The structural basis of sirtuin substrate affinity

Sirtuins comprise a family of enzymes that catalyze the deacetylation of acetyllysine side chains in a reaction that consumes NAD+. Although several crystal structures of sirtuins bound to non-native acetyl peptides have been determined, relatively little about how sirtuins discriminate among different substrates is understood. We have carried out a systematic structural and thermodynamic analysis of several peptides bound to a single sirtuin, the Sir2 homologue from Thermatoga maritima (Sir2Tm). We report structures of five different forms of Sir2Tm: two forms bound to the p53 C-terminal tail in the acetylated and unacetylated states, two forms bound to putative acetyl peptide substrates derived from the structured domains of histones H3 and H4, and one form bound to polypropylene glycol (PPG), which resembles the apoenzyme. The structures reveal previously unobserved complementary side chain interactions between Sir2Tm and the first residue N-terminal to the acetyllysine (position -1) and the second residue C-terminal to the acetyllysine (position +2). Isothermal titration calorimetry was used to compare binding constants between wild-type and mutant forms of Sir2Tm and between additional acetyl peptide substrates with substitutions at positions -1 and +2. The results are consistent with a model in which peptide positions -1 and +2 play a significant role in sirtuin substrate binding. This model provides a framework for identifying sirtuin substrates.     

Structure of Sir2Tm bound to a propionylated peptide

Lysine propionylation is a recently identified post‐translational modification that has been observed in proteins such as p53 and histones and is thought to play a role similar to acetylation in modulating protein activity. Members of the sirtuin family of deacetylases have been shown to have depropionylation activity, although the way in which the sirtuin catalytic site accommodates the bulkier propionyl group is not clear. We have determined the 1.8 Å structure of a Thermotoga maritima sirtuin, Sir2Tm, bound to a propionylated peptide derived from p53. A comparison with the structure of Sir2Tm bound to an acetylated peptide shows that hydrophobic residues in the active site shift to accommodate the bulkier propionyl group. Isothermal titration calorimetry data show that Sir2Tm binds propionylated substrates more tightly than acetylated substrates, but kinetic assays reveal that the catalytic rate of Sir2Tm deacylation of propionyl‐lysine is slightly reduced to acetyl‐lysine. These results serve to broaden our understanding of the newly identified propionyl‐lysine modification and the ability of sirtuins to depropionylate, as well as deacetylate, substrates.     

Acetylation-dependent ADP-ribosylation by Trypanosoma brucei Sir2

Sirtuins are a highly conserved family of proteins implicated in diverse cellular processes such as gene silencing, aging, and metabolic regulation. Although many sirtuins catalyze a well characterized protein/histone deacetylation reaction, there are a number of reports that suggest protein ADP-ribosyltransferase activity. Here we explored the mechanisms of ADP-ribosylation using the Trypanosoma brucei Sir2 homologue TbSIR2rp1 as a model for sirtuins that reportedly display both activities. Steady-state kinetic analysis revealed a highly active histone deacetylase (k cat = 0.1 s(-1), with Km values of 42 microm and for NAD+ and 65 microm for acetylated substrate). A series of biochemical assays revealed that TbSIR2rp1 ADP-ribosylation of protein/histone requires an acetylated substrate. The data are consistent with two distinct ADP-ribosylation pathways that involve an acetylated substrate, NAD+ and TbSIR2rp1 as follows: 1) a noncatalytic reaction between the deacetylation product O-acetyl-ADP-ribose (or its hydrolysis product ADP-ribose) and histones, and 2) a more efficient mechanism involving interception of an ADP-ribose-acetylpeptide-enzyme intermediate by a side-chain nucleophile from bound histone. However, the sum of both ADP-ribosylation reactions was approximately 5 orders of magnitude slower than histone deacetylation under identical conditions. The biological implications of these results are discussed.     

