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John M Denu

Publications and source records attributed to John M Denu.

At least 19 recordsLinked to original sources
PubMed2007

Catalytic mechanism of a MYST family histone acetyltransferase.

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-epsilon-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 Esa1, 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 x 10(-4) s(-1), or approximately 2500-fold slower than histone acetylation; kcat = 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 Esa1 was the proposed nucleophile that forms an acetyl-cysteine intermediate. Here, mutation of this cysteine (C304A) in Esa1 or within the piccolo NuA4 complex yielded an enzyme that was catalytically indistinguishable from the wild type. Similarly, a pH rate (kcat) 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 kcat approximately 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-epsilon-lysine by deprotonation. Together, the results suggest that MYST family HATs utilize a direct-attack mechanism within an Esa1 x acetyl-CoA x histone ternary complex.

Acetyltransferases↗
PubMed2006

Structure of Arabidopsis thaliana At1g77540 protein, a minimal acetyltransferase from the COG2388 family.

We describe X-ray crystal and NMR solution structures of the protein coded for by Arabidopsis thaliana gene At1g77540.1 (At1g77540). The crystal structure was determined to 1.15 A with an R factor of 14.9% (Rfree = 17.0%) by multiple-wavelength anomalous diffraction using sodium bromide derivatized crystals. The ensemble of NMR conformers was determined with protein samples labeled with 15N and 13C + 15N. The X-ray structure and NMR ensemble were closely similar with rmsd 1.4 A for residues 8-93. At1g77540 was found to adopt a fold similar to that of GCN5-related N-acetyltransferases. Enzymatic activity assays established that At1g77540 possesses weak acetyltransferase activity against histones H3 and H4. Chemical shift perturbations observed in 15N-HSQC spectra upon the addition of CoA indicated that the cofactor binds and identified its binding site. The molecular details of this interaction were further elucidated by solving the X-ray structure of the At1g77540-CoA complex. This work establishes that the domain family COG2388 represents a novel class of acetyltransferase and provides insight into possible mechanistic roles of the conserved Cys76 and His41 residues of this family.

Acetylation↗
PubMed2006

Sirtuins deacetylate and activate mammalian acetyl-CoA synthetases.

Silent Information Regulator 2 (Sir2) enzymes (or sirtuins) are NAD(+)-dependent deacetylases that modulate gene silencing, aging and energy metabolism. Previous work has implicated several transcription factors as sirtuin targets. Here, we investigated whether mammalian sirtuins could directly control the activity of metabolic enzymes. We demonstrate that mammalian Acetyl-CoA synthetases (AceCSs) are regulated by reversible acetylation and that sirtuins activate AceCSs by deacetylation. Site-specific acetylation of mouse AceCS1 on Lys-661 was identified by using mass spectrometry and a specific anti-acetyl-AceCS antibody. SIRT1 was the only member of seven human Sir2 homologues capable of deacetylating AceCS1 in cellular coexpression experiments. SIRT1 expression also led to a pronounced increase in AceCS1-dependent fatty-acid synthesis from acetate. Using purified enzymes, only SIRT1 and SIRT3 exhibited high catalytic efficiency against acetylated AceCS1. In mammals, two AceCSs have been identified: cytoplasmic AceCS1 and mitochondrial AceCS2. Because SIRT3 is localized to the mitochondria, we investigated whether AceCS2 also might be regulated by acetylation, and specifically deacetylated by mitochondrial SIRT3. AceCS2 was completely inactivated upon acetylation and was rapidly reactivated by SIRT3 deacetylation. Lys-635 of mouse AceCS2 was identified as the targeted residue. Using reversible acetylation to modulate enzyme activity, we propose a model for the control of AceCS1 by SIRT1 and of AceCS2 by SIRT3.

Acetate-CoA Ligase↗
PubMed2006

Metabolite of SIR2 reaction modulates TRPM2 ion channel.

