soft wood pulps in the paper industry. Laccases offer a potential alternative to conventional, environmental-polluting chlorine and chlorine-based bleaching and has no reductive effect on the final yield of pulp as compared to hemicellulases (xylanases and mannanases). In the last decade, reports on biobleaching with laccases are based on lab. observations only. There are several crit. challenges before this enzyme can be implemented for pulp bleaching at the industrial scale. This review discusses significant factors like redox potential, laccase mediator system (LMS)-synthetic or natural, pH, temp., stability of enzyme, unwanted grafting reactions of laccase, and cost-intensive prodn. at large scale which constitute a great hitch for the successful implementation of laccases at industrial level.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhvFOitr7K&md5=e1239d62a6213e3c88e524ee48eb893f
  12. 12
    Cañas, A. I.; Camarero, S. Laccases and Their Natural Mediators: Biotechnological Tools for Sustainable Eco-Friendly Processes. Biotechnol. Adv. 2010, 28, 694– 705,  DOI: 10.1016/j.biotechadv.2010.05.002
    Google Scholar
    12
    Laccases and their natural mediators: biotechnological tools for sustainable eco-friendly processes
    Canas, Ana I.; Camarero, Susana
    Biotechnology Advances (2010), 28 (6), 694-705CODEN: BIADDD; ISSN:0734-9750. (Elsevier)
    A review. Laccases are oxidoreductases which oxidize a variety of arom. compds. using oxygen as the electron acceptor and producing water as byproduct. The interest for these old enzymes (first described in 19th century) has progressively increased due to their outstanding biotechnol. applicability. The presence of redox mediators is required for a no. of biotechnol. applications, providing the oxidn. of complex substrates not oxidized by the enzyme alone. The efficiency of laccase-mediator systems to degrade recalcitrant compds. has been demonstrated, but still the high cost and possible toxicity of artificial mediators hamper their application at the industrial scale. Here, we present a general outlook of how alternative mediators can change this tendency. We focus on phenolic compds. related to lignin polymer that promotes the in vitro transformation of recalcitrant non-phenolic structures by laccase and are seemingly the natural mediators of laccases. The use of eco-friendly mediators easily available from lignocellulose, could contribute to the industrial implementation of laccases and the development of the 21th century biorefineries.
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  13. 13
    Maestre-Reyna, M.; Liu, W.-C.; Jeng, W.-Y.; Lee, C.-C.; Hsu, C.-A.; Wen, T.-N.; Wang, A. H.-J.; Shyur, L.-F. Structural and Functional Roles of Glycosylation in Fungal Laccase from Lentinus Sp. PLoS One 2015, 10, e0120601  DOI: 10.1371/journal.pone.0120601
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    13
    Structural and functional roles of glycosylation in fungal laccase from Lentinus sp.
    Maestre-Reyna, Manuel; Liu, Wei-Chun; Jeng, Wen-Yih; Lee, Cheng-Chung; Hsu, Chih-An; Wen, Tuan-Nan; Wang, Andrew H.-J.; Shyur, Lie-Fen
    PLoS One (2015), 10 (4), e0120601/1-e0120601/28CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
    Laccases are multi-copper oxidases that catalyze the oxidn. of various org. and inorg. compds. by reducing O2 to water. Here we report the crystal structure at 1.8 Å resoln. of a native laccase (designated nLcc4) isolated from a white-rot fungus Lentinus sp. nLcc4 is composed of three cupredoxin-like domains D1-D3 each folded into a Greek key β-barrel topol. T1 and T2/T3 copper binding sites and three N-glycosylated sites at Asn75, Asn238, and Asn458 were elucidated. Initial rate kinetic anal. revealed that the kcat, Km, and kcat/Km of nLcc4 with substrate ABTS were 3,382 s-1, 65.0 ± 6.5 μM, and 52 s-1μM-1, resp.; and the values with lignosulfonic acid detd. using isothermal titrn. calorimetry were 0.234 s-1, 56.7 ± 3.2 μM, and 0.004 s-1μM-1, resp. Endo H-deglycosylated nLcc4 (dLcc4), with only one GlcNAc residue remaining at each of the three N-glycosylation sites in the enzyme, exhibited similar kinetic efficiency and thermal stability to that of nLcc4. The isolated Lcc4 gene contains an open reading frame of 1563 bp with a deduced polypeptide of 521 amino acid residues including a predicted signaling peptide of 21 residues at the N-terminus. Recombinant wild-type Lcc4 and mutant enzymes N75D, N238D and N458D were expressed in Pichia pastoris cells to evaluate the effect on enzyme activity by single glycosylation site deficiency. The mutant enzymes secreted in the cultural media of P. pastoris cells were obsd. to maintain only 4-50% of the activity of the wild-type laccase. Mol. dynamics simulations analyses of various states of (de-)glycosylation in nLcc support the kinetic results and suggest that the local H-bond networks between the domain connecting loop D2-D3 and the glycan moieties play a crucial role in the laccase activity. This study provides new insights into the role of glycosylation in the structure and function of a Basidiomycete fungal laccase.
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  14. 14
    Rodgers, C. J.; Blanford, C. F.; Giddens, S. R.; Skamnioti, P.; Armstrong, F. A.; Gurr, S. J. Designer Laccases: A Vogue for High-Potential Fungal Enzymes?. Trends Biotechnol. 2010, 28, 63– 72,  DOI: 10.1016/j.tibtech.2009.11.001
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    14
    Designer laccases: a vogue for high-potential fungal enzymes?
    Rodgers, Caroline J.; Blanford, Christopher F.; Giddens, Stephen R.; Skamnioti, Pari; Armstrong, Fraser A.; Gurr, Sarah J.
    Trends in Biotechnology (2010), 28 (2), 63-72CODEN: TRBIDM; ISSN:0167-7799. (Elsevier B.V.)
    A review. Laccases are blue multicopper oxidases that catalyze the four-electron redn. of O2 to water coupled with the oxidn. of small org. substrates. Secreted basidiomycete white-rot fungal laccases orchestrate this with high thermodn. efficiency, making these enzymes excellent candidates for exploitation as industrial oxidants. However, these fungi are less tractable genetically than the ascomycetes, which predominantly produce lower-potential laccases. We address the state-of-play regarding expression of high redn. potential laccases in heterologous hosts, and issues regarding enzyme glycosylation status. We describe the synergistic role of structural biol., particularly in unmasking structure-function relationships following genetic modification and their collective impact on laccase yields. Such recent research draws closer the prospect of industrial quantities of designer, fit-for-purpose laccases.
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  15. 15
    Mateljak, I.; Monza, E.; Lucas, M. F.; Guallar, V.; Aleksejeva, O.; Ludwig, R.; Leech, D.; Shleev, S.; Alcalde, M. Increasing Redox Potential, Redox Mediator Activity, and Stability in a Fungal Laccase by Computer-Guided Mutagenesis and Directed Evolution. ACS Catal. 2019, 9, 4561– 4572,  DOI: 10.1021/acscatal.9b00531
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    15
    Increasing Redox Potential, Redox Mediator Activity, and Stability in a Fungal Laccase by Computer-Guided Mutagenesis and Directed Evolution
    Mateljak, Ivan; Monza, Emanuele; Lucas, Maria Fatima; Guallar, Victor; Aleksejeva, Olga; Ludwig, Roland; Leech, Donal; Shleev, Sergey; Alcalde, Miguel
    ACS Catalysis (2019), 9 (5), 4561-4572CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)
    Fungal high-redox-potential laccases (HRPLs) are multicopper oxidases with a relaxed substrate specificity that is highly dependent on their binding affinity and redox potential of the T1Cu site (ET1). In this study, we combined computational design with directed evolution to tailor an HRPL variant with increased ET1 and activity toward high-redox-potential mediators as well as enhanced stability. Laccase mutant libraries were screened in vitro using synthetic high-redox-potential mediators with different oxidn. routes and chem. natures, while computer-aided evolution expts. were run in parallel to guide benchtop mutagenesis, without compromising protein stability. Through this strategy, the ET1 of the evolved HRPL increased from 740 to 790 mV, with a concomitant improvement in thermal and acidic pH stability. The kinetic consts. for high-redox-potential mediators were markedly improved and were then successfully tested within laccase mediator systems (LMSs). Two hydrophobic substitutions surrounding the T1Cu site appeared to underlie these effects, and they were rationalized at the at. level. Together, this study represents a proof-of-concept of the joint elevation of the ET1, redox mediator activity, and stability in an HRPL, making this versatile biocatalyst a promising candidate for future LMS applications and for the development of bioelectrochem. devices.
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  16. 16
    Gomez-Fernandez, B. J.; Risso, V. A.; Rueda, A.; Sanchez-Ruiz, J. M.; Alcalde, M. Ancestral Resurrection and Directed Evolution of Fungal Mesozoic Laccases. Appl. Environ. Microbiol. 2020, 86, e00778– e00720
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    16
    Ancestral resurrection and directed evolution of fungal Mesozoic laccases
    Gomez-Fernandez, Bernardo J.; Risso, Valeria A.; Rueda, Andres; Sanchez-Ruiz, Jose M.; Alcalde, Miguel
    Applied and Environmental Microbiology (2020), 86 (14), e00778CODEN: AEMIDF; ISSN:1098-5336. (American Society for Microbiology)
    Ancestral sequence reconstruction and resurrection provides useful information for protein engineering, yet its alliance with directed evolution has been little explored. In this study, we have resurrected several ancestral nodes of fungal laccases dating back ~ 500 to 250 million years. Unlike modern laccases, the resurrected Mesozoic laccases were readily secreted by yeast, with similar kinetic parameters, a broader stability, and distinct pH activity profiles. The resurrected Agaricomycetes laccase carried 136 ancestral mutations, a mol. testimony to its origin, and it was subjected to directed evolution in order to improve the rate of 1,3-cyclopentanedione oxidn., a β-diketone initiator commonly used in vinyl polymn. reactions.
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  17. 17
    Mateljak, I.; Rice, A.; Yang, K.; Tron, T.; Alcalde, M. The Generation of Thermostable Fungal Laccase Chimeras by SCHEMA-RASPP Structure-Guided Recombination in Vivo. ACS Synth. Biol. 2019, 8, 833– 843,  DOI: 10.1021/acssynbio.8b00509
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    17
    The Generation of Thermostable Fungal Laccase Chimeras by SCHEMA-RASPP Structure-Guided Recombination in Vivo
    Mateljak, Ivan; Rice, Austin; Yang, Kevin; Tron, Thierry; Alcalde, Miguel
    ACS Synthetic Biology (2019), 8 (4), 833-843CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
    Fungal laccases are biotechnol. relevant enzymes that are capable of oxidizing a wide array of compds., using oxygen from the air and releasing water as the only byproduct. The laccase structure is comprised of three cupredoxin domains sheltering two copper centers-the T1Cu site and the T2/T3 trinuclear Cu cluster-connected to each other through a highly conserved internal electron transfer pathway. As such, the generation of laccase chimeras with high sequence diversity from different orthologs is difficult to achieve without compromising protein functionality. Here, we have obtained a diverse family of functional chimeras showing increased thermostability from three fungal laccase orthologs with ∼70% protein sequence identity. Assisted by the high frequency of homologous DNA recombination in Saccharomyces cerevisiae, computationally selected SCHEMA-RASPP blocks were spliced and cloned in a one-pot transformation. As a result of this in vivo assembly, an enriched library of laccase chimeras was rapidly generated, with multiple recombination events simultaneously occurring between and within the SCHEMA blocks. The resulting library was screened at high temp., identifying a collection of thermostable chimeras with considerable sequence diversity, which varied from their closest parent homolog by 46 amino acids on av. The most thermostable variant increased its half-life of thermal inactivation at 70 °C 5-fold (up to 108 min), whereas several chimeras also displayed improved stability at acidic pH. The two catalytic copper sites spanned different SCHEMA blocks, shedding light on the recognition of specific residues involved in substrate oxidn. In summary, this case-study, through comparison with previous laccase engineering studies, highlights the benefits of bringing together computationally guided recombination and in vivo shuffling as an invaluable strategy for laccase evolution, which can be translated to other enzyme systems.
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  18. 18
    Pardo, I.; Camarero, S. Laccase Engineering by Rational and Evolutionary Design. Cell. Mol. Life Sci. 2015, 72, 897– 910,  DOI: 10.1007/s00018-014-1824-8
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    18
    Laccase engineering by rational and evolutionary design
    Pardo, Isabel; Camarero, Susana
    Cellular and Molecular Life Sciences (2015), 72 (5), 897-910CODEN: CMLSFI; ISSN:1420-682X. (Birkhaeuser Basel)
    A review. Laccases are considered as green catalysts of great biotechnol. potential. This has attracted a great interest in designing laccases a la carte with enhanced stabilities or activities tailored to specific conditions for different fields of application. Over 20 yr, numerous efforts have been taken to engineer these multicopper oxidases and to understand their reaction mechanisms by site-directed mutagenesis, and more recently, using computational calcns. and directed evolution tools. Here, the authors review the most relevant contributions made in the field of laccase engineering, from the comprehensive study of their structure-function relations to the tailoring of outstanding biocatalysts.
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  19. 19
    Wang, J.; Li, L.; Xu, H.; Zhang, Y.; Liu, Y.; Zhang, F.; Shen, G.; Yan, L.; Wang, W.; Tang, H.; Qiu, H.; Gu, J.-D.; Wang, W. Construction of a Fungal Consortium for Effective Degradation of Rice Straw Lignin and Potential Application in Bio-Pulping. Bioresour. Technol. 2021, 344, 126168  DOI: 10.1016/j.biortech.2021.126168
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  20. 20
    Goldenzweig, A.; Goldsmith, M.; Hill, S. E.; Gertman, O.; Laurino, P.; Ashani, Y.; Dym, O.; Unger, T.; Albeck, S.; Prilusky, J.; Lieberman, R. L.; Aharoni, A.; Silman, I.; Sussman, J. L.; Tawfik, D. S.; Fleishman, S. J. Automated Structure- and Sequence-Based Design of Proteins for High Bacterial Expression and Stability. Mol. Cell 2016, 63, 337– 346,  DOI: 10.1016/j.molcel.2016.06.012
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    Automated Structure- and Sequence-Based Design of Proteins for High Bacterial Expression and Stability
    Goldenzweig, Adi; Goldsmith, Moshe; Hill, Shannon E.; Gertman, Or; Laurino, Paola; Ashani, Yacov; Dym, Orly; Unger, Tamar; Albeck, Shira; Prilusky, Jaime; Lieberman, Raquel L.; Aharoni, Amir; Silman, Israel; Sussman, Joel L.; Tawfik, Dan S.; Fleishman, Sarel J.
    Molecular Cell (2016), 63 (2), 337-346CODEN: MOCEFL; ISSN:1097-2765. (Elsevier Inc.)
    Upon heterologous overexpression, many proteins misfold or aggregate, thus resulting in low functional yields. Human acetylcholinesterase (hAChE), an enzyme mediating synaptic transmission, is a typical case of a human protein that necessitates mammalian systems to obtain functional expression. We developed a computational strategy and designed an AChE variant bearing 51 mutations that improved core packing, surface polarity, and backbone rigidity. This variant expressed at ∼2,000-fold higher levels in E. coli compared to wild-type hAChE and exhibited 20°C higher thermostability with no change in enzymic properties or in the active-site configuration as detd. by crystallog. To demonstrate broad utility, we similarly designed four other human and bacterial proteins. Testing at most three designs per protein, we obtained enhanced stability and/or higher yields of sol. and active protein in E. coli. Our algorithm requires only a 3D structure and several dozen sequences of naturally occurring homologs, and is available at http://pross.weizmann.ac.il.
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  21. 21
    Khersonsky, O.; Lipsh, R.; Avizemer, Z.; Ashani, Y.; Goldsmith, M.; Leader, H.; Dym, O.; Rogotner, S.; Trudeau, D. L.; Prilusky, J.; Amengual-Rigo, P.; Guallar, V.; Tawfik, D. S.; Fleishman, S. J. Automated Design of Efficient and Functionally Diverse Enzyme Repertoires. Mol. Cell 2018, 72, 178– 186.e5,  DOI: 10.1016/j.molcel.2018.08.033
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    Automated Design of Efficient and Functionally Diverse Enzyme Repertoires
    Khersonsky, Olga; Lipsh, Rosalie; Avizemer, Ziv; Ashani, Yacov; Goldsmith, Moshe; Leader, Haim; Dym, Orly; Rogotner, Shelly; Trudeau, Devin L.; Prilusky, Jaime; Amengual-Rigo, Pep; Guallar, Victor; Tawfik, Dan S.; Fleishman, Sarel J.
    Molecular Cell (2018), 72 (1), 178-186.e5CODEN: MOCEFL; ISSN:1097-2765. (Elsevier Inc.)
    Substantial improvements in enzyme activity demand multiple mutations at spatially proximal positions in the active site. Such mutations, however, often exhibit unpredictable epistatic (non-additive) effects on activity. Here we describe FuncLib, an automated method for designing multipoint mutations at enzyme active sites using phylogenetic anal. and Rosetta design calcns. We applied FuncLib to two unrelated enzymes, a phosphotriesterase and an acetyl-CoA synthetase. All designs were active, and most showed activity profiles that significantly differed from the wild-type and from one another. Several dozen designs with only 3-6 active-site mutations exhibited 10- to 4,000-fold higher efficiencies with a range of alternative substrates, including hydrolysis of the toxic organophosphate nerve agents soman and cyclosarin and synthesis of butyryl-CoA. FuncLib is implemented as a web server (http://FuncLib.weizmann.ac.il); it circumvents iterative, high-throughput exptl. screens and opens the way to designing highly efficient and diverse catalytic repertoires.
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  22. 22
    Weinstein, J. J.; Goldenzweig, A.; Hoch, S.-Y.; Fleishman, S. J. PROSS 2: A New Server for the Design of Stable and Highly Expressed Protein Variants. Bioinformatics 2021, 37, 123– 125,  DOI: 10.1093/bioinformatics/btaa1071
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    PROSS 2: a new server for the design of stable and highly expressed protein variants
    Weinstein, Jonathan Jacob; Goldenzweig, Adi; Hoch, Shlomoyakir; Fleishman, Sarel Jacob
    Bioinformatics (2021), 37 (1), 123-125CODEN: BOINFP; ISSN:1367-4811. (Oxford University Press)
    Summary: Many natural and designed proteins are only marginally stable limiting their usefulness in research and applications. Recently, we described an automated structure and sequence-based design method, called PROSS, for optimizing protein stability and heterologous expression levels that has since been validated on dozens of proteins. Here, we introduce improvements to the method, workflow and presentation, including more accurate sequence anal., error handling and automated anal. of the quality of the sequence alignment that is used in design calcns.
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  23. 23
    Peleg, Y.; Vincentelli, R.; Collins, B. M.; Chen, K.-E.; Livingstone, E. K.; Weeratunga, S.; Leneva, N.; Guo, Q.; Remans, K.; Perez, K.; Bjerga, G. E. K.; Larsen, Ø.; Vaněk, O.; Skořepa, O.; Jacquemin, S.; Poterszman, A.; Kjær, S.; Christodoulou, E.; Albeck, S.; Dym, O.; Ainbinder, E.; Unger, T.; Schuetz, A.; Matthes, S.; Bader, M.; de Marco, A.; Storici, P.; Semrau, M. S.; Stolt-Bergner, P.; Aigner, C.; Suppmann, S.; Goldenzweig, A.; Fleishman, S. J. Community-Wide Experimental Evaluation of the PROSS Stability-Design Method. J. Mol. Biol. 2021, 433, 166964  DOI: 10.1016/j.jmb.2021.166964
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    Community-Wide Experimental Evaluation of the PROSS Stability-Design Method
    Peleg, Yoav; Vincentelli, Renaud; Collins, Brett M.; Chen, Kai-En; Livingstone, Emma K.; Weeratunga, Saroja; Leneva, Natalya; Guo, Qian; Remans, Kim; Perez, Kathryn; Bjerga, Gro E. K.; Larsen, Oeivind; Vanek, Ondrej; Skorepa, Ondrej; Jacquemin, Sophie; Poterszman, Arnaud; Kjaer, Svend; Christodoulou, Evangelos; Albeck, Shira; Dym, Orly; Ainbinder, Elena; Unger, Tamar; Schuetz, Anja; Matthes, Susann; Bader, Michael; de Marco, Ario; Storici, Paola; Semrau, Marta S.; Stolt-Bergner, Peggy; Aigner, Christian; Suppmann, Sabine; Goldenzweig, Adi; Fleishman, Sarel J.