Sir2 protein deacetylases: evidence for chemical intermediates and functions of a conserved histidine

Sir2 NAD+-dependent protein deacetylases are implicated in a variety of cellular processes such as apoptosis, gene silencing, life-span regulation, and fatty acid metabolism. Despite this, there have been relatively few investigations into the detailed chemical mechanism. Sir2 proteins (sirtuins) catalyze the chemical conversion of NAD+ and acetylated lysine to nicotinamide, deacetylated lysine, and 2'-O-acetyl-ADP-ribose (OAADPr). In this study, Sir2-catalyzed reactions are shown to transfer an 18O label from the peptide acetyl group to the ribose 1'-position of OAADPr, providing direct evidence for the formation of a covalent alpha-1'-O-alkylamidate, whose existence is further supported by the observed methanolysis of the alpha-1'-O-alkylamidate intermediate to yield beta-1'-O-methyl-ADP-ribose in a Sir2 histidine-to-alanine mutant. This conserved histidine (His-135 in HST2) activates the ribose 2'-hydroxyl for attack on the alpha-1'-O-alkylamidate. The histidine mutant is stalled at the intermediate, allowing water and other alcohols to compete kinetically with the attacking 2'-hydroxyl. Measurement of the pH dependence of kcat and kcat/Km values for both wild-type and histidine-to-alanine mutant enzymes confirms roles of this residue in NAD+ binding and in general-base activation of the 2'-hydroxyl. Also, transfer of an 18O label from water to the carbonyl oxygen of the acetyl group in OAADPr is consistent with water addition to the proposed 1',2'-cyclic intermediate formed after 2'-hydroxyl attack on the alpha-1'-O-alkylamidate. The effect of pH and of solvent viscosity on the kcat values suggests that final product release is rate-limiting in the wild-type enzyme. Implications of this new evidence on the mechanisms of deacetylation and possible ADP-ribosylation catalyzed by Sir2 deacetylases are discussed.     

Structural insights into intermediate steps in the Sir2 deacetylation reaction

Sirtuin enzymes comprise a unique class of NAD(+)-dependent protein deacetylases. Although structures of many sirtuin complexes have been determined, structural resolution of intermediate chemical steps are needed to understand the deacetylation mechanism. We report crystal structures of the bacterial sirtuin, Sir2Tm, in complex with an S-alkylamidate intermediate, analogous to the naturally occurring O-alkylamidate intermediate, and a Sir2Tm ternary complex containing a dissociated NAD(+) analog and acetylated peptide. The structures and biochemical studies reveal critical roles for the invariant active site histidine in positioning the reaction intermediate, and for a conserved phenylalanine residue in shielding reaction intermediates from base exchange with nicotinamide. The new structural and biochemical studies provide key mechanistic insight into intermediate steps of the Sir2 deacetylation reaction.     

Insights into the sirtuin mechanism from ternary complexes containing NAD+ and acetylated peptide

Sirtuin proteins comprise a unique class of NAD+-dependent protein deacetylases. Although several structures of sirtuins have been determined, the mechanism by which NAD+ cleavage occurs has remained unclear. We report the structures of ternary complexes containing NAD+ and acetylated peptide bound to the bacterial sirtuin Sir2Tm and to a catalytic mutant (Sir2Tm(H116Y)). NAD+ in these structures binds in a conformation different from that seen in previous structures, exposing the alpha face of the nicotinamide ribose to the carbonyl oxygen of the acetyl lysine substrate. The NAD+ conformation is identical in both structures, suggesting that proper coenzyme orientation is not dependent on contacts with the catalytic histidine. We also present the structure of Sir2Tm(H116A) bound to deacteylated peptide and 3'-O-acetyl ADP ribose. Taken together, these structures suggest a mechanism for nicotinamide cleavage in which an invariant phenylalanine plays a central role in promoting formation of the O-alkylamidate reaction intermediate and preventing nicotinamide exchange.     

