Linking chromatin function with metabolic networks: Sir2 family of NAD(+)-dependent deacetylases

Chromatin remodeling enzymes rely on coenzymes derived from metabolic pathways, suggesting a tight synchronization among apparently diverse cellular processes. A unique example of this link is the recently described NAD(+)-dependent protein and/or histone deacetylases. The founding member of this family - yeast silent information regulator 2 (ySir2) - is involved in gene silencing, chromosomal stability and ageing. Sir2-like enzymes catalyze a reaction in which the cleavage of NAD(+)and histone and/or protein deacetylation are coupled to the formation of O-acetyl-ADP-ribose, a novel metabolite. The dependence of the reaction on both NAD(+) and the generation of this potential second messenger offers new clues to understanding the function and regulation of nuclear, cytoplasmic and mitochondrial Sir2-like enzymes.     

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.     

Assembly of the SIR complex and its regulation by O-acetyl-ADP-ribose, a product of NAD-dependent histone deacetylation

Assembly of silent chromatin domains in budding yeast involves the deacetylation of histone tails by Sir2 and the association of the Sir3 and Sir4 proteins with hypoacetylated histone tails. Sir2 couples deacetylation to NAD hydrolysis and the synthesis of a metabolite, O-acetyl-ADP-ribose (AAR), but the functional significance of NAD hydrolysis or AAR, if any, is unknown. Here we examine the association of the Sir2, Sir3, and Sir4 proteins with each other and histone tails. Our analysis reveals that deacetylation of histone H4-lysine 16 (K16), which is critical for silencing in vivo, is also critical for the binding of Sir3 and Sir4 to histone H4 peptides in vitro. Moreover, AAR itself promotes the association of multiple copies of Sir3 with Sir2/Sir4 and induces a dramatic structural rearrangement in the SIR complex. These results suggest that Sir2 activity modulates the assembly of the SIR complex through both histone deacetylation and AAR synthesis.     

Coenzyme specificity of Sir2 protein deacetylases: implications for physiological regulation

Sir2 (silent information regulator 2) enzymes catalyze a unique protein deacetylation reaction that requires the coenzyme NAD(+) and produces nicotinamide and a newly discovered metabolite, O-acetyl-ADP-ribose (OAADPr). Conserved from bacteria to humans, these proteins are implicated in the control of gene silencing, metabolism, apoptosis, and aging. Here we examine the role of NAD(+) metabolites/derivatives and salvage pathway intermediates as activators, inhibitors, or coenzyme substrates of Sir2 enzymes in vitro. Also, we probe the coenzyme binding site using inhibitor binding studies and alternative coenzyme derivatives as substrates. Sir2 enzymes showed an exquisite selectivity for the nicotinamide base coenzyme, with the most dramatic losses in binding affinity/reactivity resulting from relatively minor changes in the nicotinamide ring, either by reduction, as in NADH, or by converting the amide to its acid analogue. Both ends of the dinucleotide NAD(+) are shown to be critical for high selectivity and high affinity. Among the NAD(+) metabolites tested none were able to allosterically activate, although all led to various extents of inhibition, consistent with competition at the coenzyme binding site. Nicotinamide was the most potent inhibitor examined, suggesting that cellular nicotinamide levels would provide an effective small molecule regulator of protein deacetylation and generation of OAADPr. The presented findings also suggest that changes in the physiological NAD(+):NADH ratio, without a change in NAD(+), would yield little alteration in Sir2 activity. That is, NADH is an extremely ineffective inhibitor of Sir2 enzymes (average IC(50) of 17 mm). We propose that changes in both free nicotinamide and free NAD(+) afford the greatest contribution to cellular activity of Sir2 enzymes but with nicotinamide having a more dramatic effect during smaller fluctuations in concentration.     

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.     

Enzymatic activities of Sir2 and chromatin silencing

Heritable domains of generalized repression are a common feature of eukaryotic chromosomes and involve the assembly of DNA into a silenced chromatin structure. Sir2, a conserved protein required for silencing in yeast, has recently been shown to couple histone deacetylation to cleavage of a high-energy bond in nicotinamide adenine dinucleotide (NAD) and the synthesis of a novel product, O-acetyl-ADP-ribose. The deacetylase activity provides a direct link between Sir2 and the hypoacetylated state of silent chromatin. However, the unusual coupling of deacetylation to cleavage and synthesis of other bonds raises the possibility that deacetylation is not the only crucial function of Sir2.     