    Journal of Molecular Biology (2021), 433 (13), 166964CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
    Recent years have seen a dramatic improvement in protein-design methodol. Nevertheless, most methods demand expert intervention, limiting their widespread adoption. By contrast, the PROSS algorithm for improving protein stability and heterologous expression levels has been successfully applied to a range of challenging enzymes and binding proteins. Here, we benchmark the application of PROSS as a stand-alone tool for protein scientists with no or limited experience in modeling. Twelve labs. from the Protein Prodn. and Purifn. Partnership in Europe (P4EU) challenged the PROSS algorithm with 14 unrelated protein targets without support from the PROSS developers. For each target, up to six designs were evaluated for expression levels and in some cases, for thermal stability and activity. In nine targets, designs exhibited increased heterologous expression levels either in prokaryotic and/or eukaryotic expression systems under exptl. conditions that were tailored for each target protein. Furthermore, we obsd. increased thermal stability in nine of ten tested targets. In two prime examples, the human Stem Cell Factor (hSCF) and human Cadherin-Like Domain (CLD12) from the RET receptor, the wild type proteins were not expressible as sol. proteins in E. coli, yet the PROSS designs exhibited high expression levels in E. coli and HEK293 cells, resp., and improved thermal stability. We conclude that PROSS may improve stability and expressibility in diverse cases, and that improvement typically requires target-specific expression conditions. This study demonstrates the strengths of community-wide efforts to probe the generality of new methods and recommends areas for future research to advance practically useful algorithms for protein science.
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    Bengel, L. L.; Aberle, B.; Egler-Kemmerer, A.-N.; Kienzle, S.; Hauer, B.; Hammer, S. C. Engineered Enzymes Enable Selective N-Alkylation of Pyrazoles with Simple Haloalkanes. Angew. Chem., Int. Ed. Engl. 2021, 60, 5554– 5560,  DOI: 10.1002/anie.202014239
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    Engineered Enzymes Enable Selective N-Alkylation of Pyrazoles With Simple Haloalkanes
    Bengel Ludwig L; Aberle Benjamin; Egler-Kemmerer Alexander-N; Kienzle Samuel; Hauer Bernhard; Hammer Stephan C; Hammer Stephan C
    Angewandte Chemie (International ed. in English) (2021), 60 (10), 5554-5560 ISSN:.
    Selective alkylation of pyrazoles could solve a challenge in chemistry and streamline synthesis of important molecules. Here we report catalyst-controlled pyrazole alkylation by a cyclic two-enzyme cascade. In this enzymatic system, a promiscuous enzyme uses haloalkanes as precursors to generate non-natural analogs of the common cosubstrate S-adenosyl-l-methionine. A second engineered enzyme transfers the alkyl group in highly selective C-N bond formations to the pyrazole substrate. The cosubstrate is recycled and only used in catalytic amounts. Key is a computational enzyme-library design tool that converted a promiscuous methyltransferase into a small enzyme family of pyrazole-alkylating enzymes in one round of mutagenesis and screening. With this enzymatic system, pyrazole alkylation (methylation, ethylation, propylation) was achieved with unprecedented regioselectivity (>99 %), regiodivergence, and in a first example on preparative scale.
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  25. 25
    Barber-Zucker, S.; Mindel, V.; Garcia-Ruiz, E.; Weinstein, J. J.; Alcalde, M.; Fleishman, S. J. Stable and Functionally Diverse Versatile Peroxidases Designed Directly from Sequences. J. Am. Chem. Soc. 2022, 144, 3564– 3571,  DOI: 10.1021/jacs.1c12433
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    Stable and Functionally Diverse Versatile Peroxidases Designed Directly from Sequences
    Barber-Zucker, Shiran; Mindel, Vladimir; Garcia-Ruiz, Eva; Weinstein, Jonathan J.; Alcalde, Miguel; Fleishman, Sarel J.
    Journal of the American Chemical Society (2022), 144 (8), 3564-3571CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    White-rot fungi secrete a repertoire of high-redox potential oxidoreductases to efficiently decomp. lignin. Of these enzymes, versatile peroxidases (VPs) are the most promiscuous biocatalysts. VPs are attractive enzymes for research and industrial use but their recombinant prodn. is extremely challenging. To date, only a single VP has been structurally characterized and optimized for recombinant functional expression, stability, and activity. Computational enzyme optimization methods can be applied to many enzymes in parallel but they require accurate structures. Here, we demonstrate that model structures computed by deep-learning-based ab initio structure prediction methods are reliable starting points for one-shot PROSS stability-design calcns. Four designed VPs encoding as many as 43 mutations relative to the wildtype enzymes are functionally expressed in yeast, whereas their wildtype parents are not. Three of these designs exhibit substantial and useful diversity in their reactivity profiles and tolerance to environmental conditions. The reliability of the new generation of structure predictors and design methods increases the scale and scope of computational enzyme optimization, enabling efficient discovery and exploitation of the functional diversity in natural enzyme families directly from genomic databases.
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    Goldenzweig, A.; Fleishman, S. J. Principles of Protein Stability and Their Application in Computational Design. Annu. Rev. Biochem. 2018, 87, 105– 129,  DOI: 10.1146/annurev-biochem-062917-012102
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    Principles of Protein Stability and Their Application in Computational Design
    Goldenzweig, Adi; Fleishman, Sarel J.
    Annual Review of Biochemistry (2018), 87 (), 105-129CODEN: ARBOAW; ISSN:0066-4154. (Annual Reviews)
    A review. Proteins are increasingly used in basic and applied biomedical research. Many proteins, however, are only marginally stable and can be expressed in limited amts., thus hampering research and applications. Research has revealed the thermodn., cellular, and evolutionary principles and mechanisms that underlie marginal stability. With this growing understanding, computational stability design methods have advanced over the past two decades starting from methods that selectively addressed only some aspects of marginal stability. Current methods are more general and, by combining phylogenetic anal. with atomistic design, have shown drastic improvements in soly., thermal stability, and aggregation resistance while maintaining the protein's primary mol. activity. Stability design is opening the way to rational engineering of improved enzymes, therapeutics, and vaccines and to the application of protein design methodol. to large proteins and mol. activities that have proven challenging in the past.
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  27. 27
    Piontek, K.; Antorini, M.; Choinowski, T. Crystal Structure of a Laccase from the fungusTrametes Versicolor at 190-Å Resolution Containing a Full Complement of Coppers. J. Biol. Chem. 2002, 277, 37663– 37669,  DOI: 10.1074/jbc.M204571200
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    27
    Crystal structure of a laccase from the fungus Trametes versicolor at 1.90-Å resolution containing a full complement of coppers
    Piontek, Klaus; Antorini, Matteo; Choinowski, Thomas
    Journal of Biological Chemistry (2002), 277 (40), 37663-37669CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    Laccase is a polyphenol oxidase, which belongs to the family of blue multi-Cu oxidases. These enzymes catalyze the 1-electron oxidn. of 4 reducing-substrate mols. concomitant with the 4-electron redn. of O2 to H2O. Laccases oxidize a broad range of substrates, preferably phenolic compds. In the presence of mediators, fungal laccases exhibit an enlarged substrate range and are then able to oxidize compds. with a redox potential exceeding their own. Until now, only 1 crystal structure of a laccase in an inactive, type-2 Cu-depleted form has been reported. Here, the authors present the 1st crystal structure of an active laccase contg. a full complement of Cu atoms, the complete polypeptide chain together with 7 carbohydrate moieties. Despite the presence of all Cu atoms in the new structure, the folds of the 2 laccases were quite similar. The coordination of the type-3 Cu atoms, however, was distinctly different. The geometry of the trinuclear Cu cluster in T. versicolor laccase was similar to that found in ascorbate oxidase and that of mammalian ceruloplasmin structures, suggesting a common reaction mechanism for Cu oxidn. and O2 redn. In contrast to most blue-Cu proteins, the type-1 Cu atom in T. versicolor laccase had no axial ligand and was only 3-fold coordinated. Previously, a modest elevation of the redox potential was attributed to the lack of an axial ligand. Based on the present structural data and sequence comparisons, a mechanism is presented to explain how laccases could tune their redox potential by as much as 200 mV.
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  28. 28
    Polyakov, K. M.; Fedorova, T. V.; Stepanova, E. V.; Cherkashin, E. A.; Kurzeev, S. A.; Strokopytov, B. V.; Lamzin, V. S.; Koroleva, O. V. Structure of Native Laccase from Trametes Hirsuta at 18 A Resolution. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2009, 65, 611– 617,  DOI: 10.1107/S0907444909011950
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    Structure of native laccase from Trametes hirsuta at 1.8 Å resolution
    Polyakov, Konstantin M.; Fedorova, Tatyana V.; Stepanova, Elena V.; Cherkashin, Evgeny A.; Kurzeev, Sergei A.; Strokopytov, Boris V.; Lamzin, Victor S.; Koroleva, Olga V.
    Acta Crystallographica, Section D: Biological Crystallography (2009), 65 (6), 611-617CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)
    An anal. of the crystal structure of the native form of laccase from T. hirsuta at 1.8 Å resoln. is given. This structure provides a basis for the elucidation of the mechanism of catalytic action of these ubiquitous proteins. The 1.8-Å resoln. native structure provided a good level of structural detail compared with many previously reported laccase structures. A brief comparison with the active sites of other laccases is given.
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  29. 29
    Pardo, I.; Santiago, G.; Gentili, P.; Lucas, F.; Monza, E.; Medrano, F. J.; Galli, C.; Martínez, A. T.; Guallar, V.; Camarero, S. Re-Designing the Substrate Binding Pocket of Laccase for Enhanced Oxidation of Sinapic Acid. Catal. Sci. Technol. 2016, 6, 3900– 3910,  DOI: 10.1039/C5CY01725D
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    29
    Re-designing the substrate binding pocket of laccase for enhanced oxidation of sinapic acid
    Pardo, I.; Santiago, G.; Gentili, P.; Lucas, F.; Monza, E.; Medrano, F. J.; Galli, C.; Martinez, A. T.; Guallar, V.; Camarero, S.
    Catalysis Science & Technology (2016), 6 (11), 3900-3910CODEN: CSTAGD; ISSN:2044-4753. (Royal Society of Chemistry)
    Iterative satn. mutagenesis was performed over six residues delimiting the substrate binding pocket of a high redox potential chimeric laccase with the aim of enhancing its activity toward sinapic acid (SA), a lignin-related phenol of industrial interest. In total, more than 15 000 clones were screened and two selected variants, together with the parent-type laccase, were purified and characterized. The new variants presented shifted pH activity profiles and enhanced turnover rates on sinapic acid and its Me ester, whereas the oxidn. of related phenols was not significantly enhanced. Neither the enzyme's redox potential nor the mechanism of the reaction was affected. Quantum mechanics and mol. dynamics calcns. were done to rationalize the effect of the selected mutations, revealing the crit. role of the residues of the enzyme pocket to provide the precise binding of the substrate that enables an efficient electron transfer to the T1 copper. The results presented highlight the power of combining directed evolution and computational approaches to give novel solns. in enzyme engineering and to understand the mechanistic reasons behind them, offering new insights for further rational design towards specific targets.
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  30. 30
    Maté, D.; García-Burgos, C.; García-Ruiz, E.; Ballesteros, A. O.; Camarero, S.; Alcalde, M. Laboratory Evolution of High-Redox Potential Laccases. Chem. Biol. 2010, 17, 1030– 1041,  DOI: 10.1016/j.chembiol.2010.07.010
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    30
    Laboratory evolution of high-redox potential laccases
    Mate, Diana; Garcia-Burgos, Carlos; Garcia-Ruiz, Eva; Ballesteros, Antonio O.; Camarero, Susana; Alcalde, Miguel
    Chemistry & Biology (Cambridge, MA, United States) (2010), 17 (9), 1030-1041CODEN: CBOLE2; ISSN:1074-5521. (Cell Press)
    Thermostable laccases with a high-redox potential have been engineered through a strategy that combines directed evolution with rational approaches with site-directed mutation. The original laccase signal sequence was replaced by the α-factor prepro-leader, and the corresponding fusion gene was targeted for joint lab. evolution with the aim of improving kinetics and secretion by Saccharomyces cerevisiae, while retaining high thermostability. After 8 rounds of mol. evolution, the total activity of laccase of basidiomycete PM1 was enhanced 34,000-fold culminating in the OB-1 mutant as the last variant of the evolution process, a highly active and stable enzyme in terms of temp., pH range, and org. cosolvents. Mutations in the hydrophobic core of the evolved α-factor prepro-leader enhanced functional expression, whereas some mutations in the mature protein improved its catalytic capacities by altering the interactions with the surrounding residues.
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  31. 31
    Torres-Salas, P.; Mate, D. M.; Ghazi, I.; Plou, F. J.; Ballesteros, A. O.; Alcalde, M. Widening the pH Activity Profile of a Fungal Laccase by Directed Evolution. ChemBioChem 2013, 14, 934– 937,  DOI: 10.1002/cbic.201300102
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    31
    Widening the pH Activity Profile of a Fungal Laccase by Directed Evolution
    Torres-Salas, Pamela; Mate, Diana M.; Ghazi, Iraj; Plou, Francisco J.; Ballesteros, Antonio O.; Alcalde, Miguel
    ChemBioChem (2013), 14 (8), 934-937CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
    In this report, we describe the engineering of a fungal laccase with activity at neutral/alk. pH by directed evolution, by using the MtL-R2 mutant as the initial variant, and performing five cycles of in vitro evolution. The final mutant of the directed evolution expt. (variant IG-88) was purified and characterized biochem.
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  32. 32
    Ayuso-Fernández, I.; Ruiz-Dueñas, F. J.; Martínez, A. T. Evolutionary Convergence in Lignin-Degrading Enzymes. Proc. Natl. Acad. Sci. U. S. A. 2018, 115, 6428– 6433,  DOI: 10.1073/pnas.1802555115
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    32
    Evolutionary convergence in lignin-degrading enzymes
    Ayuso-Fernandez, Ivan; Ruiz-Duenas, Francisco J.; Martinez, Angel T.
    Proceedings of the National Academy of Sciences of the United States of America (2018), 115 (25), 6428-6433CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    The resurrection of ancestral enzymes of now-extinct organisms (paleogenetics) is a developing field that allows the study of evolutionary hypotheses otherwise impossible to be tested. In the present study, we target fungal peroxidases that play a key role in lignin degrdn., an essential process in the carbon cycle and often a limiting step in biobased industries. Ligninolytic peroxidases are secreted by wood-rotting fungi, the origin of which was recently established in the Carboniferous period assocd. with the appearance of these enzymes. These first peroxidases were not able to degrade lignin directly and used diffusible metal cations to attack its phenolic moiety. The phylogenetic anal. of the peroxidases of Polyporales, the order in which most extant wood-rotting fungi are included, suggests that later in evolution these enzymes would have acquired the ability to degrade nonphenolic lignin using a tryptophanyl radical interacting with the bulky polymer at the surface of the enzyme. Here, we track this powerful strategy for lignin degrdn. as a phenotypic trait in fungi and show that it is not an isolated event in the evolution of Polyporales. Using ancestral enzyme resurrection, we study the mol. changes that led to the appearance of the same surface oxidn. site in two distant peroxidase lineages. By characterization of the resurrected enzymes, we demonstrate convergent evolution at the amino acid level during the evolution of these fungi and track the different changes leading to phylogenetically distant ligninolytic peroxidases from ancestors lacking the ability to degrade nonphenolic lignin.
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  33. 33
    Jones, S. M.; Solomon, E. I. Electron Transfer and Reaction Mechanism of Laccases. Cell. Mol. Life Sci. 2015, 72, 869– 883,  DOI: 10.1007/s00018-014-1826-6
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    33
    Electron transfer and reaction mechanism of laccases
    Jones, Stephen M.; Solomon, Edward I.
    Cellular and Molecular Life Sciences (2015), 72 (5), 869-883CODEN: CMLSFI; ISSN:1420-682X. (Birkhaeuser Basel)
    A review. Laccases are part of the family of multicopper oxidases (MCOs), which couple the oxidn. of substrates to the four electron redn. of O2 to H2O. MCOs contain a min. of four Cu's divided into Type 1 (T1), Type 2 (T2), and binuclear Type 3 (T3) Cu sites that are distinguished based on unique spectroscopic features. Substrate oxidn. occurs near the T1, and electrons are transferred approx. 13 Å through the protein via the Cys-His pathway to the T2/T3 trinuclear copper cluster (TNC), where dioxygen redn. occurs. This review outlines the electron transfer (ET) process in laccases, and the mechanism of O2 redn. as elucidated through spectroscopic, kinetic, and computational data. Marcus theory is used to describe the relevant factors which impact ET rates including the driving force, reorganization energy, and electronic coupling matrix element. Then, the mechanism of O2 reaction is detailed with particular focus on the intermediates formed during the two 2e- redn. steps. The first 2e- step forms the peroxide intermediate, followed by the second 2e- step to form the native intermediate, which has been shown to be the catalytically relevant fully oxidized form of the enzyme.
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    Mate, D. M.; Gonzalez-Perez, D.; Falk, M.; Kittl, R.; Pita, M.; De Lacey, A. L.; Ludwig, R.; Shleev, S.; Alcalde, M. Blood Tolerant Laccase by Directed Evolution. Chem. Biol. 2013, 20, 223– 231,  DOI: 10.1016/j.chembiol.2013.01.001
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    34
    Blood Tolerant Laccase by Directed Evolution
    Mate, Diana M.; Gonzalez-Perez, David; Falk, Magnus; Kittl, Roman; Pita, Marcos; De Lacey, Antonio L.; Ludwig, Roland; Shleev, Sergey; Alcalde, Miguel
    Chemistry & Biology (Oxford, United Kingdom) (2013), 20 (2), 223-231CODEN: CBOLE2; ISSN:1074-5521. (Elsevier Ltd.)
    High-redox potential laccases are powerful biocatalysts with a wide range of applications in biotechnol. We have converted a thermostable laccase from a white-rot fungus into a blood tolerant laccase. Adapting the fitness of this laccase to the specific compn. of human blood (above neutral pH, high chloride concn.) required several generations of directed evolution in a surrogate complex blood medium. Our evolved laccase was tested in both human plasma and blood, displaying catalytic activity while retaining a high redox potential at the T1 copper site. Mutations introduced in the second coordination sphere of the T1 site shifted the pH activity profile and drastically reduced the inhibitory effect of chloride. This proof of concept that laccases can be adapted to function in extreme conditions opens an array of opportunities for implantable nanobiodevices, chem. syntheses, and detoxification.
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  35. 35
    Madzak, C.; Mimmi, M. C.; Caminade, E.; Brault, A.; Baumberger, S.; Briozzo, P.; Mougin, C.; Jolivalt, C. Shifting the Optimal pH of Activity for a Laccase from the Fungus Trametes Versicolor by Structure-Based Mutagenesis. Protein Eng., Des. Sel. 2006, 19, 77– 84,  DOI: 10.1093/protein/gzj004
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    35
    Shifting the optimal pH of activity for a laccase from the fungus Trametes versicolor by structure-based mutagenesis
    Madzak, C.; Mimmi, M. C.; Caminade, E.; Brault, A.; Baumberger, S.; Briozzo, P.; Mougin, C.; Jolivalt, C.
