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Protein acetylation is a widespread post-translational modification implicated in many cellular processes. Recent advances in mass spectrometry have enabled the cataloging of thousands of sites throughout the cell; however, identifying regulatory acetylation marks have proven to be a daunting task. Knowledge of the kinetics and stoichiometry of site-specific acetylation is an important factor to uncover function. Here, an improved method of quantifying acetylation stoichiometry was developed and validated, providing a detailed landscape of dynamic acetylation stoichiometry within cellular compartments. The dynamic nature of site-specific acetylation in response to serum stimulation was revealed. In two distinct human cell lines, growth factor stimulation led to site-specific, temporal acetylation changes, revealing diverse kinetic profiles that clustered into several groups. Overlap of dynamic acetylation sites among two different human cell lines suggested similar regulatory control points across major cellular pathways that include splicing, translation, and protein homeostasis. Rapid increases in acetylation on protein translational machinery suggest a positive regulatory role under progrowth conditions. Finally, higher median stoichiometry was observed in cellular compartments where active acetyltransferases are well described. Data sets can be accessed through ProteomExchange via the MassIVE repository (ProteomExchange: PXD014453; MassIVE: MSV000084029).

Posttranslational modifications of proteins control many complex biological processes, including genome expression, chromatin dynamics, metabolism, and cell division through a language of chemical modifications. Improvements in mass spectrometry-based proteomics have demonstrated protein acetylation is a widespread and dynamic modification in the cell; however, many questions remain on the regulation and downstream effects, and an assessment of the overall acetylation stoichiometry is needed. In this chapter, we describe the determination of acetylation stoichiometry using data-independent acquisition mass spectrometry to expand the number of acetylation sites quantified. However, the increased depth of data-independent acquisition is limited by the spectral library used to deconvolute fragmentation spectra. We describe a powerful approach of subcellular fractionation in conjunction with offline prefractionation to increase the depth of the spectral library. This deep interrogation of subcellular compartments provides essential insights into the compartment-specific regulation and downstream functions of protein acetylation.

Epigenetic modifications play critical roles in diverse biological processes. Histone Lys-to-Met (K-to-M) mutations act as gain-of-function mutations to inhibit a wide range of histone methyltransferases and are thought to promote tumorigenesis. However, it is largely unknown whether K-to-M mutations impact organismal development. Using Arabidopsis ( Arabidopsis thaliana ) as a model system, we discovered that a transgene exogenously expressing histone 3 Lys-36 to Met mutation (K36M) acts in a dominant-negative manner to cause global reduction of H3K36 methylation. Remarkably, this dominant repressive activity is dosage-dependent and causes strong developmental perturbations including extreme branching and early flowering by affecting the expression of genes involved in developmental and metabolic processes. Besides the established pathological roles of K-to-M mutations in tumor cells, we demonstrate a physiological outcome for K-to-M induced H3K36 hypomethylation. This study provides evidence for a conserved dominant-negative inhibitory role of histone K-to-M mutation across the plant and animal kingdoms. We also highlight the unique ability of K36M mutations to alter plant developmental processes leading to severe pleiotropic phenotypes. Finally, our data suggests K-to-M mutations may provide a useful strategy for altering epigenetic landscapes in organisms where histone methyltransferases are uncharacterized., (© 2017 The author(s). All Rights Reserved.)

The KDM4 histone demethylases are conserved epigenetic regulators linked to development, spermatogenesis and tumorigenesis. However, how the KDM4 family targets specific chromatin regions is largely unknown. Here, an extensive histone peptide microarray analysis uncovers trimethyl-lysine histone-binding preferences among the closely related KDM4 double tudor domains (DTDs). KDM4A/B DTDs bind strongly to H3K23me3, a poorly understood histone modification recently shown to be enriched in meiotic chromatin of ciliates and nematodes. The 2.28 Å co-crystal structure of KDM4A-DTD in complex with H3K23me3 peptide reveals key intermolecular interactions for H3K23me3 recognition. Furthermore, analysis of the 2.56 Å KDM4B-DTD crystal structure pinpoints the underlying residues required for exclusive H3K23me3 specificity, an interaction supported by in vivo co-localization of KDM4B and H3K23me3 at heterochromatin in mammalian meiotic and newly postmeiotic spermatocytes. In vitro demethylation assays suggest H3K23me3 binding by KDM4B stimulates H3K36 demethylation. Together, these results provide a possible mechanism whereby H3K23me3-binding by KDM4B directs localized H3K36 demethylation during meiosis and spermatogenesis., Competing Interests: J.M.D. consults for Bio-Techne and FORGE Life Science. The remaining authors declare no competing financial interests.