The transient receptor potential melastatin-related channel 2 (TRPM2) is a nonselective cation channel, whose prolonged activation by oxidative and nitrative agents leads to cell death. Here, we show that the drug puromycin selectively targets TRPM2-expressing cells, leading to cell death. Our data suggest that the silent information regulator 2 (Sir2 or sirtuin) family of enzymes mediates this susceptibility to cell death. Sirtuins are protein deacetylases that regulate gene expression, apoptosis, metabolism, and aging. These NAD+-dependent enzymes catalyze a reaction in which the acetyl group from substrate is transferred to the ADP-ribose portion of NAD+ to form deacetylated product, nicotinamide, and the metabolite OAADPr, whose functions remain elusive. Using cell-based assays and RNA interference, we show that puromycin-induced cell death is greatly diminished by nicotinamide (a potent sirtuin inhibitor), and by decreased expression of sirtuins SIRT2 and SIRT3. Furthermore, we demonstrate using channel current recordings and binding assays that OAADPr directly binds to the cytoplasmic domain of TRPM2 and activates the TRPM2 channel. ADP-ribose binds TRPM2 with similarly affinity, whereas NAD+ displays almost negligible binding. These studies provide the first evidence for the potential role of sirtuin-generated OAADPr in TRPM2 channel gating.

Adenosine Diphosphate Ribose↗
PubMed2006

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.

Acetylation↗
PubMed2006

SIRT1 top 40 hits: use of one-bead, one-compound acetyl-peptide libraries and quantum dots to probe deacetylase specificity.

A novel, high-throughput method for determining deacetylase substrate specificity was developed using a one-bead, one-compound (OBOC) acetyl-peptide library with a quantum dot tagging strategy and automated bead-sorting. A 5-mer OBOC peptide library of 104,907 unique sequences was constructed around a central epsilon-amino acetylated lysine. The library was screened using the human NAD+-dependent deacetylase SIRT1 for the most efficiently deacetylated peptide sequences. Beads preferentially deacetylated by SIRT1 were biotinylated and labeled with streptavidin-coated quantum dots. After fluorescent bead-sorting, the top 39 brightest beads were sequenced by mass spectrometry. In-solution deacetylase assays on randomly chosen hit and nonhit sequences revealed that hits correlated with increased catalytic activity by as much as 20-fold. We found that SIRT1 can discriminate peptide substrates in a context-dependent fashion.

Acetylation↗
PubMed2005

Mechanism of human SIRT1 activation by resveratrol.

The NAD+-dependent protein deacetylase family, Sir2 (or sirtuins), is important for many cellular processes including gene silencing, regulation of p53, fatty acid metabolism, cell cycle regulation, and life span extension. Resveratrol, a polyphenol found in wines and thought to harbor major health benefits, was reported to be an activator of Sir2 enzymes in vivo and in vitro. In addition, resveratrol was shown to increase life span in three model organisms through a Sir2-dependent pathway. Here, we investigated the molecular basis for Sir2 activation by resveratrol. Among the three enzymes tested (yeast Sir2, human SIRT1, and human SIRT2), only SIRT1 exhibited significant enzyme activation ( approximately 8-fold) using the commercially available Fluor de Lys kit (BioMol). To examine the requirements for resveratrol activation of SIRT1, we synthesized three p53 acetylpeptide substrates either lacking a fluorophore or containing a 7-amino-4-methylcoumarin (p53-AMC) or rhodamine 110 (p53-R110). Although SIRT1 activation was independent of the acetylpeptide sequence, resveratrol activation was completely dependent on the presence of a covalently attached fluorophore. Substrate competition studies indicated that the fluorophore decreased the binding affinity of the peptide, and, in the presence of resveratrol, fluorophore-containing substrates bound more tightly to SIRT1. Using available crystal structures, a model of SIRT1 bound to p53-AMC peptide was constructed. Without resveratrol, the coumarin of p53-AMC peptide is solvent-exposed and makes no significant contacts with SIRT1. We propose that binding of resveratrol to SIRT1 promotes a conformational change that better accommodates the attached coumarin group.

Antioxidants↗
PubMed2005

The Sir 2 family of protein deacetylases.

The importance of NAD(+)-dependent deacetylases (Sir 2 family or sirtuins) in cell survival, ageing and apoptosis has ignited a flurry of both chemical and cellular investigations aimed at understanding this unique class of enzymes. This review focuses on recent mechanistic advances that highlight structure, catalysis, substrate recognition and interactions with small-molecule effectors. Recent X-ray structures revealed binding sites for both NAD(+) and acetyl-peptide. Biochemical studies support a two-step chemical mechanism involving the initial formation of a 1'-O-alkylamidate adduct formed between the acetyl-group and the nicotinamide ribose of NAD(+). Acetyl transfer to the 2' ribose and addition of water yield deacetylated peptide and 2'-O-acetyl-ADP-ribose, a potential second messenger. Also, the molecular basis of nicotinamide inhibition was revealed, and sirtuin activators (resveratrol) and inhibitors (sirtinol and splitomicin) were identified through small-molecule library screening.