    Protein Engineering, Design & Selection (2006), 19 (2), 77-84CODEN: PEDSBR; ISSN:1741-0126. (Oxford University Press)
    Laccases are oxidizing enzymes of interest because of their potential environmental and industrial applications. We performed site-directed mutagenesis of a laccase produced by Trametes versicolor in order to improve its catalytic properties. Considering a strong interaction of the Asp residue in position 206 with the substrate xylidine, we replaced it with Glu, Ala or Asn, expressed the mutant enzymes in the yeast Yarrowia lipolytica and assayed the transformation of phenolic and non-phenolic substrates. The transformation rates remain within the same range whatever the mutation of the laccase and the type of substrate: at most a 3-fold factor increase was obtained for kcat between the wild-type and the most efficient mutant Asp206Ala with 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic) acid as a substrate. Nevertheless, the Asn mutation led to a significant shift of the pH (ΔpH = 1.4) for optimal activity against 2,6-dimethoxyphenol. This study also provides a new insight into the binding of the reducing substrate into the active T1 site and induced modifications in catalytic properties of the enzyme.
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  36. 36
    Galli, C.; Gentili, P.; Jolivalt, C.; Madzak, C.; Vadalà, R. How Is the Reactivity of Laccase Affected by Single-Point Mutations? Engineering Laccase for Improved Activity towards Sterically Demanding Substrates. Appl. Microbiol. Biotechnol. 2011, 91, 123– 131,  DOI: 10.1007/s00253-011-3240-4
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    36
    How is the reactivity of laccase affected by single-point mutations? Engineering laccase for improved activity towards sterically demanding substrates
    Galli, Carlo; Gentili, Patrizia; Jolivalt, Claude; Madzak, Catherine; Vadala, Raffaella
    Applied Microbiology and Biotechnology (2011), 91 (1), 123-131CODEN: AMBIDG; ISSN:0175-7598. (Springer)
    In spite of its broad specificity among phenols, Trametes versicolor laccase (I) hardly succeeds in oxidizing hindered substrates. To improve the oxidn. ability of this I toward bulky phenolic substrates, the authors designed a series of single-point mutants on the basis of the amino acid layout inside the reducing substrate active site known from the crystal structure of the enzyme. Here, site-directed mutagenesis of I addressed 4 Phe residues in key positions (Phe-162, Phe-265, Phe-332, and Phe-337) at the entrance of the binding pocket, as these residues appeared instrumental for docking of the substrate. These Phe residues were replaced by smaller-sized but still apolar Ala residues. A I double mutant (F162A/F332A) was also designed. Measurement of the oxidn. efficiency toward encumbered phenols showed that mutant F162A was more efficient than wild-type I. Double mutant F162A/F332A led to 98% consumption of bisphenol A in only 5 h and was more efficient than the single mutants in the aerobic oxidn. of this bulky substrate. In contrast, lack of appropriate hydrophobic interactions with the substrate possibly depressed the oxidn. outcome with mutants F265A and F332A. One explanation for the lack of reactivity of mutant F337A, supported by literature reports, is that this residue is part of the 2nd coordination shell of type 1 Cu. A mutation at this position thus leads to a drastic coordination shell destabilization. The thermostability of the mutants and their resistance in a mixed water-dioxane solvent were also investigated.
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  37. 37
    Mateljak, I.; Tron, T.; Alcalde, M. Evolved α-factor Prepro-leaders for Directed Laccase Evolution in Saccharomyces Cerevisiae. Microb. Biotechnol. 2017, 10, 1830– 1836,  DOI: 10.1111/1751-7915.12838
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    37
    Evolved α-factor prepro-leaders for directed laccase evolution in Saccharomyces cerevisiae
    Mateljak, Ivan; Tron, Thierry; Alcalde, Miguel
    Microbial Biotechnology (2017), 10 (6), 1830-1836CODEN: MBIIB2; ISSN:1751-7915. (Wiley-Blackwell)
    Summary : Although the functional expression of fungal laccases in Saccharomyces cerevisiae has proven to be complicated, the replacement of signal peptides appears to be a suitable approach to enhance secretion in directed evolution expts. In this study, twelve constructs were prepd. by fusing native and evolved α-factor prepro-leaders from S. cerevisiae to four different laccases with low-, medium- and high-redox potential (PM1L from basidiomycete PM1; PcL from Pycnoporus cinnabarinus; TspC30L from Trametes sp. strain C30; and MtL from Myceliophthora thermophila). Microcultures of the prepro-leader:laccase fusions were grown in selective expression medium that used galactose as both the sole carbon source and as the inducer of expression so that the secretion and activity were assessed with low- and high-redox potential mediators in a high-throughput screening context. With total activity improvements as high as sevenfold over those obtained with the native α-factor prepro-leader, the evolved prepro-leader from PcL (αPcL) most strongly enhanced secretion of the high- and medium-redox potential laccases PcL, PM1L and TspC30L in the microtiter format with an expression pattern driven by prepro-leaders in the order αPcL > αPM1L ∼ αnative. By contrast, the pattern of the low-redox potential MtL was αnative > αPcL > αPM1L. When produced in flask with rich medium, the evolved prepro-leaders outperformed the αnative signal peptide irresp. of the laccase attached, enhancing secretion over 50-fold. Together, these results highlight the importance of using evolved α-factor prepro-leaders for functional expression of fungal laccases in directed evolution campaigns.
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  38. 38
    Pardo, I.; Rodríguez-Escribano, D.; Aza, P.; de Salas, F.; Martínez, A. T.; Camarero, S. A Highly Stable Laccase Obtained by Swapping the Second Cupredoxin Domain. Sci. Rep. 2018, 8, 15669,  DOI: 10.1038/s41598-018-34008-3
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    38
    A highly stable laccase obtained by swapping the second cupredoxin domain
    Pardo Isabel; Rodriguez-Escribano David; Aza Pablo; de Salas Felipe; Martinez Angel T; Camarero Susana; Pardo Isabel
    Scientific reports (2018), 8 (1), 15669 ISSN:.
    The robustness of a high-redox potential laccase has been enhanced by swapping its second cupredoxin domain with that from another fungal laccase, which introduced a pool of neutral mutations in the protein sequence without affecting enzyme functionality. The new laccase showed outstanding stability to temperature, pH (2-9) and to organic solvents, while maintaining the ability to oxidize high-redox potential substrates. By engineering the signal peptide, enzyme secretion levels in Saccharomyces cerevisiae were increased, which allowed to purify the engineered enzyme for further characterization. The purified domain-swap laccase presented higher activity in the presence of ethanol or methanol, superior half-lives at 50-70 °C, improved stability at acidic pH, and similar catalytic efficiency for DMP albeit a lower one for ABTS (due to a shift in optimum pH). A new N-glycosylation site and a putative new surface salt-bridge were evaluated as possible determinants for the improved stability by site-directed mutagenesis. Although neither seemed to be strictly responsible for the improved thermostability, the new salt bridge was found to notably contribute to the high stability of the swapped enzyme in a broad pH range. Finally, the application potential of the new laccase was demonstrated with the enzymatic treatment of kraft lignin, an industrially relevant lignin stream, at high temperature, neutral pH and short incubation times.
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  39. 39
    Hildén, K.; Hakala, T. K.; Lundell, T. Thermotolerant and Thermostable Laccases. Biotechnol. Lett. 2009, 31, 1117– 1128,  DOI: 10.1007/s10529-009-9998-0
    Google Scholar
    39
    Thermotolerant and thermostable laccases
    Hilden, Kristiina; Hakala, Terhi K.; Lundell, Taina
    Biotechnology Letters (2009), 31 (8), 1117-1128CODEN: BILED3; ISSN:0141-5492. (Springer)
    A review. Laccases are phenol-oxidizing, usually 4-Cu-contg. metalloenzymes. For industrial and biotechnol. purposes, laccases have been among the 1st fungal oxidoreductases providing larger-scale applications such as removal of polyphenols in wine and beverages, conversion of toxic compds. and textile dyes in waste waters, and in bleaching and removal of lignin from wood and non-wood fibers. In order to facilitate novel and more efficient biocatalytic process applications, there is a need for laccases with improved biochem. properties, such as thermostability and thermotolerance. Here, the authors provide a current overview of the sources and characteristics of such laccases, with particular emphasis on the fungal enzymes.
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  40. 40
    Bar-Even, A.; Noor, E.; Savir, Y.; Liebermeister, W.; Davidi, D.; Tawfik, D. S.; Milo, R. The Moderately Efficient Enzyme: Evolutionary and Physicochemical Trends Shaping Enzyme Parameters. Biochemistry 2011, 50, 4402– 4410,  DOI: 10.1021/bi2002289
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    40
    The Moderately Efficient Enzyme: Evolutionary and Physicochemical Trends Shaping Enzyme Parameters
    Bar-Even, Arren; Noor, Elad; Savir, Yonatan; Liebermeister, Wolfram; Davidi, Dan; Tawfik, Dan S.; Milo, Ron
    Biochemistry (2011), 50 (21), 4402-4410CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    The kinetic parameters of enzymes are key to understanding the rate and specificity of most biol. processes. Although specific trends are frequently studied for individual enzymes, global trends are rarely addressed. We performed an anal. of kcat and KM values of several thousand enzymes collected from the literature. We found that the "av. enzyme" exhibits a kcat of ∼10 s-1 and a kcat/KM of ∼ 105 s-1 M-1, much below the diffusion limit and the characteristic textbook portrayal of kinetically superior enzymes. Why do most enzymes exhibit moderate catalytic efficiencies Maximal rates may not evolve in cases where weaker selection pressures are expected. We find, for example, that enzymes operating in secondary metab. are, on av., ∼ 30-fold slower than those of central metab. We also find indications that the physicochem. properties of substrates affect the kinetic parameters. Specifically, low mol. mass and hydrophobicity appear to limit KM optimization. In accordance, substitution with phosphate, CoA, or other large modifiers considerably lowers the KM values of enzymes utilizing the substituted substrates. It therefore appears that both evolutionary selection pressures and physicochem. constraints shape the kinetic parameters of enzymes. It also seems likely that the catalytic efficiency of some enzymes toward their natural substrates could be increased in many cases by natural or lab. evolution.
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  41. 41
    Nannemann, D. P.; Birmingham, W. R.; Scism, R. A.; Bachmann, B. O. Assessing Directed Evolution Methods for the Generation of Biosynthetic Enzymes with Potential in Drug Biosynthesis. Future Med. Chem. 2011, 3, 809– 819,  DOI: 10.4155/fmc.11.48
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    41
    Assessing directed evolution methods for the generation of biosynthetic enzymes with potential in drug biosynthesis
    Nannemann, David P.; Birmingham, William R.; Scism, Robert A.; Bachmann, Brian O.
    Future Medicinal Chemistry (2011), 3 (7), 809-819CODEN: FMCUA7; ISSN:1756-8919. (Future Science Ltd.)
    To address the synthesis of increasingly structurally diverse small-mol. drugs, methods for the generation of efficient and selective biol. catalysts are becoming increasingly important. Directed evolution is an umbrella term referring to a variety of methods for improving or altering the function of enzymes using a nature-inspired twofold strategy of mutagenesis followed by selection. This article provides an objective assessment of the effectiveness of directed evolution campaigns in generating enzymes with improved catalytic parameters for new substrates from the last decade, excluding studies that aimed to select for only improved phys. properties and those that lack kinetic characterization. An anal. of the trends of methodologies and their success rates from 81 qualifying examples in the literature reveals the av. fold improvement for k cat (or V max), K m and k cat/K m to be 366-, 12- and 2548-fold, resp., whereas the median fold improvements are 5.4, 3 and 15.6. Further anal. by enzyme class, library-generation methodol. and screening methodol. explores relationships between successful campaigns and the methodologies employed.
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  42. 42
    Hosseinzadeh, P.; Lu, Y. Design and Fine-Tuning Redox Potentials of Metalloproteins Involved in Electron Transfer in Bioenergetics. Biochim. Biophys. Acta 2016, 1857, 557– 581,  DOI: 10.1016/j.bbabio.2015.08.006
    Google Scholar
    42
    Design and fine-tuning redox potentials of metalloproteins involved in electron transfer in bioenergetics
    Hosseinzadeh, Parisa; Lu, Yi
    Biochimica et Biophysica Acta, Bioenergetics (2016), 1857 (5), 557-581CODEN: BBBEB4; ISSN:0005-2728. (Elsevier B. V.)
    Redox potentials are a major contributor in controlling the electron transfer (ET) rates and thus regulating the ET processes in the bioenergetics. To maximize the efficiency of the ET process, one needs to master the art of tuning the redox potential, esp. in metalloproteins, as they represent major classes of ET proteins. In this review, we first describe the importance of tuning the redox potential of ET centers and its role in regulating the ET in bioenergetic processes including photosynthesis and respiration. The main focus of this review is to summarize recent work in designing the ET centers, namely cupredoxins, cytochromes, and iron-sulfur proteins, and examples in design of protein networks involved these ET centers. We then discuss the factors that affect redox potentials of these ET centers including metal ion, the ligands to metal center and interactions beyond the primary ligand, esp. non-covalent secondary coordination sphere interactions. We provide examples of strategies to fine-tune the redox potential using both natural and unnatural amino acids and native and nonnative cofactors. Several case studies are used to illustrate recent successes in this area. Outlooks for future endeavors are also provided.
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  43. 43
    Bertrand, T.; Jolivalt, C.; Briozzo, P.; Caminade, E.; Joly, N.; Madzak, C.; Mougin, C. Crystal Structure of a Four-Copper Laccase Complexed with an Arylamine: Insights into Substrate Recognition and Correlation with Kinetics. Biochemistry 2002, 41, 7325– 7333,  DOI: 10.1021/bi0201318
    Google Scholar
    43
    Crystal structure of a four-copper laccase complexed with an arylamine: Insights into substrate recognition and correlation with kinetics
    Bertrand, Thomas; Jolivalt, Claude; Briozzo, Pierre; Caminade, Eliane; Joly, Nathalie; Madzak, Catherine; Mougin, Christian
    Biochemistry (2002), 41 (23), 7325-7333CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    Laccases are multi-Cu oxidases that catalyze the oxidn. of a wide range of phenols or arylamines, and their use in industrial oxidative processes is increasing. The authors previously purified from the white rot fungus, Trametes versicolor, a laccase that exists as 5 different isoenzymes, depending on glycosylation. Here, the 2.4 Å resoln. structure of the most abundant isoenzyme of the glycosylated enzyme was solved. The 4 Cu atoms were present, and it is the 1st crystal structure of a laccase in its active form. The crystd. enzyme bound 2,5-xylidine, which was used as a laccase inducer in the fungus culture. This arylamine was a very weak reducing substrate of the enzyme. The cavity enclosing 2,5-xylidine was found to be rather wide, allowing the accommodation of substrates of various sizes. Several amino acid residues made hydrophobic interactions with the arom. ring of the ligand. In addn., 2 charged or polar residues interacted with its amino group. The 1st was a His residue that also coordinated the Cu that functions as the primary electron acceptor. The 2nd was an Asp residue conserved among fungal laccases. The purified enzyme could oxidize various hydroxylated compds. of the phenylurea family of herbicides that the authors synthesized. These phenolic substrates had better affinities at pH 5 than at pH 3, which could be related to the binding of 2,5-xylidine by the Asp residue. This is the 1st high-resoln. structure of a multi-Cu oxidase complexed to a reducing substrate. It provides a model for engineering laccases that are either more efficient or with a wider substrate specificity.
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  44. 44
    Jurrus, E.; Engel, D.; Star, K.; Monson, K.; Brandi, J.; Felberg, L. E.; Brookes, D. H.; Wilson, L.; Chen, J.; Liles, K.; Chun, M.; Li, P.; Gohara, D. W.; Dolinsky, T.; Konecny, R.; Koes, D. R.; Nielsen, J. E.; Head-Gordon, T.; Geng, W.; Krasny, R.; Wei, G.-W.; Holst, M. J.; McCammon, J. A.; Baker, N. A. Improvements to the APBS Biomolecular Solvation Software Suite. Protein Sci. 2018, 27, 112– 128,  DOI: 10.1002/pro.3280
    Google Scholar
    44
    Improvements to the APBS biomolecular solvation software suite
    Jurrus, Elizabeth; Engel, Dave; Star, Keith; Monson, Kyle; Brandi, Juan; Felberg, Lisa E.; Brookes, David H.; Wilson, Leighton; Chen, Jiahui; Liles, Karina; Chun, Minju; Li, Peter; Gohara, David W.; Dolinsky, Todd; Konecny, Robert; Koes, David R.; Nielsen, Jens Erik; Head-Gordon, Teresa; Geng, Weihua; Krasny, Robert; Wei, Guo-Wei; Holst, Michael J.; McCammon, J. Andrew; Baker, Nathan A.
    Protein Science (2018), 27 (1), 112-128CODEN: PRCIEI; ISSN:1469-896X. (Wiley-Blackwell)
    The Adaptive Poisson-Boltzmann Solver (APBS) software was developed to solve the equations of continuum electrostatics for large biomol. assemblages that have provided impact in the study of a broad range of chem., biol., and biomedical applications. APBS addresses the three key technol. challenges for understanding solvation and electrostatics in biomedical applications: accurate and efficient models for biomol. solvation and electrostatics, robust and scalable software for applying those theories to biomol. systems, and mechanisms for sharing and analyzing biomol. electrostatics data in the scientific community. To address new research applications and advancing computational capabilities, we have continually updated APBS and its suite of accompanying software since its release in 2001. In this article, we discuss the models and capabilities that have recently been implemented within the APBS software package including a Poisson-Boltzmann anal. and a semi-anal. solver, an optimized boundary element solver, a geometry-based geometric flow solvation model, a graph theory-based algorithm for detg. pKa values, and an improved web-based visualization tool for viewing electrostatics.
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  45. 45
    Mehra, R.; Muschiol, J.; Meyer, A. S.; Kepp, K. P. A Structural-Chemical Explanation of Fungal Laccase Activity. Sci. Rep. 2018, 8, 17285,  DOI: 10.1038/s41598-018-35633-8
    Google Scholar
    45
    A structural-chemical explanation of fungal laccase activity
    Mehra Rukmankesh; Kepp Kasper P; Mehra Rukmankesh; Muschiol Jan; Meyer Anne S
    Scientific reports (2018), 8 (1), 17285 ISSN:.
    Fungal laccases (EC 1.10.3.2) are multi-copper oxidases that oxidize a wide variety of substrates. Despite extensive studies, the molecular basis for their diverse activity is unclear. Notably, there is no current way to rationally predict the activity of a laccase toward a given substrate. Such knowledge would greatly facilitate the rational design of new laccases for technological purposes. We report a study of three datasets of experimental Km values and activities for Trametes versicolor and Cerrena unicolor laccase, using a range of protein modeling techniques. We identify diverse binding modes of the various substrates and confirm an important role of Asp-206 and His-458 (T. versicolor laccase numbering) in guiding substrate recognition. Importantly, we demonstrate that experimental Km values correlate with binding affinities computed by MMGBSA. This confirms the common assumption that the protein-substrate affinity is a major contributor to observed Km. From quantitative structure-activity relations (QSAR) we identify physicochemical properties that correlate with observed Km and activities. In particular, the ionization potential, shape, and binding affinity of the substrate largely determine the enzyme's Km for the particular substrate. Our results suggest that Km is not just a binding constant but also contains features of the enzymatic activity. In addition, we identify QSAR models with only a few descriptors showing that phenolic substrates employ optimal hydrophobic packing to reach the T1 site, but then require additional electronic properties to engage in the subsequent electron transfer. Our results advance our ability to model laccase activity and lend promise to future rational optimization of laccases toward phenolic substrates.