Acetylation of lysine ϵ-amino groups influences many cellular processes and has been mapped to thousands of sites across many organisms. Stoichiometric information of acetylation is essential to accurately interpret biological significance. Here, we developed and employed a novel method for directly quantifying stoichiometry of site-specific acetylation in the entire proteome of Escherichia coli. By coupling isotopic labeling and a novel pairing algorithm, our approach performs an in silico enrichment of acetyl peptides, circumventing the need for immunoenrichment. We investigated the function of the sole NAD(+)-dependent protein deacetylase, CobB, on both site-specific and global acetylation. We quantified 2206 peptides from 899 proteins and observed a wide distribution of acetyl stoichiometry, ranging from less than 1% up to 98%. Bioinformatic analysis revealed that metabolic enzymes, which either utilize or generate acetyl-CoA, and proteins involved in transcriptional and translational processes displayed the highest degree of acetylation. Loss of CobB led to increased global acetylation at low stoichiometry sites and induced site-specific changes at high stoichiometry sites, and biochemical analysis revealed altered acetyl-CoA metabolism. Thus, this study demonstrates that sirtuin deacetylase deficiency leads to both site-specific and global changes in protein acetylation stoichiometry, affecting central metabolism., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Post-translational modifications of histones elicit structural and functional changes within chromatin that regulate various epigenetic processes. Epigenetic mechanisms rely on enzymes whose activities are driven by coenzymes and metabolites from intermediary metabolism. Lysine acetyltransferases (KATs) catalyze the transfer of acetyl groups from acetyl-CoA to epsilon amino groups. Utilization of this critical metabolite suggests these enzymes are modulated by the metabolic status of the cell. This review highlights studies linking KATs to metabolism. We cover newly identified acyl modifications (propionylation and butyrylation), discuss the control of KAT activity by cellular acetyl-CoA levels, and provide insights into how acetylation regulates metabolic proteins. We conclude with a discussion of the current approaches to identifying novel KATs and their metabolic substrates., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Histone lysine and arginine residues are subject to a wide array of post-translational modifications including methylation, citrullination, acetylation, ubiquitination, and sumoylation. The combinatorial action of these modifications regulates critical DNA processes including replication, repair, and transcription. In addition, enzymes that modify histone lysine and arginine residues have been correlated with a variety of human diseases including arthritis, cancer, heart disease, diabetes, and neurodegenerative disorders. Thus, it is important to fully understand the detailed kinetic and chemical mechanisms of these enzymes. Here, we review recent progress towards determining the mechanisms of histone lysine and arginine modifying enzymes. In particular, the mechanisms of S-adenosyl-methionine (AdoMet) dependent methyltransferases, FAD-dependent demethylases, iron dependent demethylases, acetyl-CoA dependent acetyltransferases, zinc dependent deacetylases, NAD(+) dependent deacetylases, and protein arginine deiminases are covered. Particular attention is paid to the conserved active-site residues necessary for catalysis and the individual chemical steps along the catalytic pathway. When appropriate, areas requiring further work are discussed.

Class III histone deacetylases (Sir2 or sirtuins) catalyze the NAD+-dependent conversion of acetyl-lysine residues to nicotinamide, 2'-O-acetyl-ADP-ribose (OAADPr), and deacetylated lysine. Class I and II HDACs utilize a different deacetylation mechanism, utilizing an active site zinc to direct hydrolysis of acetyl-lysine residues to lysine and acetate. Here, using ten acetyl-lysine analog peptides, we have probed the substrate binding pockets of sirtuins and investigated the catalytic differences among sirtuins and class I and II deacetylases. For the sirtuin Hst2, acetyl-lysine analog peptide binding correlated with the hydrophobic substituent parameter pi with a slope of -0.35 from a plot of log Kd versus pi. Interestingly, propionyl- and butyryl-lysine peptides were found to bind tighter to Hst2 compared with acetyl-lysine peptide and showed measurable rates of catalysis with Hst2, Sirt1, Sirt2, and Sirt3, suggesting propionyl- and butyryl-lysine proteins may be sirtuin substrates in vivo. Unique among the acetyl-lysine analog peptides examined, homocitrulline peptide produced ADP-ribose instead of the corresponding OAADPr analog. The electron-withdrawing nature of each acetyl analog had a profound impact on the deacylation rate between deacetylase classes. The rate of catalysis with the acetyl-lysine analog peptides varied over five orders of magnitude with the class III deacetylase Hst2, revealing a linear free energy relationship with a slope of -1.57 when plotted versus the Taft constant, sigma*. HDAC8, a class I deacetylase, displayed the opposite trend with a slope of +0.79. These results are applicable toward the development of selective substrates and other mechanistic probes of protein deacetylases.