Small molecule regulation of Sir2 protein deacetylases

The Sir2 family of histone/protein deacetylases (sirtuins) is comprised of homologues found across all kingdoms of life. These enzymes catalyse a unique reaction in which NAD+ and acetylated substrate are converted into deacetylated product, nicotinamide, and a novel metabolite O-acetyl ADP-ribose. Although the catalytic mechanism is well conserved across Sir2 family members, sirtuins display differential specificity toward acetylated substrates, which translates into an expanding range of physiological functions. These roles include control of gene expression, cell cycle regulation, apoptosis, metabolism and ageing. The dependence of sirtuin activity on NAD+ has spearheaded investigations into how these enzymes respond to metabolic signals, such as caloric restriction. In addition, NAD+ metabolites and NAD+ salvage pathway enzymes regulate sirtuin activity, supporting a link between deacetylation of target proteins and metabolic pathways. Apart from physiological regulators, forward chemical genetics and high-throughput activity screening has been used to identify sirtuin inhibitors and activators. This review focuses on small molecule regulators that control the activity and functions of this unusual family of protein deacetylases.     

Mechanism of nicotinamide inhibition and transglycosidation by Sir2 histone/protein deacetylases

Silent information regulator 2 (Sir2) enzymes catalyze NAD+-dependent protein/histone deacetylation, where the acetyl group from the lysine epsilon-amino group is transferred to the ADP-ribose moiety of NAD+, producing nicotinamide and the novel metabolite O-acetyl-ADP-ribose. Sir2 proteins have been shown to regulate gene silencing, metabolic enzymes, and life span. Recently, nicotinamide has been implicated as a direct negative regulator of cellular Sir2 function; however, the mechanism of nicotinamide inhibition was not established. Sir2 enzymes are multifunctional in that the deacetylase reaction involves the cleavage of the nicotinamide-ribosyl, cleavage of an amide bond, and transfer of the acetyl group ultimately to the 2'-ribose hydroxyl of ADP-ribose. Here we demonstrate that nicotinamide inhibition is the result of nicotinamide intercepting an ADP-ribosyl-enzyme-acetyl peptide intermediate with regeneration of NAD+ (transglycosidation). The cellular implications are discussed. A variety of 3-substituted pyridines was found to be substrates for enzyme-catalyzed transglycosidation. A Br?nsted plot of the data yielded a slope of +0.98, consistent with the development of a nearly full positive charge in the transition state, and with basicity of the attacking nucleophile as a strong predictor of reactivity. NAD+ analogues including beta-2'-deoxy-2'-fluororibo-NAD+ and a His-to-Ala mutant were used to probe the mechanism of nicotinamide-ribosyl cleavage and acetyl group transfer. We demonstrate that nicotinamide-ribosyl cleavage is distinct from acetyl group transfer to the 2'-OH ribose. The observed enzyme-catalyzed formation of a labile 1'-acetylated-ADP-fluororibose intermediate using beta-2'-deoxy-2'-fluororibo-NAD+ supports a mechanism where, after nicotinamide-ribosyl cleavage, the carbonyl oxygen of acetylated substrate attacks the C-1' ribose to form an initial iminium adduct.     

Sir2 regulation by nicotinamide results from switching between base exchange and deacetylation chemistry

Life span regulation and inhibition of gene silencing in yeast have been linked to nicotinamide effects on Sir2 enzymes. The Sir2 enzymes are NAD(+)-dependent protein deacetylases that influence gene expression by forming deacetylated proteins, nicotinamide and 2'-O-acetyl-ADPR. Nicotinamide is a base-exchange substrate as well as a biologically effective inhibitor. Characterization of the base-exchange reaction reveals that nicotinamide regulates sirtuins by switching between deacetylation and base exchange. Nicotinamide switching is quantitated for the Sir2s from Archeaglobus fulgidus (Sir2Af2), Saccharomyces cerevisiae (Sir2p), and mouse (Sir2alpha). Inhibition of deacetylation was most effective for mouse Sir2 alpha, suggesting species-dependent development of this regulatory mechanism. The Sir2s are proposed to form a relatively stable covalent intermediate between ADPR and the acetyl oxygen of the acetyllysine-protein substrate. During the lifetime of this intermediate, nicotinamide occupation of the catalytic site determines the fate of the covalent complex. Saturation of the nicotinamide site for mouse, yeast, and bacterial Sir2s causes 95, 65, and 21% of the intermediate, respectively, to return to acetylated protein. The fraction of the intermediate committed to deacetylation results from competition between the nicotinamide and the neighboring 2'-hydroxyl group at the opposite stereochemical face. Nicotinamide switching supports the previously proposed Sir2 catalytic mechanism and the existence of a 1'-O-peptidyl-ADPR.Sir2 intermediate. These findings suggest a strategy for increasing Sir2 enzyme catalytic activity in vivo by inhibition of chemical exchange but not deacetylation.     