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  46. 46
    Boerjan, W.; Ralph, J.; Baucher, M. Lignin Biosynthesis. Annu. Rev. Plant Biol. 2003, 54, 519– 546,  DOI: 10.1146/annurev.arplant.54.031902.134938
    Google Scholar
    46
    Lignin biosynthesis
    Boerjan, Wout; Ralph, John; Baucher, Marie
    Annual Review of Plant Biology (2003), 54 (), 519-546CODEN: ARPBDW ISSN:. (Annual Reviews Inc.)
    A review. The lignin biosynthetic pathway has been studied for more than a century but has undergone major revisions over the past decade. Significant progress has been made in cloning new genes by genetic and combined bioinformatics and biochem. approaches. In vitro enzymic assays and detailed analyses of mutants and transgenic plants altered in the expression of lignin biosynthesis genes have provided a solid basis for redrawing the monolignol biosynthetic pathway, and structural analyses have shown that plant cell walls can tolerate large variations in lignin content and structure. In some cases, the potential value for agriculture of transgenic plants with modified lignin structure has been demonstrated. This review presents a current picture of monolignol biosynthesis, polymn., and lignin structure.
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  47. 47
    Dashtban, M.; Schraft, H.; Syed, T. A.; Qin, W. Fungal Biodegradation and Enzymatic Modification of Lignin. Int. J. Biochem. Mol. Biol. 2010, 1, 36– 50
    Google Scholar
    47
    Fungal biodegradation and enzymatic modification of lignin
    Dashtban, Mehdi; Schraft, Heidi; Syed, Tarannum A.; Qin, Wensheng
    International Journal of Biochemistry and Molecular Biology (2010), 1 (1), 36-50CODEN: IJBMHV; ISSN:2152-4114. (e-Century Publishing Corp.)
    A review. Lignin, the most abundant arom. biopolymer on Earth, is extremely recalcitrant to degrdn. By linking to both hemicellulose and cellulose, it creates a barrier to any solns. or enzymes and prevents the penetration of lignocellulolytic enzymes into the interior lignocellulosic structure. Some basidiomycetes white-rot fungi are able to degrade lignin efficiently using a combination of extracellular ligninolytic enzymes, org. acids, mediators and accessory enzymes. This review describes ligninolytic enzyme families produced by these fungi that are involved in wood decay processes, their mol. structures, biochem. properties and the mechanisms of action which render them attractive candidates in biotechnol. applications. These enzymes include phenol oxidase (laccase) and heme peroxidases [lignin peroxidase (LiP), manganese peroxidase (MnP) and versatile peroxidase (VP)]. Accessory enzymes such as H2O2-generating oxidases and degrdn. mechanisms of plant cell-wall components in a non-enzymic manner by prodn. of free hydroxyl radicals (·OH) are also discussed.
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  48. 48
    Camarero, S.; Martínez, M. J.; Martínez, A. T. Understanding Lignin Biodegradation for the Improved Utilization of Plant Biomass in Modern Biorefineries. Biofuels, Bioprod. Biorefin. 2014, 8, 615– 625,  DOI: 10.1002/bbb.1467
    Google Scholar
    48
    Understanding lignin biodegradation for the improved utilization of plant biomass in modern biorefineries
    Camarero, Susana; Martinez, Maria Jesus; Martinez, Angel T.
    Biofuels, Bioproducts & Biorefining (2014), 8 (5), 615-625CODEN: BBBICH; ISSN:1932-104X. (John Wiley & Sons Ltd.)
    A review. Wood-rotting fungi are the sole organisms in nature able to degrade the lignin polymer making the polysaccharide components of lignocellulose fully accessible. This process has been investigated for decades as a model for biotechnol. application in the pulp and paper industry, animal feeding, and ethanol prodn. In the current lignocellulose biorefinery concept, ligninolytic fungi and the oxidoreductases (laccases and peroxidases) secreted by these fungi constitute powerful biotechnol. tools for the complete utilization of plant biomass. The evolution of mol. biol., which brings into play specifically designed biol. systems and on-demand enzymes, together with the technol. advances in processing of plant biomass, smoothes the way for a sustainable conversion of renewable feedstocks to new added-value products, with lower energy costs and less environmental impact. The present study reviews some of the main achievements attained by our group in the field of lignin biodegrdn. that have contributed to: (i) better understanding of the mechanisms by which fungi delignify the lignocellulosic materials; and (ii) assessing the applicability of these ligninolytic systems to increase the efficiency of some industrial processes and to develop new means for sustainable and environmentally sound prodn. of chems., materials, and fuels. © 2014 Society of Chem. Industry and John Wiley & Sons, Ltd.
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  49. 49
    Martínez, A. T.; Ruiz-Dueñas, F. J.; Martínez, M. J.; Del Río, J. C.; Gutiérrez, A. Enzymatic Delignification of Plant Cell Wall: From Nature to Mill. Curr. Opin. Biotechnol. 2009, 20, 348– 357,  DOI: 10.1016/j.copbio.2009.05.002
    Google Scholar
    49
    Enzymatic delignification of plant cell wall: From nature to mill
    Martinez, Angel T.; Ruiz-Duenas, Francisco J.; Martinez, Maria Jesus; del Rio, Jose C.; Gutierrez, Ana
    Current Opinion in Biotechnology (2009), 20 (3), 348-357CODEN: CUOBE3; ISSN:0958-1669. (Elsevier B.V.)
    A review. Lignin removal is a central issue in paper pulp manuf., and prodn. of other renewable chems., materials, and biofuels in future lignocellulose biorefineries. Biotechnol. can contribute to more efficient and environmentally sound deconstruction of plant cell wall by providing tailor-made biocatalysts based on the oxidative enzymes responsible for lignin attack in Nature. With this purpose, the already-known ligninolytic oxidoreductases are being improved using (rational and random-based) protein engineering, and still unknown enzymes will be identified by the application of the different "omics" technologies. Enzymic delignification will be soon at the pulp mill (combined with pitch removal) and our understanding of the reactions produced will increase by using modern techniques for lignin anal.
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  50. 50
    Sethupathy, S.; Morales, G. M.; Li, Y.; Wang, Y.; Jiang, J.; Sun, J.; Zhu, D. Harnessing Microbial Wealth for Lignocellulose Biomass Valorization through Secretomics: A Review. Biotechnol. Biofuels 2021, 14, 154,  DOI: 10.1186/s13068-021-02006-9
    Google Scholar
    50
    Harnessing microbial wealth for lignocellulose biomass valorization through secretomics: a review
    Sethupathy, Sivasamy; Morales, Gabriel Murillo; Li, Yixuan; Wang, Yongli; Jiang, Jianxiong; Sun, Jianzhong; Zhu, Daochen
    Biotechnology for Biofuels (2021), 14 (1), 154CODEN: BBIIFL; ISSN:1754-6834. (BioMed Central Ltd.)
    A review. The recalcitrance of lignocellulosic biomass is a major constraint to its high-value use at industrial scale. In nature, microbes play a crucial role in biomass degrdn., nutrient recycling and ecosystem functioning. Therefore, the use of microbes is an attractive way to transform biomass to produce clean energy and high-value compds. The microbial degrdn. of lignocelluloses is a complex process which is dependent upon multiple secreted enzymes and their synergistic activities. The availability of the cutting edge proteomics and highly sensitive mass spectrometry tools make possible for researchers to probe the secretome of microbes and microbial consortia grown on different lignocelluloses for the identification of hydrolytic enzymes of industrial interest and their substrate-dependent expression. This review summarizes the role of secretomics in identifying enzymes involved in lignocelluloses deconstruction, the development of enzyme cocktails and the construction of synthetic microbial consortia for biomass valorization, providing our perspectives to address the current challenges.
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  51. 51
    Khersonsky, O.; Fleishman, S. J. What Have We Learned from Design of Function in Large Proteins?. BioDesign Research 2022, 2022, 1– 11,  DOI: 10.34133/2022/9787581
    Google Scholar
    There is no corresponding record for this reference.
  52. 52
    Perez-Riverol, Y.; Bai, J.; Bandla, C.; García-Seisdedos, D.; Hewapathirana, S.; Kamatchinathan, S.; Kundu, D. J.; Prakash, A.; Frericks-Zipper, A.; Eisenacher, M.; Walzer, M.; Wang, S.; Brazma, A.; Vizcaíno, J. A. The PRIDE Database Resources in 2022: A Hub for Mass Spectrometry-Based Proteomics Evidences. Nucleic Acids Res. 2022, 50, D543– D552,  DOI: 10.1093/nar/gkab1038
    Google Scholar
    52
    PRIDE database resources in 2022 hub for mass spectrometry-based proteomics evidences
    Perez-Riverol, Yasset; Bai, Jingwen; Bandla, Chakradhar; Garcia-Seisdedos, David; Hewapathirana, Suresh; Kamatchinathan, Selvakumar; Kundu, Deepti J.; Prakash, Ananth; Frericks-Zipper, Anika; Eisenacher, Martin; Walzer, Mathias; Wang, Shengbo; Brazma, Alvis; Vizcaino, Juan Antonio
    Nucleic Acids Research (2022), 50 (D1), D543-D552CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
    The PRoteomics IDEntifications (PRIDE) database is the world's largest data repository of mass spectrometry-based proteomics data. PRIDE is one of the founding members of the global ProteomeXchange (PX) consortium and an ELIXIR core data resource. In this manuscript, we summarize the developments in PRIDE resources and related tools since the previous update manuscript was published in Nucleic Acids Research in 2019. The no. of submitted datasets to PRIDE Archive (the archival component of PRIDE) has reached on av. around 500 datasets per mo during 2021. In addn. to continuous improvements in PRIDE Archive data pipelines and infrastructure, the PRIDE Spectra Archive has been developed to provide direct access to the submitted mass spectra using Universal Spectrum Identifiers. As a key point, the file format MAGE-TAB for proteomics has been developed to enable the improvement of sample metadata annotation. Addnl., the resource PRIDE Peptidome provides access to aggregated peptide/protein evidences across PRIDE Archive. Furthermore, we will describe how PRIDE has increased its efforts to reuse and disseminate high-quality proteomics data into other added-value resources such as UniProt, Ensembl and Expression Atlas.
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  • Abstract

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    Figure 1

    Figure 1. The stabilize-and-diversify strategy. (A) The crystal structures of three HRPLs from Trametes versicolor, Trametes hirsuta, and basidiomycete PM1 (PDB entries 1GYC, (27)3FPX (28) and 5ANH, (29) respectively) were selected as the starting points for design. (B) First, the three HRPLs were designed for expressibility and stability using PROSS. (20) For each wild type protein, four PROSS designs were selected for experimental testing. Copper atoms (cyan) and some PROSS-designed positions (yellow) are indicated in spheres. (C) Two designs with highest activity and stability, one based on the HRPL from Trametes versicolor and the other from Trametes hirsuta, were selected for FuncLib active-site design (green spheres). (21) For each PROSS-designed progenitor, 30 designs were selected for experimental screening. (D) FuncLib designs show diverse reactivity profiles. Four designs from Trametes hirsuta (light pink, light purple, light blue, and light green) show dramatic improvements in catalytic efficiency against various HRPL substrates compared to their PROSS progenitor (purple).

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    Figure 2

    Figure 2. High thermal and pH stability in designs. (A) Kinetic thermostability (t1/2) profiles were determined by incubating yeast supernatants at 60 or 70 °C and measuring the residual activity at times 0–120 or 0–150 min, compared to the initial activity. The results are presented as the mean ± S.D. of three independent experiments. See more data and direct comparison between designs in Figure S1C. (B) pH stability profiles were determined by incubating the supernatants at 100 mM borate-citrate–phosphate buffer with pH values ranging from 2 to 9 and measuring the residual activity at times 0–168 h, compared to the initial activity at each pH. The results are presented as the mean of three independent experiments.

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    Figure 3

    Figure 3. FuncLib designs exhibit striking diversity in reactivity profiles. (A) Activities of OB-1 (gray), the Th3 PROSS progenitor (purple) and its four FuncLib designs measured against five substrates. The pH-dependent activity profiles are shown as individual graphs for each substrate where the activity for each design is normalized to the activity at optimal pH. Complete pH-dependent activity data are available in Figure S5. Three kinetic parameters: the inverse Michaelis constant (KM–1), the catalytic rate (kcat), or the catalytic efficiency (kcat/KM) are shown. Longer bars represent improved activities. The results are presented as the mean of three independent experiments. OB-1 exhibits superior kcat values relative to all the designs and is not shown in the graphs here to clarify the differences among the designs; the OB-1 values are given in gray font for each substrate and kinetic parameter. Complete kinetic data are available in Table S4. (B) The activity of OB-1, the Th3 PROSS progenitor, and its four FuncLib designs was measured against the same five substrates as in (A) but in the secreted yeast supernatant and at saturating substrate concentrations. Here, for each substrate the activity of all designs is normalized to the activity of the most active variant. In media, the designs and OB-1 exhibit similar activity levels. The results are presented as the mean ± S.D. of three independent biological replicates. (C) RB5 oxidation was determined by incubating the purified OB-1; the Th3 PROSS progenitor and its four FuncLib designs for 6 h with RB5 and with the laccase mediators VLA and 1-HBT or without mediator and measuring the residual dye absorption at times 0–6 h, compared to the initial absorption for each design. The results are presented as the mean ± S.D. of three independent experiments.

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    Figure 4

    Figure 4. Subtle changes to active-site electrostatics, solvation, and packing underlie the dramatic reactivity differences in designs. Th3 backbone, based on PDB entry 3FPX, (28) is presented as a purple cartoon. Th3 is colored in purple, Th3.1 in light pink, Th3.7 in light purple, Th3.10 in light blue, and Th3.14 in light green in all panels. The four copper atoms are presented as teal spheres and T1Cu chelating residues from Th3 are presented in teal sticks. 2,5-xylidine from the Trametes versicolor laccase structure (PDB entry 1KYA (43)) located in the T1Cu binding site is shown in yellow sticks. (A) Changes in T1Cu hydrophobicity. Substitutions Asn206Asp, Ser427Asp, and Ile455Val in Th3.7 are presented as sticks (left). Electrostatic potential maps of Th3 (middle) and Th3.7 (right) T1Cu sites were calculated using the APBS suite (44) through the PyMOL Molecular Graphics System (Version 2.4.1, Schrödinger, LLC) and are colored in scale of [−5,5] kT/e. (B) Changes in substrate accessibility. Substitutions Phe265Met and Ala393Ser in Th3.1 are presented in sticks. (C) Changes in T1Cu-chelating His395 environment. Substitution Ser427Asp in Th3.10 and neighboring positions are presented in sticks. Hydrogen bonds within the 427–429 triad and with His395 are indicated in dashed lines and colored as the designs. (D) Dense packing near the T1Cu site and obstruction of water molecules. Substitution Phe457Trp in T3.14 is shown in sticks, and residues near Th3 Phe457 are presented as purple lines. Phe457 from Trametes hirsuta laccase crystal structure (PDB entry 3FPX (28)) is presented as gray sticks and proximal water molecules as red spheres.

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    This article references 52 other publications.

    1. 1
      Davidi, L.; Moraïs, S.; Artzi, L.; Knop, D.; Hadar, Y.; Arfi, Y.; Bayer, E. A. Toward Combined Delignification and Saccharification of Wheat Straw by a Laccase-Containing Designer Cellulosome. Proc. Natl. Acad. Sci. U. S. A. 2016, 113, 10854– 10859,  DOI: 10.1073/pnas.1608012113
      1
      Toward combined delignification and saccharification of wheat straw by a laccase-containing designer cellulosome
      Davidi, Lital; Morais, Sarah; Artzi, Lior; Knop, Doriv; Hadar, Yitzhak; Arfi, Yonathan; Bayer, Edward A.
      Proceedings of the National Academy of Sciences of the United States of America (2016), 113 (39), 10854-10859CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Efficient breakdown of lignocellulose polymers into simple mols. is a key technol. bottleneck limiting the prodn. of plant-derived biofuels and chems. In nature, plant biomass degrdn. is achieved by the action of a wide range of microbial enzymes. In aerobic microorganisms, these enzymes are secreted as discrete elements in contrast to certain anaerobic bacteria, where they are assembled into large multienzyme complexes termed cellulosomes. These complexes allow for very efficient hydrolysis of cellulose and hemicellulose due to the spatial proximity of synergistically acting enzymes and to the limited diffusion of the enzymes and their products. Recently, designer cellulosomes have been developed to incorporate foreign enzymic activities in cellulosomes so as to enhance lignocellulose hydrolysis further. In this study, we complemented a cellulosome active on cellulose and hemicellulose by addn. of an enzyme active on lignin. To do so, we designed a dockerin-fused variant of a recently characterized laccase from the aerobic bacterium Thermobifida fusca. The resultant chimera exhibited activity levels similar to the wild-type enzyme and properly integrated into the designer cellulosome. The resulting complex yielded a twofold increase in the amt. of reducing sugars released from wheat straw compared with the same system lacking the laccase. The unorthodox use of aerobic enzymes in designer cellulosome machinery effects simultaneous degrdn. of the three major components of the plant cell wall (cellulose, hemicellulose, and lignin), paving the way for more efficient lignocellulose conversion into sol. sugars en route to alternative fuels prodn.
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    2. 2
      Ragauskas, A. J.; Williams, C. K.; Davison, B. H.; Britovsek, G.; Cairney, J.; Eckert, C. A.; Frederick, W. J., Jr.; Hallett, J. P.; Leak, D. J.; Liotta, C. L.; Mielenz, J. R.; Murphy, R.; Templer, R.; Tschaplinski, T. The Path Forward for Biofuels and Biomaterials. Science 2006, 311, 484– 489,  DOI: 10.1126/science.1114736
      2
      The path forward for biofuels and biomaterials
      Ragauskas, Arthur J.; Williams, Charlotte K.; Davison, Brian H.; Britovsek, George; Cairney, John; Eckert, Charles A.; Frederick, William J., Jr.; Hallett, Jason P.; Leak, David J.; Liotta, Charles L.; Mielenz, Jonathan R.; Murphy, Richard; Templer, Richard; Tschaplinski, Timothy
      Science (Washington, DC, United States) (2006), 311 (5760), 484-489CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      A review. Biomass represents an abundant carbon-neutral renewable resource for the prodn. of bioenergy and biomaterials, and its enhanced use would address several societal needs. Advances in genetics, biotechnol., process chem., and engineering are leading to a new manufg. concept for converting renewable biomass to valuable fuels and products, generally referred to as the biorefinery. The integration of agro-energy crops and biorefinery manufg. technologies offers the potential for the development of sustainable bio-power and biomaterials that will lead to a new manufg. paradigm.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XmvVylsw%253D%253D&md5=7640c8c07be990221399b3f9e2788022
    3. 3
      Tuck, C. O.; Pérez, E.; Horváth, I. T.; Sheldon, R. A.; Poliakoff, M. Valorization of Biomass: Deriving More Value from Waste. Science 2012, 337, 695– 699,  DOI: 10.1126/science.1218930
      3
      Valorization of Biomass: Deriving More Value from Waste
      Tuck, Christopher O.; Perez, Eduardo; Horvath, Istvan T.; Sheldon, Roger A.; Poliakoff, Martyn
      Science (Washington, DC, United States) (2012), 337 (6095), 695-699CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      A review is given. Most of the C-based compds. currently manufd. by the chem. industry are derived from petroleum. The rising cost and dwindling supply of oil have been focusing attention on possible routes to making chems., fuels, and solvents from biomass instead. In this context, many recent studies have assessed the relative merits of applying different dedicated crops to chem. prodn. We highlight the opportunities for diverting existing residual biomass, the byproducts of present agricultural and food-processing streams to this end.