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.

Acetylation of histone H3 on lysine 56 occurs during mitotic and meiotic S phase in fungal species. This acetylation blocks a direct electrostatic interaction between histone H3 and nucleosomal DNA, and the absence of this modification is associated with extreme sensitivity to genotoxic agents. We show here that H3-K56 acetylation is catalyzed when Rtt109, a protein that lacks significant homology to known acetyltransferases, forms an active complex with either of two histone binding proteins, Asf1 or Vps75. Rtt109 binds to both these cofactors, but not to histones alone, forming enzyme complexes with kinetic parameters similar to those of known histone acetyltransferase (HAT) enzymes. Therefore, H3-K56 acetylation is catalyzed by a previously unknown mechanism that requires a complex of two proteins: Rtt109 and a histone chaperone. Additionally, these complexes are functionally distinct, with the Rtt109/Asf1 complex, but not the Rtt109/Vps75 complex, being critical for resistance to genotoxic agents.

Summary: The mitochondrial sirtuin SIRT3 regulates metabolic homeostasis during fasting and calorie restriction. We identified mitochondrial 3-hydroxy-3-methylglutaryl CoA synthase 2 (HMGCS2) as an acetylated protein and a possible target of SIRT3 in a proteomics survey in hepatic mitochondria from Sirt3 −/− (SIRT3KO) mice. HMGCS2 is the rate-limiting step in β-hydroxybutyrate synthesis and is hyperacetylated at lysines 310, 447, and 473 in the absence of SIRT3. HMGCS2 is deacetylated by SIRT3 in response to fasting in wild-type mice, but not in SIRT3KO mice. HMGCS2 is deacetylated in vitro when incubated with SIRT3 and in vivo by overexpression of SIRT3. Deacetylation of HMGCS2 lysines 310, 447, and 473 by incubation with wild-type SIRT3 or by mutation to arginine enhances its enzymatic activity. Molecular dynamics simulations show that in silico deacetylation of these three lysines causes conformational changes of HMGCS2 near the active site. Mice lacking SIRT3 show decreased β-hydroxybutyrate levels during fasting. Our findings show SIRT3 regulates ketone body production during fasting and provide molecular insight into how protein acetylation can regulate enzymatic activity. [ABSTRACT FROM AUTHOR]

Most histone acetyltransferases (HATs) function as multisubunit complexes in which accessory proteins regulate substrate specificity and catalytic efficiency. Rttl09 is a particularly interesting example of a HAT whose specificity and catalytic activity require association with either of two histone chaperones, Vps75 or Asf 1. Here, we utilize biochemical, structural, and genetic analyses to provide the detailed molecular mechanism for activation of a HAT (Rttl09) by its activating subunit Vps75. The rate-determining step of the activated complex is the transfer of the acetyl group from acetyl C0A to the acceptor lysine residue. Vps75 stimulates catalysis (>250-fold), not by contributing a catalytic base, but by stabilizing the catalytically active conformation of Rttl09. To provide structural insight into the functional complex, we produced a molecular model of Rttl09-Vps7S based on X-ray diffraction of crystals of the complex. This model reveals distinct negative electrostatic surfaces on an Rttl09 molecule that interface with complementary electropositive ends of a symmetrical Vps75 dimer. Rttl09 variants with interface point substitutions lack the ability to be fully activated by Vps75, and one such variant displayed impaired Vps75-dependent histone acetylation functions in yeast, yet these variants showed no adverse effect on Asfidependent Rttl09 activities in vitro and in vivo. Finally, we provide evidence for a molecular model in which a 1:2 complex of Rttl09-Vps75 acetylates a heterodimer of H3-H4. The activation mechanism of Rttl09-Vps75 provides a valuable framework for understanding the molecular regulation of HATs within multisubunit complexes. [ABSTRACT FROM AUTHOR]