Sir2 regulates histone H3 lysine 9 methylation and heterochromatin assembly in fission yeast

Hypoacetylated histones are a hallmark of heterochromatin in organisms ranging from yeast to humans. Histone deacetylation is carried out by both NAD(+)-dependent and NAD(+)-independent enzymes. In the budding yeast Saccharomyces cerevisiae, deacetylation of histones in heterochromatic chromosomal domains requires Sir2, a phylogenetically conserved NAD(+)-dependent deacetylase. In the fission yeast Schizosaccharomyces pombe, NAD(+)-independent histone deacetylases are required for the formation of heterochromatin, but the role of Sir2-like deacetylases in this process has not been evaluated. Here, we show that spSir2, the S. pombe Sir2-like protein that is the most closely related to the S. cerevisiae Sir2, is an NAD(+)-dependent deacetylase that efficiently deacetylates histone H3 lysine 9 (K9) and histone H4 lysine 16 (K16) in vitro. In sir2 Delta cells, silencing at the donor mating-type loci, telomeres, and the inner centromeric repeats (imr) is abolished, while silencing at the outer centromeric repeats (otr) and rDNA is weakly reduced. Furthermore, Sir2 is required for hypoacetylation and methylation of H3-K9 and for the association of Swi6 with the above loci in vivo. Our findings suggest that the NAD(+)-dependent deacetylase Sir2 plays an important and conserved role in heterochromatin assembly in eukaryotes.     

Structural basis for nicotinamide inhibition and base exchange in Sir2 enzymes

The Sir2 family of proteins consists of broadly conserved NAD(+)-dependent deacetylases that are implicated in diverse biological processes, including DNA regulation, metabolism, and longevity. Sir2 proteins are regulated in part by the cellular concentrations of a noncompetitive inhibitor, nicotinamide, that reacts with a Sir2 reaction intermediate via a base-exchange reaction to reform NAD(+) at the expense of deacetylation. To gain a mechanistic understanding of nicotinamide inhibition in Sir2 enzymes, we captured the structure of nicotinamide bound to a Sir2 homolog, yeast Hst2, in complex with its acetyl-lysine 16 histone H4 substrate and a reaction intermediate analog, ADP-HPD. Together with related biochemical studies and structures, we identify a nicotinamide inhibition and base-exchange site that is distinct from the so-called "C pocket" binding site for the nicotinamide group of NAD(+). These results provide insights into the Sir2 mechanism of nicotinamide inhibition and have important implications for the development of Sir2-specific effectors.     

Structural identification of 2'- and 3'-O-acetyl-ADP-ribose as novel metabolites derived from the Sir2 family of beta -NAD+-dependent histone/protein deacetylases

The Sir2 (silent information regulator 2) family of histone/protein deacetylases has been implicated in a wide range of biological activities, including gene silencing, life-span extension, and chromosomal stability. Their dependence on beta-NAD(+) for activity is unique among the known classes of histone/protein deacetylase. Sir2 enzymes have been shown to couple substrate deacetylation and beta-NAD(+) cleavage to the formation of O-acetyl-ADP-ribose, a newly described metabolite. To gain a better understanding of the catalytic mechanism and of the biological implications of producing this molecule, we have performed a detailed enzymatic and structural characterization of O-acetyl-ADP-ribose. Through the use of mass spectrometry, rapid quenching techniques, and NMR structural analyses, 2'-O-acetyl-ADP-ribose and 3'-O-acetyl-ADP-ribose were found to be the solution products produced by the Sir2 family of enzymes. Rapid quenching approaches under single-turnover conditions identified 2'-O-acetyl-ADP-ribose as the enzymatic product, whereas 3'-O-acetyl-ADP-ribose was formed by intramolecular transesterification after enzymatic release into bulk solvent, where 2'- and 3'-O-acetyl-ADP-ribose exist in equilibrium (48:52). In addition to (1)H and (13)C chemical shift assignments for each regioisomer, heteronuclear multiple-bond correlation spectroscopy was used to assign unambiguously the position of the acetyl group. These findings are highly significant, because they differ from the previous conclusion, which suggested that 1'-O-acetyl-ADP-ribose was the solution product of the reaction. Possible mechanisms for the generation of 2'-O-acetyl-ADP-ribose are discussed.     

