Synthesis of isopentenyl phosphate.
Isopentenyl phosphate was prepared by coupling 2-cyanoethyl phosphate and 3-methyl-3-buten-1-ol followed by mild alkaline hydrolysis according to the method of Tener (
34). Barium 2-cyanoethylphosphate (1 mmol, 322 mg) was suspended in 1 ml of water and mixed with 2 ml of a suspension of Dowex 50W-X8 H
+ in water. The resulting slurry was poured into a column, and the 2-cyanoethyl phosphate was eluted with 1 ml water. The combined eluate was dried by evaporation of the water, dissolved in pyridine (1 ml), and evaporated from pyridine (1 ml) two additional times before finally being dissolved in 1.5 ml of pyridine. To this solution was added 100 μl of 3-methyl-3-buten-1-ol (1 mmol) followed by 660 mg of dicyclohexylcarbodiimide, and the mixture was stirred for 2 days at room temperature. After the addition of 0.5 ml of water and stirring for 30 min, an additional 5 ml of water was added and the sample was filtered to remove the 1,3-dicyclohexylurea precipitate. The resulting sample was concentrated by evaporation with nitrogen, and the 2-cyanoethyl phosphate derivative of 3-methyl-3-buten-1-ol was extracted twice with 2 ml methylene chloride. The combined extracts were dried over Na
2SO
4 and evaporated. The yield of crude 2-cyanoethyl derivative was 0.25 g. The sample was dissolved in 8 ml of 1 M aqueous cyclohexylamine, held for 12 h at 3°C, and concentrated by evaporation of the solvent with a stream of nitrogen gas, and the excess cyclohexylamine was extracted three times with 2 ml methylene chloride. The sample was concentrated and the cyclohexylamine salt crystallized from either ethanol or ethanol-water mixtures to yield 124 mg of white crystals.
1H nuclear magnetic resonance (NMR) (400 MHz; D2O) of the isopentenyl phosphate didicyclohexylamine salt showed chemical shifts, referenced to sodium 3-trimethylsilylpropionate-2,2,3,3-d4 (TMS), at the following values: IP δ, 4.844 (2H, m, vinyl hydrogens), 3.883 (2H, dt, JH-1→P = 5.66 Hz, JH-1→H-2,2′ = 7.03 Hz, C-1 methylene), 2.350 (2H, t, JH-2→H-1,1′ = 7.03 Hz, C-2 methylene), and 1.780 (3H, s, 3-methyl). In addition, resonance values of δ 3.16 (2H, m, H-C-NH3 +), 2.0 (4H, m, NH3 +-CH-CH eq), 1.82 (4H, m, NH3 +-CH-CH ax), and 1.35 (12H, m, CH2) for two equivalents of the dicyclohexylamine salt were also observed. 13C NMR (D2O) showed chemical shifts at δ 113.9 (CH2=), 65.7 (CH2OP), 33.2 (CH2), and 24.7 (3-methyl) for the isopentenyl phosphate and δ 53.0 (C-1), 33.2 (C-2 and C-2′), 27.0 (C-4), and 26.6 (C-3 and C-3′) for the cyclohexylamine salt. Proton-decoupled 31P NMR showed a single resonance at δ 4.38 (1P, s). Negative matrix-assisted laser desorption ionization (MALDI) analysis performed using a 2,5-hydroxybenzoic acid matrix showed a [M-H]− ion at m/z 165. A solution containing 1 mg of the sample in water (100 μl) and passed through a Dowex 50W-X8 H+ column with subsequent removal of the water by evaporation produced the free acid of isopentenyl phosphate. Reaction of this product with 20 μl of a mixture of pyridine, hexamethyldisilazane, and chlorotrimethylsilane (9:3:1 [vol/vol/vol]) for 2 min at 100°C followed by electron impact mass spectral analysis showed M+ = 310 m/z, M+-15 (CH3) = 295 m/z, M+-67 = 243 m/z, M+-15 -68 (CH2=C(CH3)-CH=CH2) = 227 m/z, and M+-15 -84 (CH2=C(CH3)-CH2-CH=O) = 211 m/z for the di-TMS derivative.
Cloning and heterologous expression of MJ0044.