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    4. 4
      Ragauskas, A. J.; Beckham, G. T.; Biddy, M. J.; Chandra, R.; Chen, F.; Davis, M. F.; Davison, B. H.; Dixon, R. A.; Gilna, P.; Keller, M.; Langan, P.; Naskar, A. K.; Saddler, J. N.; Tschaplinski, T. J.; Tuskan, G. A.; Wyman, C. E. Lignin Valorization: Improving Lignin Processing in the Biorefinery. Science 2014, 344, 1246843  DOI: 10.1126/science.1246843
      4
      Lignin valorization: improving lignin processing in the biorefinery
      Ragauskas Arthur J; Beckham Gregg T; Biddy Mary J; Chandra Richard; Saddler Jack N; Chen Fang; Dixon Richard A; Davis Mark F; Davison Brian H; Gilna Paul; Tschaplinski Timothy J; Tuskan Gerald A; Keller Martin; Langan Paul; Naskar Amit K; Wyman Charles E
      Science (New York, N.Y.) (2014), 344 (6185), 1246843 ISSN:.
      Research and development activities directed toward commercial production of cellulosic ethanol have created the opportunity to dramatically increase the transformation of lignin to value-added products. Here, we highlight recent advances in this lignin valorization effort. Discovery of genetic variants in native populations of bioenergy crops and direct manipulation of biosynthesis pathways have produced lignin feedstocks with favorable properties for recovery and downstream conversion. Advances in analytical chemistry and computational modeling detail the structure of the modified lignin and direct bioengineering strategies for future targeted properties. Refinement of biomass pretreatment technologies has further facilitated lignin recovery, and this coupled with genetic engineering will enable new uses for this biopolymer, including low-cost carbon fibers, engineered plastics and thermoplastic elastomers, polymeric foams, fungible fuels, and commodity chemicals.
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    5. 5
      Alcalde, M. Engineering the Ligninolytic Enzyme Consortium. Trends Biotechnol. 2015, 33, 155– 162,  DOI: 10.1016/j.tibtech.2014.12.007
      5
      Engineering the ligninolytic enzyme consortium
      Alcalde, Miguel
      Trends in Biotechnology (2015), 33 (3), 155-162CODEN: TRBIDM; ISSN:0167-7799. (Elsevier Ltd.)
      A review, with commentary. The ligninolytic enzyme consortium is one of the most-efficient oxidative systems found in nature, playing a pivotal role during wood decay and coal formation. Typically formed by high redox-potential oxidoreductases, this array of enzymes can be used within the emerging lignocellulose biorefineries in processes that range from the prodn. of bioenergy to that of biomaterials. To ensure that these versatile enzymes meet industry stds. and needs, they have been subjected to directed evolution and hybrid approaches that surpass the limits imposed by nature. This Opinion article analyzes recent achievements in this field, including the incipient groundbreaking research into the evolution of resurrected enzymes, and the engineering of ligninolytic secretomes to create consolidated bioprocessing microbes with synthetic biol. applications.
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    6. 6
      Chen, C.-C.; Dai, L.; Ma, L.; Guo, R.-T. Enzymatic Degradation of Plant Biomass and Synthetic Polymers. Nat. Rev. Chem. 2020, 4, 114– 126,  DOI: 10.1038/s41570-020-0163-6
      There is no corresponding record for this reference.
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    7. 7
      Ruiz-Dueñas, F. J.; Morales, M.; García, E.; Miki, Y.; Martínez, M. J.; Martínez, A. T. Substrate Oxidation Sites in Versatile Peroxidase and Other Basidiomycete Peroxidases. J. Exp. Bot. 2009, 60, 441– 452,  DOI: 10.1093/jxb/ern261
      7
      Substrate oxidation sites in versatile peroxidase and other basidiomycete peroxidases
      Ruiz-Duenas, Francisco J.; Morales, Maria; Garcia, Eva; Miki, Yuta; Martinez, Maria Jesus; Martinez, Angel T.
      Journal of Experimental Botany (2009), 60 (2), 441-452CODEN: JEBOA6; ISSN:0022-0957. (Oxford University Press)
      A review. Versatile peroxidase (VP) is defined by its capabilities to oxidize the typical substrates of other basidiomycete peroxidases: (i) Mn2+, the manganese peroxidase (MnP) substrate (Mn3+ being able to oxidize phenols and initiate lipid peroxidn. reactions); (ii) veratryl alc. (VA), the typical lignin peroxidase (LiP) substrate; and (iii) simple phenols, which are the substrates of Coprinopsis cinerea peroxidase (CIP). Crystallog., spectroscopic, directed mutagenesis, and kinetic studies showed that these hybrid properties are due to the coexistence in a single protein of different catalytic sites reminiscent of those present in the other basidiomycete peroxidase families. Crystal structures of wild and recombinant VP, and kinetics of mutated variants, revealed certain differences in its Mn-oxidn. site compared with MnP. These result in efficient Mn2+ oxidn. in the presence of only two of the three acidic residues forming its binding site. A solvent-exposed tryptophan is the catalytically-active residue in VA oxidn., initiating an electron transfer pathway to heme (two other putative pathways were discarded by mutagenesis). Formation of a tryptophanyl radical after VP activation by peroxide was detected using ESR. This was the first time that a protein radical was directly demonstrated in a ligninolytic peroxidase. In contrast with LiP, the VP catalytic tryptophan is not β-hydroxylated under hydrogen peroxide excess. It was also shown that the tryptophan environment affected catalysis, its modification introducing some LiP properties in VP. Moreover, some phenols and dyes are oxidized by VP at the edge of the main heme access channel, as found in CIP. Finally, the biotechnol. interest of VP is discussed.
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    8. 8
      Valderrama, B.; Ayala, M.; Vazquez-Duhalt, R. Suicide Inactivation of Peroxidases and the Challenge of Engineering More Robust Enzymes. Chem. Biol. 2002, 9, 555– 565,  DOI: 10.1016/S1074-5521(02)00149-7
      8
      Suicide inactivation of peroxidases and the challenge of engineering more robust enzymes
      Valderrama, Brenda; Ayala, Marcela; Vazquez-Duhalt, Rafael
      Chemistry & Biology (2002), 9 (5), 555-565CODEN: CBOLE2; ISSN:1074-5521. (Cell Press)
      A review with 171 refs. As the no. of industrial applications for proteins continues to expand, the exploitation of protein engineering becomes crit. It is predicted that protein engineering can generate enzymes with new catalytic properties and create desirable, high-value, products at lower prodn. costs. Peroxidases are ubiquitous enzymes that catalyze a variety of O2-transfer reactions and are thus potentially useful for industrial and biomedical applications. However, peroxidases are unstable and are readily inactivated by their substrate, H2O2. Researchers rely on the powerful tools of mol. biol. to improve the stability of these enzymes, either by protecting residues sensitive to oxidn. or by devising more efficient intramol. pathways for free-radical allocation. Here, the authors discuss the catalytic cycle of peroxidases and the mechanism of the suicide inactivation process to establish a broad knowledge base for future rational protein engineering.
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    9. 9
      Mate, D. M.; Alcalde, M. Laccase Engineering: From Rational Design to Directed Evolution. Biotechnol. Adv. 2015, 33, 25– 40,  DOI: 10.1016/j.biotechadv.2014.12.007
      9
      Laccase engineering: From rational design to directed evolution
      Mate, Diana M.; Alcalde, Miguel
      Biotechnology Advances (2015), 33 (1), 25-40CODEN: BIADDD; ISSN:0734-9750. (Elsevier)
      A review. Laccases are multicopper oxidoreductases considered by many in the biotechnol. field as the ultimate "green catalysts". This is mainly due to their broad substrate specificity and relative autonomy (they use mol. oxygen from air as an electron acceptor and they only produce water as byproduct), making them suitable for a wide array of applications: biofuel prodn., bioremediation, org. synthesis, pulp biobleaching, textiles, the beverage and food industries, biosensor and biofuel cell development. Since the beginning of the 21st century, specific features of bacterial and fungal laccases have been exhaustively adapted in order to reach the industrial demands for high catalytic activity and stability in conjunction with reduced prodn. cost. Among the goals established for laccase engineering, heterologous functional expression, improved activity and thermostability, tolerance to non-natural media (org. solvents, ionic liqs., physiol. fluids) and resistance to different types of inhibitors are all challenges that have been met, while obtaining a more comprehensive understanding of laccase structure-function relationships. In this review we examine the most significant advances in this exciting research area in which rational, semi-rational and directed evolution approaches have been employed to ultimately convert laccases into high value-added biocatalysts.
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    10. 10
      Solomon, E. I.; Sundaram, U. M.; Machonkin, T. E. Multicopper Oxidases and Oxygenases. Chem. Rev. 1996, 96, 2563– 2606,  DOI: 10.1021/cr950046o
      10
      Multicopper Oxidases and Oxygenases
      Solomon, Edward I.; Sundaram, Uma M.; Machonkin, Timothy E.
      Chemical Reviews (Washington, D. C.) (1996), 96 (7), 2563-2605CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
      A review with 428 refs. on copper binding site and other characteristics of multicopper oxidases and oxygenases such as; tyrosinase, laccase, ascorbate oxidase, ceruloplasmin.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28Xmt1Gnu7k%253D&md5=d063642db6b6652a7611b11cb1266357
    11. 11
      Singh, G.; Kaur, K.; Puri, S.; Sharma, P. Critical Factors Affecting Laccase-Mediated Biobleaching of Pulp in Paper Industry. Appl. Microbiol. Biotechnol. 2015, 99, 155– 164,  DOI: 10.1007/s00253-014-6219-0
      11
      Critical factors affecting laccase-mediated biobleaching of pulp in paper industry
      Singh, Gursharan; Kaur, Kavleen; Puri, Sanjeev; Sharma, Prince
      Applied Microbiology and Biotechnology (2015), 99 (1), 155-164CODEN: AMBIDG; ISSN:0175-7598. (Springer)
      Next to xylanases, laccases from fungi and alkali-tolerant bacteria are the most important biocatalysts that can be employed for eco-friendly biobleaching of hard and soft wood pulps in the paper industry. Laccases offer a potential alternative to conventional, environmental-polluting chlorine and chlorine-based bleaching and has no reductive effect on the final yield of pulp as compared to hemicellulases (xylanases and mannanases). In the last decade, reports on biobleaching with laccases are based on lab. observations only. There are several crit. challenges before this enzyme can be implemented for pulp bleaching at the industrial scale. This review discusses significant factors like redox potential, laccase mediator system (LMS)-synthetic or natural, pH, temp., stability of enzyme, unwanted grafting reactions of laccase, and cost-intensive prodn. at large scale which constitute a great hitch for the successful implementation of laccases at industrial level.
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    12. 12
      Cañas, A. I.; Camarero, S. Laccases and Their Natural Mediators: Biotechnological Tools for Sustainable Eco-Friendly Processes. Biotechnol. Adv. 2010, 28, 694– 705,  DOI: 10.1016/j.biotechadv.2010.05.002
      12
      Laccases and their natural mediators: biotechnological tools for sustainable eco-friendly processes
      Canas, Ana I.; Camarero, Susana
      Biotechnology Advances (2010), 28 (6), 694-705CODEN: BIADDD; ISSN:0734-9750. (Elsevier)
      A review. Laccases are oxidoreductases which oxidize a variety of arom. compds. using oxygen as the electron acceptor and producing water as byproduct. The interest for these old enzymes (first described in 19th century) has progressively increased due to their outstanding biotechnol. applicability. The presence of redox mediators is required for a no. of biotechnol. applications, providing the oxidn. of complex substrates not oxidized by the enzyme alone. The efficiency of laccase-mediator systems to degrade recalcitrant compds. has been demonstrated, but still the high cost and possible toxicity of artificial mediators hamper their application at the industrial scale. Here, we present a general outlook of how alternative mediators can change this tendency. We focus on phenolic compds. related to lignin polymer that promotes the in vitro transformation of recalcitrant non-phenolic structures by laccase and are seemingly the natural mediators of laccases. The use of eco-friendly mediators easily available from lignocellulose, could contribute to the industrial implementation of laccases and the development of the 21th century biorefineries.
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    13. 13
      Maestre-Reyna, M.; Liu, W.-C.; Jeng, W.-Y.; Lee, C.-C.; Hsu, C.-A.; Wen, T.-N.; Wang, A. H.-J.; Shyur, L.-F. Structural and Functional Roles of Glycosylation in Fungal Laccase from Lentinus Sp. PLoS One 2015, 10, e0120601  DOI: 10.1371/journal.pone.0120601
      13
      Structural and functional roles of glycosylation in fungal laccase from Lentinus sp.
      Maestre-Reyna, Manuel; Liu, Wei-Chun; Jeng, Wen-Yih; Lee, Cheng-Chung; Hsu, Chih-An; Wen, Tuan-Nan; Wang, Andrew H.-J.; Shyur, Lie-Fen
      PLoS One (2015), 10 (4), e0120601/1-e0120601/28CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
      Laccases are multi-copper oxidases that catalyze the oxidn. of various org. and inorg. compds. by reducing O2 to water. Here we report the crystal structure at 1.8 Å resoln. of a native laccase (designated nLcc4) isolated from a white-rot fungus Lentinus sp. nLcc4 is composed of three cupredoxin-like domains D1-D3 each folded into a Greek key β-barrel topol. T1 and T2/T3 copper binding sites and three N-glycosylated sites at Asn75, Asn238, and Asn458 were elucidated. Initial rate kinetic anal. revealed that the kcat, Km, and kcat/Km of nLcc4 with substrate ABTS were 3,382 s-1, 65.0 ± 6.5 μM, and 52 s-1μM-1, resp.; and the values with lignosulfonic acid detd. using isothermal titrn. calorimetry were 0.234 s-1, 56.7 ± 3.2 μM, and 0.004 s-1μM-1, resp. Endo H-deglycosylated nLcc4 (dLcc4), with only one GlcNAc residue remaining at each of the three N-glycosylation sites in the enzyme, exhibited similar kinetic efficiency and thermal stability to that of nLcc4. The isolated Lcc4 gene contains an open reading frame of 1563 bp with a deduced polypeptide of 521 amino acid residues including a predicted signaling peptide of 21 residues at the N-terminus. Recombinant wild-type Lcc4 and mutant enzymes N75D, N238D and N458D were expressed in Pichia pastoris cells to evaluate the effect on enzyme activity by single glycosylation site deficiency. The mutant enzymes secreted in the cultural media of P. pastoris cells were obsd. to maintain only 4-50% of the activity of the wild-type laccase. Mol. dynamics simulations analyses of various states of (de-)glycosylation in nLcc support the kinetic results and suggest that the local H-bond networks between the domain connecting loop D2-D3 and the glycan moieties play a crucial role in the laccase activity. This study provides new insights into the role of glycosylation in the structure and function of a Basidiomycete fungal laccase.
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    14. 14
      Rodgers, C. J.; Blanford, C. F.; Giddens, S. R.; Skamnioti, P.; Armstrong, F. A.; Gurr, S. J. Designer Laccases: A Vogue for High-Potential Fungal Enzymes?. Trends Biotechnol. 2010, 28, 63– 72,  DOI: 10.1016/j.tibtech.2009.11.001
      14
      Designer laccases: a vogue for high-potential fungal enzymes?
      Rodgers, Caroline J.; Blanford, Christopher F.; Giddens, Stephen R.; Skamnioti, Pari; Armstrong, Fraser A.; Gurr, Sarah J.
      Trends in Biotechnology (2010), 28 (2), 63-72CODEN: TRBIDM; ISSN:0167-7799. (Elsevier B.V.)
      A review. Laccases are blue multicopper oxidases that catalyze the four-electron redn. of O2 to water coupled with the oxidn. of small org. substrates. Secreted basidiomycete white-rot fungal laccases orchestrate this with high thermodn. efficiency, making these enzymes excellent candidates for exploitation as industrial oxidants. However, these fungi are less tractable genetically than the ascomycetes, which predominantly produce lower-potential laccases. We address the state-of-play regarding expression of high redn. potential laccases in heterologous hosts, and issues regarding enzyme glycosylation status. We describe the synergistic role of structural biol., particularly in unmasking structure-function relationships following genetic modification and their collective impact on laccase yields. Such recent research draws closer the prospect of industrial quantities of designer, fit-for-purpose laccases.
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    15. 15
      Mateljak, I.; Monza, E.; Lucas, M. F.; Guallar, V.; Aleksejeva, O.; Ludwig, R.; Leech, D.; Shleev, S.; Alcalde, M. Increasing Redox Potential, Redox Mediator Activity, and Stability in a Fungal Laccase by Computer-Guided Mutagenesis and Directed Evolution. ACS Catal. 2019, 9, 4561– 4572,  DOI: 10.1021/acscatal.9b00531
      15
      Increasing Redox Potential, Redox Mediator Activity, and Stability in a Fungal Laccase by Computer-Guided Mutagenesis and Directed Evolution
      Mateljak, Ivan; Monza, Emanuele; Lucas, Maria Fatima; Guallar, Victor; Aleksejeva, Olga; Ludwig, Roland; Leech, Donal; Shleev, Sergey; Alcalde, Miguel
      ACS Catalysis (2019), 9 (5), 4561-4572CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)
      Fungal high-redox-potential laccases (HRPLs) are multicopper oxidases with a relaxed substrate specificity that is highly dependent on their binding affinity and redox potential of the T1Cu site (ET1). In this study, we combined computational design with directed evolution to tailor an HRPL variant with increased ET1 and activity toward high-redox-potential mediators as well as enhanced stability. Laccase mutant libraries were screened in vitro using synthetic high-redox-potential mediators with different oxidn. routes and chem. natures, while computer-aided evolution expts. were run in parallel to guide benchtop mutagenesis, without compromising protein stability. Through this strategy, the ET1 of the evolved HRPL increased from 740 to 790 mV, with a concomitant improvement in thermal and acidic pH stability. The kinetic consts. for high-redox-potential mediators were markedly improved and were then successfully tested within laccase mediator systems (LMSs). Two hydrophobic substitutions surrounding the T1Cu site appeared to underlie these effects, and they were rationalized at the at. level. Together, this study represents a proof-of-concept of the joint elevation of the ET1, redox mediator activity, and stability in an HRPL, making this versatile biocatalyst a promising candidate for future LMS applications and for the development of bioelectrochem. devices.
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    16. 16
      Gomez-Fernandez, B. J.; Risso, V. A.; Rueda, A.; Sanchez-Ruiz, J. M.; Alcalde, M. Ancestral Resurrection and Directed Evolution of Fungal Mesozoic Laccases. Appl. Environ. Microbiol. 2020, 86, e00778– e00720
      16
      Ancestral resurrection and directed evolution of fungal Mesozoic laccases
      Gomez-Fernandez, Bernardo J.; Risso, Valeria A.; Rueda, Andres; Sanchez-Ruiz, Jose M.; Alcalde, Miguel
      Applied and Environmental Microbiology (2020), 86 (14), e00778CODEN: AEMIDF; ISSN:1098-5336. (American Society for Microbiology)
      Ancestral sequence reconstruction and resurrection provides useful information for protein engineering, yet its alliance with directed evolution has been little explored. In this study, we have resurrected several ancestral nodes of fungal laccases dating back ~ 500 to 250 million years. Unlike modern laccases, the resurrected Mesozoic laccases were readily secreted by yeast, with similar kinetic parameters, a broader stability, and distinct pH activity profiles. The resurrected Agaricomycetes laccase carried 136 ancestral mutations, a mol. testimony to its origin, and it was subjected to directed evolution in order to improve the rate of 1,3-cyclopentanedione oxidn., a β-diketone initiator commonly used in vinyl polymn. reactions.
