Sirtuins: NAD+-dependent deacetylase mechanism and regulation
Highlights
► Sirtuin NAD+-dependent deacetylation proceeds via a novel enzymatic mechanism. ► Deacetylation via an ADPR-imidate enables regulation by NAD+ and nicotinamide. ► Chemical probes of sirtuins have increased understanding of the imidate mechanism. ► Lysine acyl modifications beyond acetylation are deacylated by select sirtuins.
Introduction
Sirtuins are NAD+-dependent deacetylases that catalyze deacetylation of modified protein substrates [1]. Deacetylation of proteins is analogous to the dephosphorylation of phosphorylated proteins by phosphatases, reversing a post-translational modification which can have a putative regulatory function. Protein lysine acetylation is likely to regulate a large number of biologic proteins and has been identified on over a thousand distinct protein sequences in mammalian cells [2••, 3]. Mammalian sirtuins (SIRT1-7, also called Class III KDACs) represent one arm of the protein deacetylase family, with 11 additional Zn2+-dependent Class I, II and IV protein lysine deacetylases. Mechanistically, the two kinds of protein deacetylases are distinct and are regulated differently. Sirtuins use NAD+ stoichiometrically to accomplish deacetylation in a complex chemical reaction [1]. The requirement of NAD+ in sirtuin catalyzed reactions is fundamental to their regulation and enables sirtuins to directly respond to changes in intracellular metabolism.
Sirtuins are broadly distributed in biology. They exist in all major phyla of life [4, 5], including some viruses [6]. This distribution indicates ancient origins and strong biological conservation, in spite of functional redundancy with Zn2+-dependent deacetylases. Available data indicate that sirtuin activities are responsive to reduced dietary intake in yeast, worms, flies and rodents. Sirtuins enable adaptation to dietary stress (reviewed extensively elsewhere, e.g. [7, 8]). Although sirtuins have garnered attention for studies showing that increased sirtuin dosage increases lifespan in yeast, flies and worms (some of the primary evidence has recently become controversial [9]), it is currently unclear if lifespan regulation is their fundamental role in organisms. The role of sirtuins in facilitating adaptation to nutrient availability might be more crucial. This role of sirtuins is clearly manifested in mammalian biology, where sirtuins regulate fat oxidation, mitochondrial biogenesis, insulin secretion, glycolysis, urea cycle, acetate utilization (see Figure 1 for a conserved example of sirtuin metabolic control) and gluconeogenesis (see [7, 8, 10]).
This review presents a chemical perspective on sirtuin function, emphasizing their uniqueness versus Zn+2-dependent deacetylases and with attention to their engagement with NAD+ metabolism as a source of regulation. Their unusual mechanism enables sensitivity to changes in NAD+ and nicotinamide concentrations to modulate their biological functions. We also discuss new developments including: expanded acyl-specificity for select sirtuins, small molecule probes and thioacetyl-modulators of sirtuins.
Section snippets
Deacetylation chemistry of sirtuins and proposed mechanism of catalysis
Sirtuin catalyzed deacetylation of proteins was first reported in 2000 by laboratories of Guarente [11], Sternglanz [12] and Boeke [4]. The full stoichiometry of the reaction is shown in Figure 1. NAD+ is reacted with an acetyllysine substrate to produce nicotinamide, the deacetylated substrate, and a unusual compound called 2′-O-acetyl-ADPribose (2′-AADPR) [13]. The substrate role of NAD+ in sirtuin chemistry is not typical of its normal functions. NAD+ is a redox-active metabolite that
NAD+ binding and reaction
The reaction of sirtuins is initiated by binding of NAD+ to the catalytic site. This binding occurs with NAD+ in an extended conformation, and the C1′ of NAD+ is positioned at the junction of a channel that accommodates the acetyllysine [16]. A termolecular X-ray structure from the Wolberger laboratory establishes that a close approach of the acetyllysine carbonyl oxygen with the C1′ of NAD+ is achieved during catalysis [23]. The sirtuin reaction provides an example of reactant preorganization
Imidate reactions
The ADPR-peptidyl imidate is the predicted consequence of the reaction of NAD+ with acetyllysine and presents a novel regulatory intermediate unknown on any other signaling enzyme [1]. Direct characterization of a sirtuin imidate has not been reported. The imidate is proposed to possess bifunctional reactivity. A regulatory reaction is mediated by the equilibrative binding of nicotinamide to the active site, where it reacts at the C1′-anomeric carbon, to reverse the imidate to NAD+ (Figure 2).
Chemical probes of the imidate mechanism
Replacement of the acetyl-oxygen with sulfur changes the chemistry of the enzyme sufficiently to lead to imidate stabilization. The laboratory of Zheng synthesized several thioacetyllysine containing peptides, and showed they inhibit sirtuin catalyzed deacetylation [44]. The effect was sirtuin specific, since Class I and II enzymes were able to de-thioacetylate the substrates [44]. The origin of the inhibition on sirtuins was determined to be via formation of a thioimidate as shown by X-ray
Sirtuins as deacetylases and deacylases
Sirtuins could be important in the regulation of post-translational modifications beyond acetylation. Such modifications include propionylation [53•], butyrylation [53•], crotonylation, malonylation and succinylation – as reviewed recently [54•]. Sirtuins might regulate such deacylations because select sirtuins prefer these modified substrates over prototypical lysine acetylated substrates. For example, PfSir2, which has already been discussed, was recently shown to remove medium and long chain
Conclusions
Sirtuins regulate acetylation of cellular proteins through a complex mechanism involving NAD+ consumption. This mechanism provides sensitivity to cellular NAD+ levels and feedback via nicotinamide inhibition. A likely chemical mechanism of these enzymes involves the formation of an enzyme-stabilized ADPR-peptidyl imidate. Direct observation of the imidate species on a sirtuin enzyme remains to be obtained, but would be of tremendous value to cementing this intermediate as responsible for the
References and recommended reading
Papers of particular interest, published within the period of review, have been highlighted as:
• of special interest
•• of outstanding interest
References (59)
- et al.
A phylogenetically conserved NAD+-dependent protein deacetylase activity in the Sir2 protein family
Proc Natl Acad Sci USA
(2000) - et al.
Chemistry of gene silencing: the mechanism of NAD+-dependent deacetylation reactions
Biochemistry
(2001) - et al.
Crystal structure of a SIR2 homolog-NAD complex
Cell
(2001) - et al.
Carboxonium compounds in carbohydrate chemistry. 7. Valence isomerism in acyloxonium cations of 1,2,3-triols. Simple synthesis of acyloxonium salts
Angew Chem Int Ed
(1969) - et al.
Structural insights into intermediate steps in the Sir2 deacetylation reaction
Structure
(2008) A view at the millennium: the efficiency of enzymatic catalysis
Acc Chem Res
(2002)- et al.
Sir2 deacetylases exhibit nucleophilic participation of acetyl-lysine in NAD+ cleavage
J Am Chem Soc
(2007) - et al.
Plasmodium falciparum Sir2 is an NAD+-dependent deacetylase and an acetyllysine-dependent and acetyllysine-independent NAD+ glycohydrolase
Biochemistry
(2008) - et al.
Nicotinamide and PNC1 govern lifespan extension by calorie restriction in Saccharomyces cerevisiae
Nature
(2003) - et al.
Inhibition of silencing and accelerated aging by nicotinamide, a putative negative regulator of yeast sir2 and human SIRT1
J Biol Chem
(2002)