The
M. jannaschii gene MJ0044 (Swiss-Prot accession number Q60352) was amplified by PCR from genomic DNA by use of the following oligonucleotide primers synthesized by Invitrogen: for MJ0044-F, 5′-GGTCATATGCTAACCATATTAAAATTAGG-3′, and for MJ0044-R, 5′-GCTGGATCCTTATTCTGAAAAATC-3′. PCR was performed as described previously using a 55°C annealing temperature (
15). The amplified PCR product was purified by use of QIAquick spin column (QIAGEN), digested with restriction enzymes NdeI and BamHI, and then ligated into the compatible sites in plasmid pT7-7 (USB). DNA sequences were verified by dye-terminator sequencing at the Virginia Bioinformatics Institute's DNA facility. The resulting plasmid, pMJ0044, was transformed into
Escherichia coli BL21-CodonPlus (DE3)-RIL cells (Stratagene). The transformed cells were grown in Luria-Bertani medium (Difco) (200 ml) supplemented with 100 μg/ml ampicillin at 37°C with shaking until they reached an optical density at 600 nm of 1.0. Recombinant protein production was induced by addition of lactose to a final concentration of 28 mM. After an additional 2 h of culture, the cells were harvested by centrifugation (4,000 ×
g, 5 min) and frozen at −20°C. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of total cellular proteins by use of a Tris-glycine buffer system confirmed induction of the desired protein.
E. coli cells expressing recombinant protein were resuspended in 4 ml extraction buffer [50 mM
N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (pH 7.0), 10 mM MgCl
2, 20 mM dithiothreitol] and lysed by sonication. After the majority of
E. coli proteins was precipitated by heating of the cell lysate to 80°C for 10 min, the MJ0044-derived protein was purified by anion exchange chromatography on a MonoQ HR column (Amersham Bioscience) (1 by 8 cm) with a linear gradient of 0 to 1 M NaCl in 25 mM Tris (pH 7.5) (over 55 ml) at 1 ml/min. The resulting MJ0044-derived protein ran as a single band at approximately 30 kDa and was >98% pure, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Coomassie blue staining. Protein concentration was determined by Bradford analysis (
8).
Enzymatic analysis of MJ0044 gene product.
Activity of IP kinase was determined by measuring the generation of ADP through a coupled assay with pyruvate kinase and lactate dehydrogenase (
1). An initial continuous assay run at room temperature included 7 units pyruvate kinase, 8 units lactate dehydrogenase, 50 mM Tris buffer (pH 7.5), 4 mM ATP, 0.8 mM IP, 7 mM MgCl
2, 3 mM 2-mercaptoethanol, 5 mM phosphoenolpyruvate, and 0.25 mM NADH in a final volume of 1 ml. The reaction was initiated with the addition of IP kinase (0.56 μg), and an initial linear decrease in absorbance at 340 nm was observed. The reaction was allowed to continue until complete consumption of NADH occurred (∼30 min). A discontinuous assay was used in subsequent experiments to allow for incubation of IP kinase reactions at the higher operating temperatures of the thermophilic MJ0044-derived protein. IP kinase (0.14 μg) was incubated for 20 min at 55°C in a reaction mixture containing 4 mM ATP, 0.8 mM IP, 7 mM MgCl
2, 3 mM 2-mercaptoethanol, and 150 mM Tris buffer (pH 7.5) in a final volume of 1 ml. Following incubation, samples were cooled on ice. For measurement of ADP in the reaction mixture, 200 to 1,000 μl of the reaction mixture was transferred to a cuvette containing water to give a final volume of 1,000 μl. Phosphoenolpyruvate and NADH were added to concentrations of 5 mM and 0.25 mM, respectively. Enzyme activity was measured at room temperature by monitoring the total decrease in absorbance at 340 nm following the addition of 7 units pyruvate kinase and 8 units lactate dehydrogenase. Replicate analysis of individual incubation mixtures following extended incubation on ice showed that there was no significant change in the amount of ADP detected over time. This indicated that IP kinase activity was minimal at 0°C. Although ADP is the preferred nucleotide substrate for pyruvate kinase, it also utilizes a number of alternate nucleotides, including GDP (
5,
29), thus allowing us to determine whether IP kinase could utilize GTP in place of ATP. For kinetic assays, the IP concentration was adjusted from 0.01 mM to 2 mM. Kinetic parameters were estimated from the slope and intercepts of the Lineweaver-Burk plot using Microsoft Excel software.