Abstract: Sirtuins catalyze the NAD+-dependent deacetylation of target proteins, which are regulated by this reversible lysine modification. During deacetylation, the glycosidic bond of the nicotinamide ribose is cleaved to yield nicotinamide and the ribose accepts the acetyl group from substrate to produce O-acetyl-ADP-ribose (OAADPr), which exists as an ∼50:50 mixture of 2′ and 3′ isomers at neutral pH. Discovery of this metabolite has fueled the idea that OAADPr may play an important role in the biology associated with sirtuins, acting as a signaling molecule and/or an important substrate for downstream enzymatic processes. Evidence for OAADPr-metabolizing enzymes indicates that at least three distinct activities exist that could modulate the cellular levels of this NAD+-derived metabolite. In Sacc ha romyces cerevisiae, NUDIX hydrolase Ysa1 cleaves OAADPr to AMP and 2- and 3-O-acetylribose-5-phosphate, lowering the cellular levels of OAADPr. A buildup of OAADPr and ADPr has been linked to a metabolic shift that lowers endogenous reactive oxygen species and diverts glucose towards preventing oxidative damage. In vitro, the mammalian enzyme ARH3 hydrolyzes OAADPr to acetate and ADPr. A third nuclear-localized activity appears to utilize OAADPr to transfer the acetyl-group to another small molecule, whose identity remains unknown. Recent studies suggest that OAADPr may regulate gene silencing by facilitating the assembly and loading of the Sir2–4 silencing complex onto nucleosomes. In mammalian cells, the Trpm2 cation channel is gated by both OAADPr and ADP-ribose. Binding is mediated by the NUDIX homology (NudT9H) domain found within the intracellular portion of the channel. OAADPr is capable of binding the Macro domain of splice variants from histone protein MacroH2A, which is highly enriched at heterochromatic regions. With recently developed tools, the pace of new discoveries of OAADPr-dependent processes should facilitate new molecular insight into the diverse biological processes modulated by sirtuins. [Copyright &y& Elsevier]

Abstract: Histone lysine and arginine residues are subject to a wide array of post-translational modifications including methylation, citrullination, acetylation, ubiquitination, and sumoylation. The combinatorial action of these modifications regulates critical DNA processes including replication, repair, and transcription. In addition, enzymes that modify histone lysine and arginine residues have been correlated with a variety of human diseases including arthritis, cancer, heart disease, diabetes, and neurodegenerative disorders. Thus, it is important to fully understand the detailed kinetic and chemical mechanisms of these enzymes. Here, we review recent progress towards determining the mechanisms of histone lysine and arginine modifying enzymes. In particular, the mechanisms of S-adenosyl-methionine (AdoMet) dependent methyltransferases, FAD-dependent demethylases, iron dependent demethylases, acetyl-CoA dependent acetyltransferases, zinc dependent deacetylases, NAD+ dependent deacetylases, and protein arginine deiminases are covered. Particular attention is paid to the conserved active-site residues necessary for catalysis and the individual chemical steps along the catalytic pathway. When appropriate, areas requiring further work are discussed. [Copyright &y& Elsevier]

Reversible protein acetylation is controlled by the opposing actions of protein lysine acetyltransferases and deacetylations. Recent developments on the structure and biochemical mechanisms of histone acetyltransferases (HATs) have provided new insight into catalysis and substrate selection. Diverse families of HATs appear to perform a conserved mechanism of acetyl transfer, where the lysine-containing substrate directly attacks enzyme-bound acetyl-CoA. The ability of HATs to form distinct multi-subunit complexes provides a means to regulate HAT activity by altering substrate specificity, targeting to specific loci, enhancing acetyltransferase activity, restricting access of non-target proteins, and coordinating the multiple enzyme activities of the complex. In the case of newly discovered Rtt109 HAT, association with distinct histone chaperones directs substrate selection between N-terminal lysines (H3K9, H3K23) and those (H3K56) within the histone fold domain. Moreover, the ability of some HATs to utilize longer chain acyl-CoA (i.e. propionyl-CoA) as alternative substrates suggests a potential direct link between the metabolic state of the cell and transcriptional regulation. [Copyright &y& Elsevier]

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 α-1'-O-alkylamidate, whose existence is further supported by the observed methanolysis of the α-1'-O-alkylamidate intermediate to yield β-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 α-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 α-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. [ABSTRACT FROM AUTHOR]

Deals with studies which illustrate the spectrum of structures and activities that can be found within a well-defined enzyme family and re-emphasize the need for combined structural, biochemical and genetic approaches. Discussion on the identification and analysis of histone lysine methyltransferases; Architecture of SET domain lysine methyltransferases; Anatomy of the active site of the Esa1 and Gcn5-like histone acetyltransferases.