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    17. 17
      Mateljak, I.; Rice, A.; Yang, K.; Tron, T.; Alcalde, M. The Generation of Thermostable Fungal Laccase Chimeras by SCHEMA-RASPP Structure-Guided Recombination in Vivo. ACS Synth. Biol. 2019, 8, 833– 843,  DOI: 10.1021/acssynbio.8b00509
      17
      The Generation of Thermostable Fungal Laccase Chimeras by SCHEMA-RASPP Structure-Guided Recombination in Vivo
      Mateljak, Ivan; Rice, Austin; Yang, Kevin; Tron, Thierry; Alcalde, Miguel
      ACS Synthetic Biology (2019), 8 (4), 833-843CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
      Fungal laccases are biotechnol. relevant enzymes that are capable of oxidizing a wide array of compds., using oxygen from the air and releasing water as the only byproduct. The laccase structure is comprised of three cupredoxin domains sheltering two copper centers-the T1Cu site and the T2/T3 trinuclear Cu cluster-connected to each other through a highly conserved internal electron transfer pathway. As such, the generation of laccase chimeras with high sequence diversity from different orthologs is difficult to achieve without compromising protein functionality. Here, we have obtained a diverse family of functional chimeras showing increased thermostability from three fungal laccase orthologs with ∼70% protein sequence identity. Assisted by the high frequency of homologous DNA recombination in Saccharomyces cerevisiae, computationally selected SCHEMA-RASPP blocks were spliced and cloned in a one-pot transformation. As a result of this in vivo assembly, an enriched library of laccase chimeras was rapidly generated, with multiple recombination events simultaneously occurring between and within the SCHEMA blocks. The resulting library was screened at high temp., identifying a collection of thermostable chimeras with considerable sequence diversity, which varied from their closest parent homolog by 46 amino acids on av. The most thermostable variant increased its half-life of thermal inactivation at 70 °C 5-fold (up to 108 min), whereas several chimeras also displayed improved stability at acidic pH. The two catalytic copper sites spanned different SCHEMA blocks, shedding light on the recognition of specific residues involved in substrate oxidn. In summary, this case-study, through comparison with previous laccase engineering studies, highlights the benefits of bringing together computationally guided recombination and in vivo shuffling as an invaluable strategy for laccase evolution, which can be translated to other enzyme systems.
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    18. 18
      Pardo, I.; Camarero, S. Laccase Engineering by Rational and Evolutionary Design. Cell. Mol. Life Sci. 2015, 72, 897– 910,  DOI: 10.1007/s00018-014-1824-8
      18
      Laccase engineering by rational and evolutionary design
      Pardo, Isabel; Camarero, Susana
      Cellular and Molecular Life Sciences (2015), 72 (5), 897-910CODEN: CMLSFI; ISSN:1420-682X. (Birkhaeuser Basel)
      A review. Laccases are considered as green catalysts of great biotechnol. potential. This has attracted a great interest in designing laccases a la carte with enhanced stabilities or activities tailored to specific conditions for different fields of application. Over 20 yr, numerous efforts have been taken to engineer these multicopper oxidases and to understand their reaction mechanisms by site-directed mutagenesis, and more recently, using computational calcns. and directed evolution tools. Here, the authors review the most relevant contributions made in the field of laccase engineering, from the comprehensive study of their structure-function relations to the tailoring of outstanding biocatalysts.
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    19. 19
      Wang, J.; Li, L.; Xu, H.; Zhang, Y.; Liu, Y.; Zhang, F.; Shen, G.; Yan, L.; Wang, W.; Tang, H.; Qiu, H.; Gu, J.-D.; Wang, W. Construction of a Fungal Consortium for Effective Degradation of Rice Straw Lignin and Potential Application in Bio-Pulping. Bioresour. Technol. 2021, 344, 126168  DOI: 10.1016/j.biortech.2021.126168
      There is no corresponding record for this reference.
    20. 20
      Goldenzweig, A.; Goldsmith, M.; Hill, S. E.; Gertman, O.; Laurino, P.; Ashani, Y.; Dym, O.; Unger, T.; Albeck, S.; Prilusky, J.; Lieberman, R. L.; Aharoni, A.; Silman, I.; Sussman, J. L.; Tawfik, D. S.; Fleishman, S. J. Automated Structure- and Sequence-Based Design of Proteins for High Bacterial Expression and Stability. Mol. Cell 2016, 63, 337– 346,  DOI: 10.1016/j.molcel.2016.06.012
      20
      Automated Structure- and Sequence-Based Design of Proteins for High Bacterial Expression and Stability
      Goldenzweig, Adi; Goldsmith, Moshe; Hill, Shannon E.; Gertman, Or; Laurino, Paola; Ashani, Yacov; Dym, Orly; Unger, Tamar; Albeck, Shira; Prilusky, Jaime; Lieberman, Raquel L.; Aharoni, Amir; Silman, Israel; Sussman, Joel L.; Tawfik, Dan S.; Fleishman, Sarel J.
      Molecular Cell (2016), 63 (2), 337-346CODEN: MOCEFL; ISSN:1097-2765. (Elsevier Inc.)
      Upon heterologous overexpression, many proteins misfold or aggregate, thus resulting in low functional yields. Human acetylcholinesterase (hAChE), an enzyme mediating synaptic transmission, is a typical case of a human protein that necessitates mammalian systems to obtain functional expression. We developed a computational strategy and designed an AChE variant bearing 51 mutations that improved core packing, surface polarity, and backbone rigidity. This variant expressed at ∼2,000-fold higher levels in E. coli compared to wild-type hAChE and exhibited 20°C higher thermostability with no change in enzymic properties or in the active-site configuration as detd. by crystallog. To demonstrate broad utility, we similarly designed four other human and bacterial proteins. Testing at most three designs per protein, we obtained enhanced stability and/or higher yields of sol. and active protein in E. coli. Our algorithm requires only a 3D structure and several dozen sequences of naturally occurring homologs, and is available at http://pross.weizmann.ac.il.
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    21. 21
      Khersonsky, O.; Lipsh, R.; Avizemer, Z.; Ashani, Y.; Goldsmith, M.; Leader, H.; Dym, O.; Rogotner, S.; Trudeau, D. L.; Prilusky, J.; Amengual-Rigo, P.; Guallar, V.; Tawfik, D. S.; Fleishman, S. J. Automated Design of Efficient and Functionally Diverse Enzyme Repertoires. Mol. Cell 2018, 72, 178– 186.e5,  DOI: 10.1016/j.molcel.2018.08.033
      21
      Automated Design of Efficient and Functionally Diverse Enzyme Repertoires
      Khersonsky, Olga; Lipsh, Rosalie; Avizemer, Ziv; Ashani, Yacov; Goldsmith, Moshe; Leader, Haim; Dym, Orly; Rogotner, Shelly; Trudeau, Devin L.; Prilusky, Jaime; Amengual-Rigo, Pep; Guallar, Victor; Tawfik, Dan S.; Fleishman, Sarel J.
      Molecular Cell (2018), 72 (1), 178-186.e5CODEN: MOCEFL; ISSN:1097-2765. (Elsevier Inc.)
      Substantial improvements in enzyme activity demand multiple mutations at spatially proximal positions in the active site. Such mutations, however, often exhibit unpredictable epistatic (non-additive) effects on activity. Here we describe FuncLib, an automated method for designing multipoint mutations at enzyme active sites using phylogenetic anal. and Rosetta design calcns. We applied FuncLib to two unrelated enzymes, a phosphotriesterase and an acetyl-CoA synthetase. All designs were active, and most showed activity profiles that significantly differed from the wild-type and from one another. Several dozen designs with only 3-6 active-site mutations exhibited 10- to 4,000-fold higher efficiencies with a range of alternative substrates, including hydrolysis of the toxic organophosphate nerve agents soman and cyclosarin and synthesis of butyryl-CoA. FuncLib is implemented as a web server (http://FuncLib.weizmann.ac.il); it circumvents iterative, high-throughput exptl. screens and opens the way to designing highly efficient and diverse catalytic repertoires.
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    22. 22
      Weinstein, J. J.; Goldenzweig, A.; Hoch, S.-Y.; Fleishman, S. J. PROSS 2: A New Server for the Design of Stable and Highly Expressed Protein Variants. Bioinformatics 2021, 37, 123– 125,  DOI: 10.1093/bioinformatics/btaa1071
      22
      PROSS 2: a new server for the design of stable and highly expressed protein variants
      Weinstein, Jonathan Jacob; Goldenzweig, Adi; Hoch, Shlomoyakir; Fleishman, Sarel Jacob
      Bioinformatics (2021), 37 (1), 123-125CODEN: BOINFP; ISSN:1367-4811. (Oxford University Press)
      Summary: Many natural and designed proteins are only marginally stable limiting their usefulness in research and applications. Recently, we described an automated structure and sequence-based design method, called PROSS, for optimizing protein stability and heterologous expression levels that has since been validated on dozens of proteins. Here, we introduce improvements to the method, workflow and presentation, including more accurate sequence anal., error handling and automated anal. of the quality of the sequence alignment that is used in design calcns.
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    23. 23
      Peleg, Y.; Vincentelli, R.; Collins, B. M.; Chen, K.-E.; Livingstone, E. K.; Weeratunga, S.; Leneva, N.; Guo, Q.; Remans, K.; Perez, K.; Bjerga, G. E. K.; Larsen, Ø.; Vaněk, O.; Skořepa, O.; Jacquemin, S.; Poterszman, A.; Kjær, S.; Christodoulou, E.; Albeck, S.; Dym, O.; Ainbinder, E.; Unger, T.; Schuetz, A.; Matthes, S.; Bader, M.; de Marco, A.; Storici, P.; Semrau, M. S.; Stolt-Bergner, P.; Aigner, C.; Suppmann, S.; Goldenzweig, A.; Fleishman, S. J. Community-Wide Experimental Evaluation of the PROSS Stability-Design Method. J. Mol. Biol. 2021, 433, 166964  DOI: 10.1016/j.jmb.2021.166964
      23
      Community-Wide Experimental Evaluation of the PROSS Stability-Design Method
      Peleg, Yoav; Vincentelli, Renaud; Collins, Brett M.; Chen, Kai-En; Livingstone, Emma K.; Weeratunga, Saroja; Leneva, Natalya; Guo, Qian; Remans, Kim; Perez, Kathryn; Bjerga, Gro E. K.; Larsen, Oeivind; Vanek, Ondrej; Skorepa, Ondrej; Jacquemin, Sophie; Poterszman, Arnaud; Kjaer, Svend; Christodoulou, Evangelos; Albeck, Shira; Dym, Orly; Ainbinder, Elena; Unger, Tamar; Schuetz, Anja; Matthes, Susann; Bader, Michael; de Marco, Ario; Storici, Paola; Semrau, Marta S.; Stolt-Bergner, Peggy; Aigner, Christian; Suppmann, Sabine; Goldenzweig, Adi; Fleishman, Sarel J.
      Journal of Molecular Biology (2021), 433 (13), 166964CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
      Recent years have seen a dramatic improvement in protein-design methodol. Nevertheless, most methods demand expert intervention, limiting their widespread adoption. By contrast, the PROSS algorithm for improving protein stability and heterologous expression levels has been successfully applied to a range of challenging enzymes and binding proteins. Here, we benchmark the application of PROSS as a stand-alone tool for protein scientists with no or limited experience in modeling. Twelve labs. from the Protein Prodn. and Purifn. Partnership in Europe (P4EU) challenged the PROSS algorithm with 14 unrelated protein targets without support from the PROSS developers. For each target, up to six designs were evaluated for expression levels and in some cases, for thermal stability and activity. In nine targets, designs exhibited increased heterologous expression levels either in prokaryotic and/or eukaryotic expression systems under exptl. conditions that were tailored for each target protein. Furthermore, we obsd. increased thermal stability in nine of ten tested targets. In two prime examples, the human Stem Cell Factor (hSCF) and human Cadherin-Like Domain (CLD12) from the RET receptor, the wild type proteins were not expressible as sol. proteins in E. coli, yet the PROSS designs exhibited high expression levels in E. coli and HEK293 cells, resp., and improved thermal stability. We conclude that PROSS may improve stability and expressibility in diverse cases, and that improvement typically requires target-specific expression conditions. This study demonstrates the strengths of community-wide efforts to probe the generality of new methods and recommends areas for future research to advance practically useful algorithms for protein science.
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    24. 24
      Bengel, L. L.; Aberle, B.; Egler-Kemmerer, A.-N.; Kienzle, S.; Hauer, B.; Hammer, S. C. Engineered Enzymes Enable Selective N-Alkylation of Pyrazoles with Simple Haloalkanes. Angew. Chem., Int. Ed. Engl. 2021, 60, 5554– 5560,  DOI: 10.1002/anie.202014239
      24
      Engineered Enzymes Enable Selective N-Alkylation of Pyrazoles With Simple Haloalkanes
      Bengel Ludwig L; Aberle Benjamin; Egler-Kemmerer Alexander-N; Kienzle Samuel; Hauer Bernhard; Hammer Stephan C; Hammer Stephan C
      Angewandte Chemie (International ed. in English) (2021), 60 (10), 5554-5560 ISSN:.
      Selective alkylation of pyrazoles could solve a challenge in chemistry and streamline synthesis of important molecules. Here we report catalyst-controlled pyrazole alkylation by a cyclic two-enzyme cascade. In this enzymatic system, a promiscuous enzyme uses haloalkanes as precursors to generate non-natural analogs of the common cosubstrate S-adenosyl-l-methionine. A second engineered enzyme transfers the alkyl group in highly selective C-N bond formations to the pyrazole substrate. The cosubstrate is recycled and only used in catalytic amounts. Key is a computational enzyme-library design tool that converted a promiscuous methyltransferase into a small enzyme family of pyrazole-alkylating enzymes in one round of mutagenesis and screening. With this enzymatic system, pyrazole alkylation (methylation, ethylation, propylation) was achieved with unprecedented regioselectivity (>99 %), regiodivergence, and in a first example on preparative scale.
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    25. 25
      Barber-Zucker, S.; Mindel, V.; Garcia-Ruiz, E.; Weinstein, J. J.; Alcalde, M.; Fleishman, S. J. Stable and Functionally Diverse Versatile Peroxidases Designed Directly from Sequences. J. Am. Chem. Soc. 2022, 144, 3564– 3571,  DOI: 10.1021/jacs.1c12433
      25
      Stable and Functionally Diverse Versatile Peroxidases Designed Directly from Sequences
      Barber-Zucker, Shiran; Mindel, Vladimir; Garcia-Ruiz, Eva; Weinstein, Jonathan J.; Alcalde, Miguel; Fleishman, Sarel J.
      Journal of the American Chemical Society (2022), 144 (8), 3564-3571CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      White-rot fungi secrete a repertoire of high-redox potential oxidoreductases to efficiently decomp. lignin. Of these enzymes, versatile peroxidases (VPs) are the most promiscuous biocatalysts. VPs are attractive enzymes for research and industrial use but their recombinant prodn. is extremely challenging. To date, only a single VP has been structurally characterized and optimized for recombinant functional expression, stability, and activity. Computational enzyme optimization methods can be applied to many enzymes in parallel but they require accurate structures. Here, we demonstrate that model structures computed by deep-learning-based ab initio structure prediction methods are reliable starting points for one-shot PROSS stability-design calcns. Four designed VPs encoding as many as 43 mutations relative to the wildtype enzymes are functionally expressed in yeast, whereas their wildtype parents are not. Three of these designs exhibit substantial and useful diversity in their reactivity profiles and tolerance to environmental conditions. The reliability of the new generation of structure predictors and design methods increases the scale and scope of computational enzyme optimization, enabling efficient discovery and exploitation of the functional diversity in natural enzyme families directly from genomic databases.
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    26. 26
      Goldenzweig, A.; Fleishman, S. J. Principles of Protein Stability and Their Application in Computational Design. Annu. Rev. Biochem. 2018, 87, 105– 129,  DOI: 10.1146/annurev-biochem-062917-012102
      26
      Principles of Protein Stability and Their Application in Computational Design
      Goldenzweig, Adi; Fleishman, Sarel J.
      Annual Review of Biochemistry (2018), 87 (), 105-129CODEN: ARBOAW; ISSN:0066-4154. (Annual Reviews)
      A review. Proteins are increasingly used in basic and applied biomedical research. Many proteins, however, are only marginally stable and can be expressed in limited amts., thus hampering research and applications. Research has revealed the thermodn., cellular, and evolutionary principles and mechanisms that underlie marginal stability. With this growing understanding, computational stability design methods have advanced over the past two decades starting from methods that selectively addressed only some aspects of marginal stability. Current methods are more general and, by combining phylogenetic anal. with atomistic design, have shown drastic improvements in soly., thermal stability, and aggregation resistance while maintaining the protein's primary mol. activity. Stability design is opening the way to rational engineering of improved enzymes, therapeutics, and vaccines and to the application of protein design methodol. to large proteins and mol. activities that have proven challenging in the past.
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    27. 27
      Piontek, K.; Antorini, M.; Choinowski, T. Crystal Structure of a Laccase from the fungusTrametes Versicolor at 190-Å Resolution Containing a Full Complement of Coppers. J. Biol. Chem. 2002, 277, 37663– 37669,  DOI: 10.1074/jbc.M204571200
      27
      Crystal structure of a laccase from the fungus Trametes versicolor at 1.90-Å resolution containing a full complement of coppers
      Piontek, Klaus; Antorini, Matteo; Choinowski, Thomas
      Journal of Biological Chemistry (2002), 277 (40), 37663-37669CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Laccase is a polyphenol oxidase, which belongs to the family of blue multi-Cu oxidases. These enzymes catalyze the 1-electron oxidn. of 4 reducing-substrate mols. concomitant with the 4-electron redn. of O2 to H2O. Laccases oxidize a broad range of substrates, preferably phenolic compds. In the presence of mediators, fungal laccases exhibit an enlarged substrate range and are then able to oxidize compds. with a redox potential exceeding their own. Until now, only 1 crystal structure of a laccase in an inactive, type-2 Cu-depleted form has been reported. Here, the authors present the 1st crystal structure of an active laccase contg. a full complement of Cu atoms, the complete polypeptide chain together with 7 carbohydrate moieties. Despite the presence of all Cu atoms in the new structure, the folds of the 2 laccases were quite similar. The coordination of the type-3 Cu atoms, however, was distinctly different. The geometry of the trinuclear Cu cluster in T. versicolor laccase was similar to that found in ascorbate oxidase and that of mammalian ceruloplasmin structures, suggesting a common reaction mechanism for Cu oxidn. and O2 redn. In contrast to most blue-Cu proteins, the type-1 Cu atom in T. versicolor laccase had no axial ligand and was only 3-fold coordinated. Previously, a modest elevation of the redox potential was attributed to the lack of an axial ligand. Based on the present structural data and sequence comparisons, a mechanism is presented to explain how laccases could tune their redox potential by as much as 200 mV.
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    28. 28
      Polyakov, K. M.; Fedorova, T. V.; Stepanova, E. V.; Cherkashin, E. A.; Kurzeev, S. A.; Strokopytov, B. V.; Lamzin, V. S.; Koroleva, O. V. Structure of Native Laccase from Trametes Hirsuta at 18 A Resolution. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2009, 65, 611– 617,  DOI: 10.1107/S0907444909011950
      28
      Structure of native laccase from Trametes hirsuta at 1.8 Å resolution
      Polyakov, Konstantin M.; Fedorova, Tatyana V.; Stepanova, Elena V.; Cherkashin, Evgeny A.; Kurzeev, Sergei A.; Strokopytov, Boris V.; Lamzin, Victor S.; Koroleva, Olga V.
      Acta Crystallographica, Section D: Biological Crystallography (2009), 65 (6), 611-617CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)
      An anal. of the crystal structure of the native form of laccase from T. hirsuta at 1.8 Å resoln. is given. This structure provides a basis for the elucidation of the mechanism of catalytic action of these ubiquitous proteins. The 1.8-Å resoln. native structure provided a good level of structural detail compared with many previously reported laccase structures. A brief comparison with the active sites of other laccases is given.
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    29. 29
      Pardo, I.; Santiago, G.; Gentili, P.; Lucas, F.; Monza, E.; Medrano, F. J.; Galli, C.; Martínez, A. T.; Guallar, V.; Camarero, S. Re-Designing the Substrate Binding Pocket of Laccase for Enhanced Oxidation of Sinapic Acid. Catal. Sci. Technol. 2016, 6, 3900– 3910,  DOI: 10.1039/C5CY01725D
      29
      Re-designing the substrate binding pocket of laccase for enhanced oxidation of sinapic acid
      Pardo, I.; Santiago, G.; Gentili, P.; Lucas, F.; Monza, E.; Medrano, F. J.; Galli, C.; Martinez, A. T.; Guallar, V.; Camarero, S.