The activity of IP kinase was determined in 0.5 pH increments between pH 5 and pH 10 by use of a three-component buffer system (
28) consisting of 15 mM Bis-Tris, 7.6 mM
N-2-hydroxyethylpiperazine-
N′-3-propanesulfonic acid, and 7.6 mM 2-[
N-cyclohexylamino]ethanesulfonic acid in place of Tris buffer used in the standard assays. Following incubation at 55°C for 20 min, 250 μl of the incubation mixture was combined with 500 μl 0.5 M Tris buffer (pH 7.5)-250 μl H
2O-50 μl 0.1 M phosphoenolpyruvate-2.5 μl 0.1 M NADH. The total decrease in absorbance at 340 nm was measured following the addition of 7 units pyruvate kinase and 8 units lactate dehydrogenase.
Analysis of enzymatically generated reaction product.
For mass spectral determination of IP kinase products, 4 mM ATP, 4 mM MgCl2, and 1 mM 2-mercaptoethanol were combined in 870 μl water and the pH was adjusted to ∼7.0 with 1 M NaOH. The solution was made 1.5 mM with IP, 0.14 μg of enzyme was added to a final volume of 1 ml, and the mixture was incubated for 1 h at 55°C. The control reaction contained no enzyme. Following incubation the samples were processed by two different methods for mass spectral analysis. One involved the analysis of the TMS derivatives of the reaction mixtures and the other MALDI analysis. Negative-ion MALDI analysis of 0.1 μl of 10× concentrated reaction mixture, by use of a 2,5-hydroxybenzoic acid matrix, showed an [M-H]− ion at m/z 245, corresponding to IPP, from the sample but not from the control.
For the analysis of the TMS derivatives, each sample was passed through a Dowex 50W-X8 pyridinium column, concentrated to dryness by evaporation with nitrogen gas, and then reacted with the TMS reagent (pyridine, hexamethyldisilazane, and chlorotrimethylsilane) (9:3:1 [vol/vol/vol]) for 10 min at 100°C. Mass spectral analysis (70 eV) of samples converted into the TMS derivative by gas chromatography-mass spectrometry and direct injection showed that only the sample incubated with enzyme contained pyrophosphate. This was confirmed by the measurement of M+ = 466 and M+-15 = 451 m/z for the tetra-TMS derivative, which was identical to a known TMS derivative of pyrophosphate. The pyrophosphate was observed with samples but not with the controls by both gas chromatography-mass spectrometry and direct injection. We propose that the pyrophosphate originated with the elimination of PPi from IPP during preparation of the TMS derivatives. Both of these measurements confirm the presence of IPP as the product of the enzymatic reaction.
For NMR analysis of the isopentenyl phosphate kinase reaction, 0.28 μg of purified enzyme was mixed with 250 mM Tris buffer (pH 7.5) containing 2.25 mM ATP, 2.25 mM MgCl2, 2.25 mM IP, and 0.5 mM 2-mercaptoethanol in a final volume of 2 ml. The control was identical to the reaction mixture but contained no enzyme. The samples were incubated for 30 min at 55°C and then concentrated to 0.5 ml under a stream of nitrogen gas. 31P NMR spectra were obtained on a Varian Unity-400 NMR spectrometer at ambient temperature. The following 31P NMR assignments were made referenced to 85% H3PO4 for the proton-decoupled spectrum: ATP δ, −5.1 (1P, d, JγP→βP = 15 Hz, γP), −10.2 (1P, d, JαP→βP = 15 Hz, αP), and −19.2 (1P, m, βP); ADP δ, −5.54 (1P, d, JβP→αP = 19.9 Hz, βP) and −9.7 (1P, d, JαP→βP = 19.9 Hz, αP); IPP δ, −5.48 (1P, d, JβP→αP = 19.9 Hz, βP) and −9.3 (1P, d, JαP→βP = 19.9 Hz, αP); and IP δ, 4.38 (1P, s).