Substrate specificity of SIRT1-catalyzed lysine N-deacetylation reaction probed with the side chain modified N-acetyl-lysine analogs

Peptides containing l-N^ε-acetyl-lysine (l-AcK) or its side chain modified analogs were prepared and assayed using SIRT1, the prototypical human silent information regulator 2 (Sir2) enzyme. While previous studies showed that the side chain acetyl group of l-AcK can be extended to bulkier acyl groups for Sir2 (including SIRT1)-catalyzed lysine N^ε-deacylation reaction, our current study suggested that SIRT1-catalyzed deacetylation reaction had a very stringent requirement for the distance between the α-carbon and the side chain acetamido group, with that found in l-AcK being optimal. Moreover, our current study showed that SIRT1 catalyzed the stereospecific deacetylation of l-AcK versus its d-isomer. The results from our current study shall constitute another piece of important information to be considered when designing inhibitors for SIRT1 and Sir2 enzymes in general.     

Substrate specificity and kinetic mechanism of the Sir2 family of NAD+-dependent histone/protein deacetylases

The Silent information regulator 2 (Sir2) family of enzymes consists of NAD(+)-dependent histone/protein deacetylases that tightly couple the hydrolysis of NAD(+) and the deacetylation of an acetylated substrate to form nicotinamide, the deacetylated product, and the novel metabolite O-acetyl-ADP-ribose (OAADPR). In this paper, we analyzed the substrate specificity of the yeast Sir2 (ySir2), the yeast HST2, and the human SIRT2 homologues toward various monoacetylated histone H3 and H4 peptides, determined the basic kinetic mechanism, and resolved individual chemical steps of the Sir2 reaction. Using steady-state kinetic analysis, we have shown that ySir2, HST2, and SIRT2 exhibit varying catalytic efficiencies and display a preference among the monoacetylated peptide substrates. Bisubstrate kinetic analysis indicates that Sir2 enzymes follow a sequential mechanism, where both the acetylated substrate and NAD(+) must bind to form a ternary complex, prior to any catalytic step. Using rapid-kinetic analysis, we have shown that after ternary complex formation, nicotinamide cleavage occurs first, followed by the transfer of the acetyl group from the donor substrate to the ADP-ribose portion of NAD(+) to form OAADPr and the deacetylated product. Product and dead-end inhibition analyses revealed that nicotinamide is the first product released followed by random release of OAADPr and the deacetylated product.     

A link between transcription and intermediary metabolism: a role for Sir2 in the control of acetyl-coenzyme A synthetase

The silent information regulator protein (Sir2) and its homologs (collectively known as sirtuins) are NAD+-dependent deacetylase enzymes involved in chromosome stability, gene silencing and cell aging in eukaryotes and archaea. The discovery that sirtuin-dependent protein deacetylation is a NAD+-consuming reaction established a link with the energy generation systems of the cell. This link to metabolism was recently extended to the post-translational control of the activity of short-chain fatty acyl-coenzyme A (adenosine monophosphate-forming) synthetases in bacteria and yeast. The crystal structure of the Sir protein complexed with a peptide of a protein substrate provided insights into how sirtuins interact with their protein substrates.     