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.     

Analysis of O-acetyl-ADP-ribose as a target for Nudix ADP-ribose hydrolases

The Sir2 family of NAD(+)-dependent histone/protein deacetylases has been implicated in a wide range of biological activities, including gene silencing, life span extension, and chromosomal stability. Recent evidence has indicated that these proteins produce a novel metabolite O-acetyl-ADP-ribose (OAADPr) during deacetylation. Cellular studies have demonstrated that this metabolite exhibits biological effects when microinjected in living cells. However, the molecular targets of OAADPr remain to be identified. Here we have analyzed the ADP-ribose-specific Nudix family of hydrolases as potential in vivo metabolizing enzymes of OAADPr. In vitro, we found that the ADP-ribose hydrolases (yeast YSA1, mouse NudT5, and human NUDT9) cleaved OAADPr to the products AMP and acetylated ribose 5'-phosphate. Steady-state kinetic analyses revealed that YSA1 and NudT5 hydrolyzed OAADPr with similar kinetic constants to those obtained with ADP-ribose as substrate. In dramatic contrast, human NUDT9 was 500-fold less efficient (k(cat)/K(m) values) at hydrolyzing OAADPr compared with ADP-ribose. The inability of OAADPr to inhibit the reaction of NUDT9 with ADP-ribose suggests that NUDT9 binds OAADPr with low affinity, likely due to steric considerations of the additional acetylated-ribose moiety. We next explored whether Nudix hydrolytic activities against OAADPr could be observed in cell extracts from yeast and human. Using a detailed analysis of the products generated during the consumption of OAADPr in extracts, we identified two robust enzymatic activities that were not consistent with the known Nudix hydrolases. Instead, we identified cytoplasmic esterase activities that hydrolyze OAADPr to acetate and ADP-ribose, whereas a distinct activity residing in the nucleus is consistent with an OAADPr-specific acetyltransferase. These findings establish for the first time that select members of the ADP-ribose hydrolases are potential targets of OAADPr metabolism. However, the predominate endogenous activities observed from diverse cell extracts represent novel enzymes.     

组蛋白去乙酰化酶SIR2与染色质沉默

DNA的大部分区域通过包装成特殊的染色质结构而失去活性称为染色质沉默。这些特殊的染色质结构在维持染色体结构稳定和基因调控中起重要作用。有实验表明,沉默染色质的组蛋白H3和H4的的氨基末端尾部相对于基因组的其他区域是低乙酰化的。组蛋白去乙酰化酶SIR2（silent information regulator2）是参与染色质沉默的一种重要的蛋白质。SIR2具有两种相关联的酶活性,组蛋白去乙酰化酶活性和NAD高能骨架的断裂活性,并在酶反应过程中产生一种新的产物氧代乙酰基ADP核糖基（O-acetyl-ADP-ribose）。SIR2的组蛋白去乙酰化酶活性为研究SIR2与沉默染色质的组蛋白低乙酰化状态的关系提供了直接证据。而SIR2的这两种酶活性的关系也表明,组蛋白去乙酰化酶活性不是SIR2惟一的功能。SIR2的NAD水解酶活性和O-acetyl-ADP-ribose的合成过程也可能是染色质沉默机制所必需的。 Abstract:Chromatin silencing is the inactivation of large domains of DNA by packaging them into a specialized inaccessible chromatin structure.This type of inactivation is involved in the regulation of gene expression and is also associated with the chromosome structures required for chromosome maintenance and inheritance.Silent information protein 2(SIR2) is one of the important proteins involved in chromatin silencing.It is clear that SIR2 has two coupled enzymatic activities,histone deacetylation and NAD breakdown activities,and produces a novel compound,O-acetyl-ADP-ribose in the enzymatic reactions.The histone deacetylation activity of SIR2 provides the direct link between SIR2 and the hypoacetylation of silent chromatin.Moreover,the relationship between the NAD cleavage and the deacetylase activity of SIR2 shows that the histone deacetylase activity is not its only crucial function.The breakdown of NAD C-N bond and the synthesis of O-acetyl-ADP-ribose may also be involved in chromatin silencing.     

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.     

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.