Sirtuins catalyze the NAD+-dependent protein deacetylation of lysine residues. In this issue of Structure, report the structure of a sirtuin homolog with both substrates bound, providing new insight into the catalytic mechanism. [Copyright &y& Elsevier]

Esa1, an essential MYST histone acetyltransferase found in the yeast piccolo NuA4 complex (picNuA4), is responsible for genome-wide histone H4 and histone H2A acetylation. picNuA4 uniquely catalyzes the rapid tetra-acetylation of nucleosomal H4, though the molecular determinants driving picNuA4 efficiency and specificity have not been defined. Here, we show through rapid substrate trapping experiments that picNuA4 utilizes a nonprocessive mechanism in which picNuA4 dissociates from the substrate after each acetylation event. Quantitative mass spectral analyses indicate that picNuA4 randomly acetylates free and nucleosomal H4, with a small preference for lysines 5, 8, and 12 over lysine 16. Using a series of 24 histone mutants of H4 and H2A, we investigated the parameters affecting catalytic efficiency. Most strikingly, removal of lysine residues did not substantially affect the ability of picNuA4 to acetylate remaining sites, and insertion of an additional lysine into the H4 tail led to rapid quintuple acetylation. Conversion of the native H2A tail to an H4-like sequence resulted in enhanced multisite acetylation. Collectively, the results suggest picNuA4's site selectivity is dictated by accessibility on the nucleosome surface, the relative proximity from the histone fold domain, and a preference for intervening glycine residues with a minimal (n + 2) spacing between lysines. Functionally distinct from other HAT families, the proposed model for picNuA4 represents a unique mechanism of substrate recognition and multisite acetylation. [ABSTRACT FROM AUTHOR]

Rtt109 is a histone acetyltransferase (HAT) involved in promoting genomic stability. DNA repair. and transcriptional regulation. Rtt109 associates with the NAPI family histone chaperone Vps75 and stimulates histone acetylation. Here we explore the mechanism of histone acetylation and report a detailed kinetic investigation of the Rtt109-Vps75 complex. Rtt109 and Vps75 form a stable complex with nanomolar binding affinity (Kd = 10 ± 2 nM). Steady-state kinetic analysis reveals evidence of a sequential kinetic mechanism whereby the Rtt109-Vps75 complex, AcCoA. and histone H3 substrates form a complex prior to chemical catalysis. Product inhibition studies demonstrate that CoA binds competitively with AcCoA. and equilibrium measurements reveal AcCoA or CoA binding is not stimulated in the presence of H3 substrate. Additionally, the Rtt109-Vps75 complex binds H3 substrates in the absence AcCoA. Pre-steady-state kinetic analysis suggests the chemical attack of substrate lysine on the bound AcCoA is the rate-limiting step of catalysis, while the pH profile of kcat reveals a critical ionization with a pKa of 8.5 that must be unprotonated for catalysis. Amino acid substitution at D287 and D288 did not substantially change the shape of the kcat-pH profile, suggesting these conserved residues do not function as base catalysts for histone acetylation. However, the D288N mutant revealed a dramatic 1000-fold decrease in kcat/Km for AcCoA. consistent with a role in AcCoA binding. Together, these data support a sequential mechanism in which AcCoA and H3 bind to the Rtt 1 09-Vps75 complex without obligate order, followed by the direct aitack of the unprotonated c-amino group on AcCoA, transferring the acetyl group to H3 lysine residues. [ABSTRACT FROM AUTHOR]

Lysine acetylation is rapidly becoming established as a key post-translational modification for regulating mitochondrial metabolism. Nonetheless, distinguishing regulatory sites from among the thousands identified by mass spectrometry and elucidating how these modifications alter enzyme function remain primary challenges. Here, we performed multiplexed quantitative mass spectrometry to measure changes in the mouse liver mitochondrial acetylproteome in response to acute and chronic alterations in nutritional status, and integrated these data sets with our compendium of predicted Sirt3 targets. These analyses highlight a subset of mitochondrial proteins with dynamic acetylation sites, including acetyl-CoA acetyltransferase 1 (Acat1), an enzyme central to multiple metabolic pathways. We performed in vitro biochemistry and molecular modeling to demonstrate that acetylation of Acat1 decreases its activity by disrupting the binding of coenzyme A. Collectively, our data reveal an important new target of regulatory acetylation and provide a foundation for investigating the role of select mitochondrial protein acetylation sites in mediating acute and chronic metabolic transitions. [ABSTRACT FROM AUTHOR]