      Catalysis Science & Technology (2016), 6 (11), 3900-3910CODEN: CSTAGD; ISSN:2044-4753. (Royal Society of Chemistry)
      Iterative satn. mutagenesis was performed over six residues delimiting the substrate binding pocket of a high redox potential chimeric laccase with the aim of enhancing its activity toward sinapic acid (SA), a lignin-related phenol of industrial interest. In total, more than 15 000 clones were screened and two selected variants, together with the parent-type laccase, were purified and characterized. The new variants presented shifted pH activity profiles and enhanced turnover rates on sinapic acid and its Me ester, whereas the oxidn. of related phenols was not significantly enhanced. Neither the enzyme's redox potential nor the mechanism of the reaction was affected. Quantum mechanics and mol. dynamics calcns. were done to rationalize the effect of the selected mutations, revealing the crit. role of the residues of the enzyme pocket to provide the precise binding of the substrate that enables an efficient electron transfer to the T1 copper. The results presented highlight the power of combining directed evolution and computational approaches to give novel solns. in enzyme engineering and to understand the mechanistic reasons behind them, offering new insights for further rational design towards specific targets.
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    30. 30
      Maté, D.; García-Burgos, C.; García-Ruiz, E.; Ballesteros, A. O.; Camarero, S.; Alcalde, M. Laboratory Evolution of High-Redox Potential Laccases. Chem. Biol. 2010, 17, 1030– 1041,  DOI: 10.1016/j.chembiol.2010.07.010
      30
      Laboratory evolution of high-redox potential laccases
      Mate, Diana; Garcia-Burgos, Carlos; Garcia-Ruiz, Eva; Ballesteros, Antonio O.; Camarero, Susana; Alcalde, Miguel
      Chemistry & Biology (Cambridge, MA, United States) (2010), 17 (9), 1030-1041CODEN: CBOLE2; ISSN:1074-5521. (Cell Press)
      Thermostable laccases with a high-redox potential have been engineered through a strategy that combines directed evolution with rational approaches with site-directed mutation. The original laccase signal sequence was replaced by the α-factor prepro-leader, and the corresponding fusion gene was targeted for joint lab. evolution with the aim of improving kinetics and secretion by Saccharomyces cerevisiae, while retaining high thermostability. After 8 rounds of mol. evolution, the total activity of laccase of basidiomycete PM1 was enhanced 34,000-fold culminating in the OB-1 mutant as the last variant of the evolution process, a highly active and stable enzyme in terms of temp., pH range, and org. cosolvents. Mutations in the hydrophobic core of the evolved α-factor prepro-leader enhanced functional expression, whereas some mutations in the mature protein improved its catalytic capacities by altering the interactions with the surrounding residues.
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    31. 31
      Torres-Salas, P.; Mate, D. M.; Ghazi, I.; Plou, F. J.; Ballesteros, A. O.; Alcalde, M. Widening the pH Activity Profile of a Fungal Laccase by Directed Evolution. ChemBioChem 2013, 14, 934– 937,  DOI: 10.1002/cbic.201300102
      31
      Widening the pH Activity Profile of a Fungal Laccase by Directed Evolution
      Torres-Salas, Pamela; Mate, Diana M.; Ghazi, Iraj; Plou, Francisco J.; Ballesteros, Antonio O.; Alcalde, Miguel
      ChemBioChem (2013), 14 (8), 934-937CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
      In this report, we describe the engineering of a fungal laccase with activity at neutral/alk. pH by directed evolution, by using the MtL-R2 mutant as the initial variant, and performing five cycles of in vitro evolution. The final mutant of the directed evolution expt. (variant IG-88) was purified and characterized biochem.
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    32. 32
      Ayuso-Fernández, I.; Ruiz-Dueñas, F. J.; Martínez, A. T. Evolutionary Convergence in Lignin-Degrading Enzymes. Proc. Natl. Acad. Sci. U. S. A. 2018, 115, 6428– 6433,  DOI: 10.1073/pnas.1802555115
      32
      Evolutionary convergence in lignin-degrading enzymes
      Ayuso-Fernandez, Ivan; Ruiz-Duenas, Francisco J.; Martinez, Angel T.
      Proceedings of the National Academy of Sciences of the United States of America (2018), 115 (25), 6428-6433CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      The resurrection of ancestral enzymes of now-extinct organisms (paleogenetics) is a developing field that allows the study of evolutionary hypotheses otherwise impossible to be tested. In the present study, we target fungal peroxidases that play a key role in lignin degrdn., an essential process in the carbon cycle and often a limiting step in biobased industries. Ligninolytic peroxidases are secreted by wood-rotting fungi, the origin of which was recently established in the Carboniferous period assocd. with the appearance of these enzymes. These first peroxidases were not able to degrade lignin directly and used diffusible metal cations to attack its phenolic moiety. The phylogenetic anal. of the peroxidases of Polyporales, the order in which most extant wood-rotting fungi are included, suggests that later in evolution these enzymes would have acquired the ability to degrade nonphenolic lignin using a tryptophanyl radical interacting with the bulky polymer at the surface of the enzyme. Here, we track this powerful strategy for lignin degrdn. as a phenotypic trait in fungi and show that it is not an isolated event in the evolution of Polyporales. Using ancestral enzyme resurrection, we study the mol. changes that led to the appearance of the same surface oxidn. site in two distant peroxidase lineages. By characterization of the resurrected enzymes, we demonstrate convergent evolution at the amino acid level during the evolution of these fungi and track the different changes leading to phylogenetically distant ligninolytic peroxidases from ancestors lacking the ability to degrade nonphenolic lignin.
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    33. 33
      Jones, S. M.; Solomon, E. I. Electron Transfer and Reaction Mechanism of Laccases. Cell. Mol. Life Sci. 2015, 72, 869– 883,  DOI: 10.1007/s00018-014-1826-6
      33
      Electron transfer and reaction mechanism of laccases
      Jones, Stephen M.; Solomon, Edward I.
      Cellular and Molecular Life Sciences (2015), 72 (5), 869-883CODEN: CMLSFI; ISSN:1420-682X. (Birkhaeuser Basel)
      A review. Laccases are part of the family of multicopper oxidases (MCOs), which couple the oxidn. of substrates to the four electron redn. of O2 to H2O. MCOs contain a min. of four Cu's divided into Type 1 (T1), Type 2 (T2), and binuclear Type 3 (T3) Cu sites that are distinguished based on unique spectroscopic features. Substrate oxidn. occurs near the T1, and electrons are transferred approx. 13 Å through the protein via the Cys-His pathway to the T2/T3 trinuclear copper cluster (TNC), where dioxygen redn. occurs. This review outlines the electron transfer (ET) process in laccases, and the mechanism of O2 redn. as elucidated through spectroscopic, kinetic, and computational data. Marcus theory is used to describe the relevant factors which impact ET rates including the driving force, reorganization energy, and electronic coupling matrix element. Then, the mechanism of O2 reaction is detailed with particular focus on the intermediates formed during the two 2e- redn. steps. The first 2e- step forms the peroxide intermediate, followed by the second 2e- step to form the native intermediate, which has been shown to be the catalytically relevant fully oxidized form of the enzyme.
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    34. 34
      Mate, D. M.; Gonzalez-Perez, D.; Falk, M.; Kittl, R.; Pita, M.; De Lacey, A. L.; Ludwig, R.; Shleev, S.; Alcalde, M. Blood Tolerant Laccase by Directed Evolution. Chem. Biol. 2013, 20, 223– 231,  DOI: 10.1016/j.chembiol.2013.01.001
      34
      Blood Tolerant Laccase by Directed Evolution
      Mate, Diana M.; Gonzalez-Perez, David; Falk, Magnus; Kittl, Roman; Pita, Marcos; De Lacey, Antonio L.; Ludwig, Roland; Shleev, Sergey; Alcalde, Miguel
      Chemistry & Biology (Oxford, United Kingdom) (2013), 20 (2), 223-231CODEN: CBOLE2; ISSN:1074-5521. (Elsevier Ltd.)
      High-redox potential laccases are powerful biocatalysts with a wide range of applications in biotechnol. We have converted a thermostable laccase from a white-rot fungus into a blood tolerant laccase. Adapting the fitness of this laccase to the specific compn. of human blood (above neutral pH, high chloride concn.) required several generations of directed evolution in a surrogate complex blood medium. Our evolved laccase was tested in both human plasma and blood, displaying catalytic activity while retaining a high redox potential at the T1 copper site. Mutations introduced in the second coordination sphere of the T1 site shifted the pH activity profile and drastically reduced the inhibitory effect of chloride. This proof of concept that laccases can be adapted to function in extreme conditions opens an array of opportunities for implantable nanobiodevices, chem. syntheses, and detoxification.
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    35. 35
      Madzak, C.; Mimmi, M. C.; Caminade, E.; Brault, A.; Baumberger, S.; Briozzo, P.; Mougin, C.; Jolivalt, C. Shifting the Optimal pH of Activity for a Laccase from the Fungus Trametes Versicolor by Structure-Based Mutagenesis. Protein Eng., Des. Sel. 2006, 19, 77– 84,  DOI: 10.1093/protein/gzj004
      35
      Shifting the optimal pH of activity for a laccase from the fungus Trametes versicolor by structure-based mutagenesis
      Madzak, C.; Mimmi, M. C.; Caminade, E.; Brault, A.; Baumberger, S.; Briozzo, P.; Mougin, C.; Jolivalt, C.
      Protein Engineering, Design & Selection (2006), 19 (2), 77-84CODEN: PEDSBR; ISSN:1741-0126. (Oxford University Press)
      Laccases are oxidizing enzymes of interest because of their potential environmental and industrial applications. We performed site-directed mutagenesis of a laccase produced by Trametes versicolor in order to improve its catalytic properties. Considering a strong interaction of the Asp residue in position 206 with the substrate xylidine, we replaced it with Glu, Ala or Asn, expressed the mutant enzymes in the yeast Yarrowia lipolytica and assayed the transformation of phenolic and non-phenolic substrates. The transformation rates remain within the same range whatever the mutation of the laccase and the type of substrate: at most a 3-fold factor increase was obtained for kcat between the wild-type and the most efficient mutant Asp206Ala with 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic) acid as a substrate. Nevertheless, the Asn mutation led to a significant shift of the pH (ΔpH = 1.4) for optimal activity against 2,6-dimethoxyphenol. This study also provides a new insight into the binding of the reducing substrate into the active T1 site and induced modifications in catalytic properties of the enzyme.
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    36. 36
      Galli, C.; Gentili, P.; Jolivalt, C.; Madzak, C.; Vadalà, R. How Is the Reactivity of Laccase Affected by Single-Point Mutations? Engineering Laccase for Improved Activity towards Sterically Demanding Substrates. Appl. Microbiol. Biotechnol. 2011, 91, 123– 131,  DOI: 10.1007/s00253-011-3240-4
      36
      How is the reactivity of laccase affected by single-point mutations? Engineering laccase for improved activity towards sterically demanding substrates
      Galli, Carlo; Gentili, Patrizia; Jolivalt, Claude; Madzak, Catherine; Vadala, Raffaella
      Applied Microbiology and Biotechnology (2011), 91 (1), 123-131CODEN: AMBIDG; ISSN:0175-7598. (Springer)
      In spite of its broad specificity among phenols, Trametes versicolor laccase (I) hardly succeeds in oxidizing hindered substrates. To improve the oxidn. ability of this I toward bulky phenolic substrates, the authors designed a series of single-point mutants on the basis of the amino acid layout inside the reducing substrate active site known from the crystal structure of the enzyme. Here, site-directed mutagenesis of I addressed 4 Phe residues in key positions (Phe-162, Phe-265, Phe-332, and Phe-337) at the entrance of the binding pocket, as these residues appeared instrumental for docking of the substrate. These Phe residues were replaced by smaller-sized but still apolar Ala residues. A I double mutant (F162A/F332A) was also designed. Measurement of the oxidn. efficiency toward encumbered phenols showed that mutant F162A was more efficient than wild-type I. Double mutant F162A/F332A led to 98% consumption of bisphenol A in only 5 h and was more efficient than the single mutants in the aerobic oxidn. of this bulky substrate. In contrast, lack of appropriate hydrophobic interactions with the substrate possibly depressed the oxidn. outcome with mutants F265A and F332A. One explanation for the lack of reactivity of mutant F337A, supported by literature reports, is that this residue is part of the 2nd coordination shell of type 1 Cu. A mutation at this position thus leads to a drastic coordination shell destabilization. The thermostability of the mutants and their resistance in a mixed water-dioxane solvent were also investigated.
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    37. 37
      Mateljak, I.; Tron, T.; Alcalde, M. Evolved α-factor Prepro-leaders for Directed Laccase Evolution in Saccharomyces Cerevisiae. Microb. Biotechnol. 2017, 10, 1830– 1836,  DOI: 10.1111/1751-7915.12838
      37
      Evolved α-factor prepro-leaders for directed laccase evolution in Saccharomyces cerevisiae
      Mateljak, Ivan; Tron, Thierry; Alcalde, Miguel
      Microbial Biotechnology (2017), 10 (6), 1830-1836CODEN: MBIIB2; ISSN:1751-7915. (Wiley-Blackwell)
      Summary : Although the functional expression of fungal laccases in Saccharomyces cerevisiae has proven to be complicated, the replacement of signal peptides appears to be a suitable approach to enhance secretion in directed evolution expts. In this study, twelve constructs were prepd. by fusing native and evolved α-factor prepro-leaders from S. cerevisiae to four different laccases with low-, medium- and high-redox potential (PM1L from basidiomycete PM1; PcL from Pycnoporus cinnabarinus; TspC30L from Trametes sp. strain C30; and MtL from Myceliophthora thermophila). Microcultures of the prepro-leader:laccase fusions were grown in selective expression medium that used galactose as both the sole carbon source and as the inducer of expression so that the secretion and activity were assessed with low- and high-redox potential mediators in a high-throughput screening context. With total activity improvements as high as sevenfold over those obtained with the native α-factor prepro-leader, the evolved prepro-leader from PcL (αPcL) most strongly enhanced secretion of the high- and medium-redox potential laccases PcL, PM1L and TspC30L in the microtiter format with an expression pattern driven by prepro-leaders in the order αPcL > αPM1L ∼ αnative. By contrast, the pattern of the low-redox potential MtL was αnative > αPcL > αPM1L. When produced in flask with rich medium, the evolved prepro-leaders outperformed the αnative signal peptide irresp. of the laccase attached, enhancing secretion over 50-fold. Together, these results highlight the importance of using evolved α-factor prepro-leaders for functional expression of fungal laccases in directed evolution campaigns.
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    38. 38
      Pardo, I.; Rodríguez-Escribano, D.; Aza, P.; de Salas, F.; Martínez, A. T.; Camarero, S. A Highly Stable Laccase Obtained by Swapping the Second Cupredoxin Domain. Sci. Rep. 2018, 8, 15669,  DOI: 10.1038/s41598-018-34008-3
      38
      A highly stable laccase obtained by swapping the second cupredoxin domain
      Pardo Isabel; Rodriguez-Escribano David; Aza Pablo; de Salas Felipe; Martinez Angel T; Camarero Susana; Pardo Isabel
      Scientific reports (2018), 8 (1), 15669 ISSN:.
      The robustness of a high-redox potential laccase has been enhanced by swapping its second cupredoxin domain with that from another fungal laccase, which introduced a pool of neutral mutations in the protein sequence without affecting enzyme functionality. The new laccase showed outstanding stability to temperature, pH (2-9) and to organic solvents, while maintaining the ability to oxidize high-redox potential substrates. By engineering the signal peptide, enzyme secretion levels in Saccharomyces cerevisiae were increased, which allowed to purify the engineered enzyme for further characterization. The purified domain-swap laccase presented higher activity in the presence of ethanol or methanol, superior half-lives at 50-70 °C, improved stability at acidic pH, and similar catalytic efficiency for DMP albeit a lower one for ABTS (due to a shift in optimum pH). A new N-glycosylation site and a putative new surface salt-bridge were evaluated as possible determinants for the improved stability by site-directed mutagenesis. Although neither seemed to be strictly responsible for the improved thermostability, the new salt bridge was found to notably contribute to the high stability of the swapped enzyme in a broad pH range. Finally, the application potential of the new laccase was demonstrated with the enzymatic treatment of kraft lignin, an industrially relevant lignin stream, at high temperature, neutral pH and short incubation times.
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    39. 39
      Hildén, K.; Hakala, T. K.; Lundell, T. Thermotolerant and Thermostable Laccases. Biotechnol. Lett. 2009, 31, 1117– 1128,  DOI: 10.1007/s10529-009-9998-0
      39
      Thermotolerant and thermostable laccases
      Hilden, Kristiina; Hakala, Terhi K.; Lundell, Taina
      Biotechnology Letters (2009), 31 (8), 1117-1128CODEN: BILED3; ISSN:0141-5492. (Springer)
      A review. Laccases are phenol-oxidizing, usually 4-Cu-contg. metalloenzymes. For industrial and biotechnol. purposes, laccases have been among the 1st fungal oxidoreductases providing larger-scale applications such as removal of polyphenols in wine and beverages, conversion of toxic compds. and textile dyes in waste waters, and in bleaching and removal of lignin from wood and non-wood fibers. In order to facilitate novel and more efficient biocatalytic process applications, there is a need for laccases with improved biochem. properties, such as thermostability and thermotolerance. Here, the authors provide a current overview of the sources and characteristics of such laccases, with particular emphasis on the fungal enzymes.
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    40. 40
      Bar-Even, A.; Noor, E.; Savir, Y.; Liebermeister, W.; Davidi, D.; Tawfik, D. S.; Milo, R. The Moderately Efficient Enzyme: Evolutionary and Physicochemical Trends Shaping Enzyme Parameters. Biochemistry 2011, 50, 4402– 4410,  DOI: 10.1021/bi2002289
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      The Moderately Efficient Enzyme: Evolutionary and Physicochemical Trends Shaping Enzyme Parameters
      Bar-Even, Arren; Noor, Elad; Savir, Yonatan; Liebermeister, Wolfram; Davidi, Dan; Tawfik, Dan S.; Milo, Ron
      Biochemistry (2011), 50 (21), 4402-4410CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      The kinetic parameters of enzymes are key to understanding the rate and specificity of most biol. processes. Although specific trends are frequently studied for individual enzymes, global trends are rarely addressed. We performed an anal. of kcat and KM values of several thousand enzymes collected from the literature. We found that the "av. enzyme" exhibits a kcat of ∼10 s-1 and a kcat/KM of ∼ 105 s-1 M-1, much below the diffusion limit and the characteristic textbook portrayal of kinetically superior enzymes. Why do most enzymes exhibit moderate catalytic efficiencies Maximal rates may not evolve in cases where weaker selection pressures are expected. We find, for example, that enzymes operating in secondary metab. are, on av., ∼ 30-fold slower than those of central metab. We also find indications that the physicochem. properties of substrates affect the kinetic parameters. Specifically, low mol. mass and hydrophobicity appear to limit KM optimization. In accordance, substitution with phosphate, CoA, or other large modifiers considerably lowers the KM values of enzymes utilizing the substituted substrates. It therefore appears that both evolutionary selection pressures and physicochem. constraints shape the kinetic parameters of enzymes. It also seems likely that the catalytic efficiency of some enzymes toward their natural substrates could be increased in many cases by natural or lab. evolution.
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    41. 41
      Nannemann, D. P.; Birmingham, W. R.; Scism, R. A.; Bachmann, B. O. Assessing Directed Evolution Methods for the Generation of Biosynthetic Enzymes with Potential in Drug Biosynthesis. Future Med. Chem. 2011, 3, 809– 819,  DOI: 10.4155/fmc.11.48
      41
      Assessing directed evolution methods for the generation of biosynthetic enzymes with potential in drug biosynthesis
      Nannemann, David P.; Birmingham, William R.; Scism, Robert A.; Bachmann, Brian O.