The activity of HDAC8 depends on local and distal sequences of its peptide substrates

This paper introduces a flexible assay for characterizing the activities of the histone deacetylase enzymes. The approach combines mass spectrometry with self-assembled monolayers that present acetylated peptides and enables a label-free and one-step assay of this biochemical activity. The assay was used to characterize the activity of HDAC8 toward peptides taken from the N-terminal tail of the H4 histone and reveals that a distal region of the peptide substrate interacts with the deacetylase at an exosite and contributes to the activity of the substrate. Specifically, a peptide corresponding to residues 8-19 of H4 and having lysine 12 acetylated is an active substrate, but removal of the KRHR (residues 16-19) sequence abolishes activity. Mutation of glycine 11 to arginine in the peptide lacking the KRHR sequence restores activity, demonstrating that both local and distal sequences act synergistically to regulate the activity of the HDAC. Assays with peptides bearing multiply acetylated residues, but in which each acetyl group is isotopically labeled, permit studies of the processive deacetylation of peptides. Peptide substrates having an extended sequence that includes K20 were used to demonstrate that methylation of this residue directly affects HDAC8 activity at K12. This work provides a mechanistic basis for the regulation of HDAC activities by distal sequences and may contribute to studies aimed at evaluating the role of the histone code in regulating gene expression.     

Amino acid sequence of histone H1 at the ADP-ribose-accepting site and ADP-ribose X histone-H1 adduct as an inhibitor of cyclic-AMP-dependent phosphorylation

The ADP-ribosylation site of histone H1 from calf thymus by purified hen liver nuclear ADP-ribosyltransferase was determined and effects of the ADP-ribose X histone-H1 adduct on cAMP-dependent phosphorylation of the histone H1 were investigated. ADP-ribosylated histone H1 was prepared by incubation of histone H1, 1 mM [adenylate-32P]NAD and the purified ADP-ribosyltransferase. N-Bromosuccinimide-directed bisection of ADP-ribosylated histone H1 showed that the NH2-terminal fragment (Mr = 6000) was modified and contained serine residue 38, the site of phosphorylation by cAMP-dependent protein kinase. Digestion of the NH2-terminal fragment with cathepsin D and trypsin, and purification of this fragment, using high-performance liquid chromatography, yielded a radiolabelled single peptide corresponding to residues 29-34 of histone H1, containing the arginine residue as the ADP-ribosylation site. These results indicate that ADP-ribosylation of histone H1 occurs at the arginine residue 34, sequenced at the NH2-terminal side of the phosphate-accepting serine residue 38. Phosphorylation of histone H1 from calf thymus by cAMP-dependent protein kinase was markedly reduced when histone H1 was ADP-ribosylated. Kinetic studies of phosphorylation revealed that ADP-ribosylated histone H1 was a linear competitive inhibitor of histone H1 and a linear non-competitive inhibitor of ATP.     

A fluorometric assay of SIRT1 deacetylation activity through quantification of nicotinamide adenine dinucleotide

Sirtuins are nicotinamide adenine dinucleotide (NAD⁺)-dependent deacetylases that catalyze the deacetylation of proteins such as histones and p53. A sensitive and convenient fluorometric assay for evaluating the SIRT1 enzymatic activity was developed here. Specifically, the remaining NAD⁺ after the deacetylation was determined by converting NAD⁺ to a highly fluorescent cyclized α-adduct compound. By this assay, we found that nicotinamide, Cu²⁺, and Zn²⁺ antagonize the activity of SIRT1. Resveratrol stimulates the enzymatic activity specifically with 7-amino-4-methylcoumarin (AMC)-labeled acetylated peptide. Epigallocatechin galate (EGCG) inhibits SIRT1 activity with both AMC-labeled and unlabeled peptide. However, a combination of vitamin C with EGCG can reverse the inhibition of EGCG with the unlabeled peptide or stimulate the deacetylation of AMC-labeled peptide by SIRT1. The assay does not require any isotopic material and thus is biologically safe. It can be adapted to a 96-well microplate for high-throughput screening. Notably, the acetylated peptides with or without fluorescent labels may be used in the assay, which facilitates the substrate specificity study of SIRT1 activators or inhibitors in vitro.     