Post-translational modification of proteins/histones by lysine acylation has profound effects on the physiological function of modified proteins. Deacylation by NAD+-dependent sirtuin reactions yields as a product O-acyl-ADP-ribose, which has been implicated as a signaling molecule in modulating cellular processes. Macrodomain-containing proteins are reported to bind NAD+-derived metabolites. Here, we describe the structure and function of an orphan macrodomain protein, human C6orf130. This unique 17-kDa protein is a stand-alone macrodomain protein that occupies a distinct branch in the phylogenic tree. We demonstrate that C6orf130 catalyzes the efficient deacylation of O-acetyl-ADP-ribose, O-propionyl-ADP-ribose, and O-butyryl-ADP-ribose to produce ADP-ribose (ADPr) and acetate, propionate, and butyrate, respectively. Using NMR spectroscopy, we solved the structure of C6orf130 in the presence and absence of ADPr. The structures showed a canonical fold with a deep ligand (ADPr)-binding cleft. Structural comparisons of apo-C6orf130 and the ADPr-C6orf130 complex revealed fluctuations of the β5-α4 loop that covers the bound ADPr, suggesting that the β5-α4 loop functions as a gate to sequester substrate and offer flexibility to accommodate alternative substrates. The ADPr-C6orf130 complex identified amino acid residues involved in substrate binding and suggested residues that function in catalysis. Site-specific mutagenesis and steady-state kinetic analyses revealed two critical catalytic residues, Ser-35 and Asp-125. We propose a catalytic mechanism for deacylation of O-acyl-ADP-ribose by C6orf130 and discuss the biological implications in the context of reversible protein acylation at lysine residues. [ABSTRACT FROM AUTHOR]

Sirtuins are a family of protein lysine deacetylases, which regulate gene silencing, metabolism, life span, and chromatin structure. Sirtuins utilize NAD+ to deacetylate proteins, yielding O-acetyl-ADP-ribose (OAADPr) as a reaction product. The macrodomain is a ubiquitous protein module known to bind ADP-ribose derivatives, which diverged through evolution to support many different protein functions and pathways. The observation that some sirtuins and macrodomains are physically linked as fusion proteins or genetically coupled through the same operon, provided a clue that their functions might be connected. Indeed, here we demonstrate that the product of the sirtuin reaction OAADPr is a substrate for several related macrodomain proteins: human MacroD1, human MacroD2, Escherichia coli YmdB, and the sirtuin-linked MacroD-like protein from Staphylococcus aureus. In addition, we show that the cell extracts derived from MacroD-deficient Neurospora crassa strain exhibit a major reduction in the ability to hydrolyze OAADPr. Our data support a novel function of macrodomains as OAADPr deacetylases and potential in vivo regulators of cellular OAADPr produced by NADtdependent deacetylation. [ABSTRACT FROM AUTHOR]

Distinct catalytic mechanisms have been proposed for the Gcn5 and MYST histone acetyltransferase (HAT) families. Gcn5-like HATs utilize an ordered sequential mechanism involving direct nucleophilic attack of the N-ϵ-lysine on the enzyme-bound acetyl-CoA. Recently, MYST enzymes were reported to employ a ping-pong route of catalysis via an acetyl—cysteine intermediate. Here, using the prototypical MYST family member Esal, and its physiological complex (piccolo NuA4), steady-state kinetic analyses revealed a kinetic mechanism that requires the formation of a ternary complex prior to catalysis, where acetyl-CoA binds first and CoA is the last product released. In the absence of histone acceptor, slow rates of enzyme auto-acetylation (7 × 10-4 s-4, or ∼2500-fold slower than histone acetylation; κcat = 1.6 s-1) and of CoA formation (0.0021 s-1) were inconsistent with a kinetically competent acetyl—enzyme intermediate. Previously, Cys-304 of Esal was the proposed nucleophile that forms an acetyl—cysteine intermediate. Here, mutation of this cysteine (C304A) in Esal or within the piccolo NuA4 complex yielded an enzyme that was catalytically indistinguishable from the wild type. Similarly, a pH rate (κcat) analysis of the wild type and C304A revealed an ionization (pKa = 7.6-7.8) that must be unprotonated. Mutation of a conserved active-site glutamate (E338Q) reduced κcat ∼200-fold at pH 7.5; however, at higher pH, E338Q exhibited nearly wild-type activity. These data are consistent with Glu-338 (general base) activating the N-ϵ-lysine by deprotonation. Together, the results suggest that MYST family HATs utilize a direct-attack mechanism within an Esal· acetyl-CoA·histone ternary complex. [ABSTRACT FROM AUTHOR]