      Future Medicinal Chemistry (2011), 3 (7), 809-819CODEN: FMCUA7; ISSN:1756-8919. (Future Science Ltd.)
      To address the synthesis of increasingly structurally diverse small-mol. drugs, methods for the generation of efficient and selective biol. catalysts are becoming increasingly important. Directed evolution is an umbrella term referring to a variety of methods for improving or altering the function of enzymes using a nature-inspired twofold strategy of mutagenesis followed by selection. This article provides an objective assessment of the effectiveness of directed evolution campaigns in generating enzymes with improved catalytic parameters for new substrates from the last decade, excluding studies that aimed to select for only improved phys. properties and those that lack kinetic characterization. An anal. of the trends of methodologies and their success rates from 81 qualifying examples in the literature reveals the av. fold improvement for k cat (or V max), K m and k cat/K m to be 366-, 12- and 2548-fold, resp., whereas the median fold improvements are 5.4, 3 and 15.6. Further anal. by enzyme class, library-generation methodol. and screening methodol. explores relationships between successful campaigns and the methodologies employed.
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    42. 42
      Hosseinzadeh, P.; Lu, Y. Design and Fine-Tuning Redox Potentials of Metalloproteins Involved in Electron Transfer in Bioenergetics. Biochim. Biophys. Acta 2016, 1857, 557– 581,  DOI: 10.1016/j.bbabio.2015.08.006
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      Design and fine-tuning redox potentials of metalloproteins involved in electron transfer in bioenergetics
      Hosseinzadeh, Parisa; Lu, Yi
      Biochimica et Biophysica Acta, Bioenergetics (2016), 1857 (5), 557-581CODEN: BBBEB4; ISSN:0005-2728. (Elsevier B. V.)
      Redox potentials are a major contributor in controlling the electron transfer (ET) rates and thus regulating the ET processes in the bioenergetics. To maximize the efficiency of the ET process, one needs to master the art of tuning the redox potential, esp. in metalloproteins, as they represent major classes of ET proteins. In this review, we first describe the importance of tuning the redox potential of ET centers and its role in regulating the ET in bioenergetic processes including photosynthesis and respiration. The main focus of this review is to summarize recent work in designing the ET centers, namely cupredoxins, cytochromes, and iron-sulfur proteins, and examples in design of protein networks involved these ET centers. We then discuss the factors that affect redox potentials of these ET centers including metal ion, the ligands to metal center and interactions beyond the primary ligand, esp. non-covalent secondary coordination sphere interactions. We provide examples of strategies to fine-tune the redox potential using both natural and unnatural amino acids and native and nonnative cofactors. Several case studies are used to illustrate recent successes in this area. Outlooks for future endeavors are also provided.
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    43. 43
      Bertrand, T.; Jolivalt, C.; Briozzo, P.; Caminade, E.; Joly, N.; Madzak, C.; Mougin, C. Crystal Structure of a Four-Copper Laccase Complexed with an Arylamine: Insights into Substrate Recognition and Correlation with Kinetics. Biochemistry 2002, 41, 7325– 7333,  DOI: 10.1021/bi0201318
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      Crystal structure of a four-copper laccase complexed with an arylamine: Insights into substrate recognition and correlation with kinetics
      Bertrand, Thomas; Jolivalt, Claude; Briozzo, Pierre; Caminade, Eliane; Joly, Nathalie; Madzak, Catherine; Mougin, Christian
      Biochemistry (2002), 41 (23), 7325-7333CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      Laccases are multi-Cu oxidases that catalyze the oxidn. of a wide range of phenols or arylamines, and their use in industrial oxidative processes is increasing. The authors previously purified from the white rot fungus, Trametes versicolor, a laccase that exists as 5 different isoenzymes, depending on glycosylation. Here, the 2.4 Å resoln. structure of the most abundant isoenzyme of the glycosylated enzyme was solved. The 4 Cu atoms were present, and it is the 1st crystal structure of a laccase in its active form. The crystd. enzyme bound 2,5-xylidine, which was used as a laccase inducer in the fungus culture. This arylamine was a very weak reducing substrate of the enzyme. The cavity enclosing 2,5-xylidine was found to be rather wide, allowing the accommodation of substrates of various sizes. Several amino acid residues made hydrophobic interactions with the arom. ring of the ligand. In addn., 2 charged or polar residues interacted with its amino group. The 1st was a His residue that also coordinated the Cu that functions as the primary electron acceptor. The 2nd was an Asp residue conserved among fungal laccases. The purified enzyme could oxidize various hydroxylated compds. of the phenylurea family of herbicides that the authors synthesized. These phenolic substrates had better affinities at pH 5 than at pH 3, which could be related to the binding of 2,5-xylidine by the Asp residue. This is the 1st high-resoln. structure of a multi-Cu oxidase complexed to a reducing substrate. It provides a model for engineering laccases that are either more efficient or with a wider substrate specificity.
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    44. 44
      Jurrus, E.; Engel, D.; Star, K.; Monson, K.; Brandi, J.; Felberg, L. E.; Brookes, D. H.; Wilson, L.; Chen, J.; Liles, K.; Chun, M.; Li, P.; Gohara, D. W.; Dolinsky, T.; Konecny, R.; Koes, D. R.; Nielsen, J. E.; Head-Gordon, T.; Geng, W.; Krasny, R.; Wei, G.-W.; Holst, M. J.; McCammon, J. A.; Baker, N. A. Improvements to the APBS Biomolecular Solvation Software Suite. Protein Sci. 2018, 27, 112– 128,  DOI: 10.1002/pro.3280
      44
      Improvements to the APBS biomolecular solvation software suite
      Jurrus, Elizabeth; Engel, Dave; Star, Keith; Monson, Kyle; Brandi, Juan; Felberg, Lisa E.; Brookes, David H.; Wilson, Leighton; Chen, Jiahui; Liles, Karina; Chun, Minju; Li, Peter; Gohara, David W.; Dolinsky, Todd; Konecny, Robert; Koes, David R.; Nielsen, Jens Erik; Head-Gordon, Teresa; Geng, Weihua; Krasny, Robert; Wei, Guo-Wei; Holst, Michael J.; McCammon, J. Andrew; Baker, Nathan A.
      Protein Science (2018), 27 (1), 112-128CODEN: PRCIEI; ISSN:1469-896X. (Wiley-Blackwell)
      The Adaptive Poisson-Boltzmann Solver (APBS) software was developed to solve the equations of continuum electrostatics for large biomol. assemblages that have provided impact in the study of a broad range of chem., biol., and biomedical applications. APBS addresses the three key technol. challenges for understanding solvation and electrostatics in biomedical applications: accurate and efficient models for biomol. solvation and electrostatics, robust and scalable software for applying those theories to biomol. systems, and mechanisms for sharing and analyzing biomol. electrostatics data in the scientific community. To address new research applications and advancing computational capabilities, we have continually updated APBS and its suite of accompanying software since its release in 2001. In this article, we discuss the models and capabilities that have recently been implemented within the APBS software package including a Poisson-Boltzmann anal. and a semi-anal. solver, an optimized boundary element solver, a geometry-based geometric flow solvation model, a graph theory-based algorithm for detg. pKa values, and an improved web-based visualization tool for viewing electrostatics.
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    45. 45
      Mehra, R.; Muschiol, J.; Meyer, A. S.; Kepp, K. P. A Structural-Chemical Explanation of Fungal Laccase Activity. Sci. Rep. 2018, 8, 17285,  DOI: 10.1038/s41598-018-35633-8
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      A structural-chemical explanation of fungal laccase activity
      Mehra Rukmankesh; Kepp Kasper P; Mehra Rukmankesh; Muschiol Jan; Meyer Anne S
      Scientific reports (2018), 8 (1), 17285 ISSN:.
      Fungal laccases (EC 1.10.3.2) are multi-copper oxidases that oxidize a wide variety of substrates. Despite extensive studies, the molecular basis for their diverse activity is unclear. Notably, there is no current way to rationally predict the activity of a laccase toward a given substrate. Such knowledge would greatly facilitate the rational design of new laccases for technological purposes. We report a study of three datasets of experimental Km values and activities for Trametes versicolor and Cerrena unicolor laccase, using a range of protein modeling techniques. We identify diverse binding modes of the various substrates and confirm an important role of Asp-206 and His-458 (T. versicolor laccase numbering) in guiding substrate recognition. Importantly, we demonstrate that experimental Km values correlate with binding affinities computed by MMGBSA. This confirms the common assumption that the protein-substrate affinity is a major contributor to observed Km. From quantitative structure-activity relations (QSAR) we identify physicochemical properties that correlate with observed Km and activities. In particular, the ionization potential, shape, and binding affinity of the substrate largely determine the enzyme's Km for the particular substrate. Our results suggest that Km is not just a binding constant but also contains features of the enzymatic activity. In addition, we identify QSAR models with only a few descriptors showing that phenolic substrates employ optimal hydrophobic packing to reach the T1 site, but then require additional electronic properties to engage in the subsequent electron transfer. Our results advance our ability to model laccase activity and lend promise to future rational optimization of laccases toward phenolic substrates.
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    46. 46
      Boerjan, W.; Ralph, J.; Baucher, M. Lignin Biosynthesis. Annu. Rev. Plant Biol. 2003, 54, 519– 546,  DOI: 10.1146/annurev.arplant.54.031902.134938
      46
      Lignin biosynthesis
      Boerjan, Wout; Ralph, John; Baucher, Marie
      Annual Review of Plant Biology (2003), 54 (), 519-546CODEN: ARPBDW ISSN:. (Annual Reviews Inc.)
      A review. The lignin biosynthetic pathway has been studied for more than a century but has undergone major revisions over the past decade. Significant progress has been made in cloning new genes by genetic and combined bioinformatics and biochem. approaches. In vitro enzymic assays and detailed analyses of mutants and transgenic plants altered in the expression of lignin biosynthesis genes have provided a solid basis for redrawing the monolignol biosynthetic pathway, and structural analyses have shown that plant cell walls can tolerate large variations in lignin content and structure. In some cases, the potential value for agriculture of transgenic plants with modified lignin structure has been demonstrated. This review presents a current picture of monolignol biosynthesis, polymn., and lignin structure.
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    47. 47
      Dashtban, M.; Schraft, H.; Syed, T. A.; Qin, W. Fungal Biodegradation and Enzymatic Modification of Lignin. Int. J. Biochem. Mol. Biol. 2010, 1, 36– 50
      47
      Fungal biodegradation and enzymatic modification of lignin
      Dashtban, Mehdi; Schraft, Heidi; Syed, Tarannum A.; Qin, Wensheng
      International Journal of Biochemistry and Molecular Biology (2010), 1 (1), 36-50CODEN: IJBMHV; ISSN:2152-4114. (e-Century Publishing Corp.)
      A review. Lignin, the most abundant arom. biopolymer on Earth, is extremely recalcitrant to degrdn. By linking to both hemicellulose and cellulose, it creates a barrier to any solns. or enzymes and prevents the penetration of lignocellulolytic enzymes into the interior lignocellulosic structure. Some basidiomycetes white-rot fungi are able to degrade lignin efficiently using a combination of extracellular ligninolytic enzymes, org. acids, mediators and accessory enzymes. This review describes ligninolytic enzyme families produced by these fungi that are involved in wood decay processes, their mol. structures, biochem. properties and the mechanisms of action which render them attractive candidates in biotechnol. applications. These enzymes include phenol oxidase (laccase) and heme peroxidases [lignin peroxidase (LiP), manganese peroxidase (MnP) and versatile peroxidase (VP)]. Accessory enzymes such as H2O2-generating oxidases and degrdn. mechanisms of plant cell-wall components in a non-enzymic manner by prodn. of free hydroxyl radicals (·OH) are also discussed.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXitlOrurs%253D&md5=e29660b235d6306ce814933a9fbc6fc1
    48. 48
      Camarero, S.; Martínez, M. J.; Martínez, A. T. Understanding Lignin Biodegradation for the Improved Utilization of Plant Biomass in Modern Biorefineries. Biofuels, Bioprod. Biorefin. 2014, 8, 615– 625,  DOI: 10.1002/bbb.1467
      48
      Understanding lignin biodegradation for the improved utilization of plant biomass in modern biorefineries
      Camarero, Susana; Martinez, Maria Jesus; Martinez, Angel T.
      Biofuels, Bioproducts & Biorefining (2014), 8 (5), 615-625CODEN: BBBICH; ISSN:1932-104X. (John Wiley & Sons Ltd.)
      A review. Wood-rotting fungi are the sole organisms in nature able to degrade the lignin polymer making the polysaccharide components of lignocellulose fully accessible. This process has been investigated for decades as a model for biotechnol. application in the pulp and paper industry, animal feeding, and ethanol prodn. In the current lignocellulose biorefinery concept, ligninolytic fungi and the oxidoreductases (laccases and peroxidases) secreted by these fungi constitute powerful biotechnol. tools for the complete utilization of plant biomass. The evolution of mol. biol., which brings into play specifically designed biol. systems and on-demand enzymes, together with the technol. advances in processing of plant biomass, smoothes the way for a sustainable conversion of renewable feedstocks to new added-value products, with lower energy costs and less environmental impact. The present study reviews some of the main achievements attained by our group in the field of lignin biodegrdn. that have contributed to: (i) better understanding of the mechanisms by which fungi delignify the lignocellulosic materials; and (ii) assessing the applicability of these ligninolytic systems to increase the efficiency of some industrial processes and to develop new means for sustainable and environmentally sound prodn. of chems., materials, and fuels. © 2014 Society of Chem. Industry and John Wiley & Sons, Ltd.
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    49. 49
      Martínez, A. T.; Ruiz-Dueñas, F. J.; Martínez, M. J.; Del Río, J. C.; Gutiérrez, A. Enzymatic Delignification of Plant Cell Wall: From Nature to Mill. Curr. Opin. Biotechnol. 2009, 20, 348– 357,  DOI: 10.1016/j.copbio.2009.05.002
      49
      Enzymatic delignification of plant cell wall: From nature to mill
      Martinez, Angel T.; Ruiz-Duenas, Francisco J.; Martinez, Maria Jesus; del Rio, Jose C.; Gutierrez, Ana
      Current Opinion in Biotechnology (2009), 20 (3), 348-357CODEN: CUOBE3; ISSN:0958-1669. (Elsevier B.V.)
      A review. Lignin removal is a central issue in paper pulp manuf., and prodn. of other renewable chems., materials, and biofuels in future lignocellulose biorefineries. Biotechnol. can contribute to more efficient and environmentally sound deconstruction of plant cell wall by providing tailor-made biocatalysts based on the oxidative enzymes responsible for lignin attack in Nature. With this purpose, the already-known ligninolytic oxidoreductases are being improved using (rational and random-based) protein engineering, and still unknown enzymes will be identified by the application of the different "omics" technologies. Enzymic delignification will be soon at the pulp mill (combined with pitch removal) and our understanding of the reactions produced will increase by using modern techniques for lignin anal.
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    50. 50
      Sethupathy, S.; Morales, G. M.; Li, Y.; Wang, Y.; Jiang, J.; Sun, J.; Zhu, D. Harnessing Microbial Wealth for Lignocellulose Biomass Valorization through Secretomics: A Review. Biotechnol. Biofuels 2021, 14, 154,  DOI: 10.1186/s13068-021-02006-9
      50
      Harnessing microbial wealth for lignocellulose biomass valorization through secretomics: a review
      Sethupathy, Sivasamy; Morales, Gabriel Murillo; Li, Yixuan; Wang, Yongli; Jiang, Jianxiong; Sun, Jianzhong; Zhu, Daochen
      Biotechnology for Biofuels (2021), 14 (1), 154CODEN: BBIIFL; ISSN:1754-6834. (BioMed Central Ltd.)
      A review. The recalcitrance of lignocellulosic biomass is a major constraint to its high-value use at industrial scale. In nature, microbes play a crucial role in biomass degrdn., nutrient recycling and ecosystem functioning. Therefore, the use of microbes is an attractive way to transform biomass to produce clean energy and high-value compds. The microbial degrdn. of lignocelluloses is a complex process which is dependent upon multiple secreted enzymes and their synergistic activities. The availability of the cutting edge proteomics and highly sensitive mass spectrometry tools make possible for researchers to probe the secretome of microbes and microbial consortia grown on different lignocelluloses for the identification of hydrolytic enzymes of industrial interest and their substrate-dependent expression. This review summarizes the role of secretomics in identifying enzymes involved in lignocelluloses deconstruction, the development of enzyme cocktails and the construction of synthetic microbial consortia for biomass valorization, providing our perspectives to address the current challenges.
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    51. 51
      Khersonsky, O.; Fleishman, S. J. What Have We Learned from Design of Function in Large Proteins?. BioDesign Research 2022, 2022, 1– 11,  DOI: 10.34133/2022/9787581
      There is no corresponding record for this reference.
    52. 52
      Perez-Riverol, Y.; Bai, J.; Bandla, C.; García-Seisdedos, D.; Hewapathirana, S.; Kamatchinathan, S.; Kundu, D. J.; Prakash, A.; Frericks-Zipper, A.; Eisenacher, M.; Walzer, M.; Wang, S.; Brazma, A.; Vizcaíno, J. A. The PRIDE Database Resources in 2022: A Hub for Mass Spectrometry-Based Proteomics Evidences. Nucleic Acids Res. 2022, 50, D543– D552,  DOI: 10.1093/nar/gkab1038
      52
      PRIDE database resources in 2022 hub for mass spectrometry-based proteomics evidences
      Perez-Riverol, Yasset; Bai, Jingwen; Bandla, Chakradhar; Garcia-Seisdedos, David; Hewapathirana, Suresh; Kamatchinathan, Selvakumar; Kundu, Deepti J.; Prakash, Ananth; Frericks-Zipper, Anika; Eisenacher, Martin; Walzer, Mathias; Wang, Shengbo; Brazma, Alvis; Vizcaino, Juan Antonio
      Nucleic Acids Research (2022), 50 (D1), D543-D552CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
      The PRoteomics IDEntifications (PRIDE) database is the world's largest data repository of mass spectrometry-based proteomics data. PRIDE is one of the founding members of the global ProteomeXchange (PX) consortium and an ELIXIR core data resource. In this manuscript, we summarize the developments in PRIDE resources and related tools since the previous update manuscript was published in Nucleic Acids Research in 2019. The no. of submitted datasets to PRIDE Archive (the archival component of PRIDE) has reached on av. around 500 datasets per mo during 2021. In addn. to continuous improvements in PRIDE Archive data pipelines and infrastructure, the PRIDE Spectra Archive has been developed to provide direct access to the submitted mass spectra using Universal Spectrum Identifiers. As a key point, the file format MAGE-TAB for proteomics has been developed to enable the improvement of sample metadata annotation. Addnl., the resource PRIDE Peptidome provides access to aggregated peptide/protein evidences across PRIDE Archive. Furthermore, we will describe how PRIDE has increased its efforts to reuse and disseminate high-quality proteomics data into other added-value resources such as UniProt, Ensembl and Expression Atlas.
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  • Supporting Information

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    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acscatal.2c03006.

    • A detailed description of the computational methods, materials, and experimental procedures; amino acid sequences of the wild type, PROSS designs and characterized FuncLib designs; Tables S1–S3 include the selected sequences’ origins, lengths, PDB entries and their PROSS-designed mutational loads, and the FuncLib mutations in all screened designs; Table S4 reports the full kinetic data; Figures S1–S5 show the full stability and activity profiles of PROSS and FuncLib designs and purification analysis of the Th3 designs. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (52) partner repository with the data set identifier PXD034630 and 10.6019/PXD034630. Plasmids encoding Th3, Th3.1, Th3.7, Th3.10, Th314, and Tv9nL are available from AddGene (IDs 188064–188069) (PDF)


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