Mechanism-based inhibition of Sir2 deacetylases by thioacetyl-lysine peptide

Sir2 protein deacetylases (or sirtuins) catalyze NAD+-dependent conversion of epsilon-amino-acetylated lysine residues to deacetylated lysine, nicotinamide, and 2'-O-acetyl-ADP-ribose. Small-molecule modulation of sirtuin activity might treat age-associated diseases, such as type II diabetes, obesity, and neurodegenerative disorders. Here, we have evaluated the mechanisms of sirtuin inhibition of histone peptides containing thioacetyl or mono-, di-, and trifluoroacetyl groups at the epsilon-amino of lysine. Although all substituted peptides yielded inhibition of the deacetylation reaction, the thioacetyl-lysine peptide exhibited exceptionally potent inhibition of sirtuins Sirt1, Sirt2, Sirt3, and Hst2. Using Hst2 as a representative sirtuin, the trifluoroacetyl-lysine peptide displayed competitive inhibition with acetyl-lysine substrate and yielded an inhibition constant (Kis) of 4.8 microM, similar to its Kd value of 3.3 microM. In contrast, inhibition by thioacetyl-lysine peptide yielded an inhibition constant (Kis) of 0.017 microM, 280-fold lower than its Kd value of 4.7 microM. Examination of thioacetyl-lysine peptide as an alternative sirtuin substrate revealed conserved production of deacetylated peptide and 1'-SH-2'-O-acetyl-ADP-ribose. Pre-steady-state and steady-state analysis of the thioacetyl-lysine peptide showed rapid nicotinamide formation (4.5 s-1) but slow overall turnover (0.0024 s-1), indicating that the reaction stalled at an intermediate after nicotinamide formation. Mass spectral analysis yielded a novel species (m/z 1754.3) that is consistent with an ADP-ribose-peptidyl adduct (1'-S-alkylamidate) as the stalled intermediate. Additional experiments involving solvent isotope effects, general base mutational analysis, and density functional calculations are consistent with impaired 2'-hydroxyl attack on the ADP-ribose-peptidyl intermediate. These results have implications for the development of mechanism-based inhibitors of Sir2 deacetylases.     

N-lysine propionylation controls the activity of propionyl-CoA synthetase

Reversible protein acetylation is a ubiquitous means for the rapid control of diverse cellular processes. Acetyltransferase enzymes transfer the acetyl group from acetyl-CoA to lysine residues, while deacetylase enzymes catalyze removal of the acetyl group by hydrolysis or by an NAD(+)-dependent reaction. Propionyl-coenzyme A (CoA), like acetyl-CoA, is a high energy product of fatty acid metabolism and is produced through a similar chemical reaction. Because acetyl-CoA is the donor molecule for protein acetylation, we investigated whether proteins can be propionylated in vivo, using propionyl-CoA as the donor molecule. We report that the Salmonella enterica propionyl-CoA synthetase enzyme PrpE is propionylated in vivo at lysine 592; propionylation inactivates PrpE. The propionyl-lysine modification is introduced by bacterial Gcn-5-related N-acetyltransferase enzymes and can be removed by bacterial and human Sir2 enzymes (sirtuins). Like the sirtuin deacetylation reaction, sirtuin-catalyzed depropionylation is NAD(+)-dependent and produces a byproduct, O-propionyl ADP-ribose, analogous to the O-acetyl ADP-ribose sirtuin product of deacetylation. Only a subset of the human sirtuins with deacetylase activity could also depropionylate substrate. The regulation of cellular propionyl-CoA by propionylation of PrpE parallels regulation of acetyl-CoA by acetylation of acetyl-CoA synthetase and raises the possibility that propionylation may serve as a regulatory modification in higher organisms.     

Role of NAD(+) in the deacetylase activity of the SIR2-like proteins

In this report we describe the role of NAD(+) in the deacetylation reaction catalyzed by the SIR2 family of enzymes. We first show that the products of the reaction detected by HPLC analysis are ADP-ribose, nicotinamide, and a deacetylated peptide substrate. These products are in a 1:1:1 molar ratio, indicating that deacetylation involves the hydrolysis of one NAD(+) to ADP-ribose and nicotinamide for each acetyl group removed. Three results suggest that deacetylation requires an enzyme-ADP-ribose intermediate. First, the enzyme can promote an NAD(+) if nicotinamide exchange reaction that depends on an acetylated substrate. Second, a non-hydrolyzable NAD(+) analog is a competitive inhibitor of the enzyme, and, third, nicotinamide shows product inhibition of deacetylase activity.