Acetylation↗
PubMed2005

Vitamin B3 and sirtuin function.

Sirtuins are NAD(+)-dependent protein deacetylases that are involved in transcriptional regulation, metabolism, apoptosis, differentiation and ageing. These unique enzymes are inhibited by nicotinamide, which is a form of vitamin B3. Recent studies have uncovered the molecular basis for nicotinamide inhibition, and provided the framework to understand the physiological processes mediated by sirtuins and to develop strategies to modulate their cellular activity.

Aging↗
PubMed2005

Assays for mechanistic investigations of protein/histone acetyltransferases.

Protein/histone acetyltransferases (PATs/HATs) have been implicated in a number of cellular functions including gene regulation, DNA synthesis, and repair. This paper reviews methods that can be used to quantitatively determine the activity and ultimately the catalytic/kinetic mechanism of PAT/HATs in vitro. Two methods will be described in detail. The first method is a filter-binding assay that measures the transfer of radiolabeled acetate from acetyl-CoA to protein. The second method is a continuous, spectroscopic, enzyme-coupled assay that links the PAT/HAT reaction to the reduction of NAD+ by pyruvate or alpha-ketoglutarate dehydrogenase. Both methods are highly applicable in determining steady-state reaction rates, and obtaining the kinetic constants Vmax, Km, and V/K from substrate saturation curves. We describe a new application of the filter-binding assay to determine the kinetic parameters for HATs using low concentrations of nucleosomal substrates.

Acetyl Coenzyme A↗
PubMed2005

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.

Enzyme Activation↗
PubMed2004

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.

Acetylation↗
PubMed2004

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.

Binding Sites↗
PubMed2004

Probing the transition-state structure of dual-specificity protein phosphatases using a physiological substrate mimic.

Dual-specificity phosphatases (DSPs) belong to the large family of protein tyrosine phosphatases that contain the active-site motif (H/V)CxxGxxR(S/T), but unlike the tyrosine-specific enzymes, DSPs are able to catalyze the efficient hydrolysis of both phosphotyrosine and phosphoserine/threonine found on signaling proteins, as well as a variety of small-molecule aryl and alkyl phosphates. It is unclear how DSPs accomplish similar reaction rates for phosphoesters, whose reactivity (i.e., pK(a) of the leaving group) can vary by more than 10(8). Here, we utilize the alkyl phosphate m-nitrobenzyl phosphate (mNBP), leaving-group pK(a) = 14.9, as a physiological substrate mimic to probe the mechanism and transition state of the DSP, Vaccinia H1-related (VHR). Detailed pH and kinetic isotope effects of the V/K value for mNBP indicates that VHR reacts with the phosphate dianion of mNBP and that the nonbridge phosphate oxygen atoms are unprotonated in the transition state. (18)O and solvent isotope effects indicate differences in the respective timing of the proton transfer to the leaving group and P-O fission; with the alkyl ester substrate, protonation is ahead of P-O fission, while with the aryl substrate, the two processes are more synchronous. Kinetic analysis of the general-acid mutant D92N with mNBP was consistent with the requirement of Asp-92 in protonating the ester oxygen, either in a step prior to significant P-O bond cleavage or in a concerted but asynchronous mechanism in which protonation is ahead of P-O bond fission. Collectively, the data indicate that VHR and likely all DSPs can match leaving-group potential with the timing of the proton transfer to the ester oxygen, such that diverse aryl and alkyl phosphoesters are turned over with similar catalytic efficiency.

Aniline Compounds↗
PubMed2003

Intramolecular dephosphorylation of ERK by MKP3.

The dual specificity mitogen-activated protein kinase phosphatase MKP3 downregulates mitogenic signaling through dephosphorylation of extracellular signal-regulated kinase (ERK). Like other MKPs, MKP3 consists of a noncatalytic N-terminal domain and a catalytic C-terminal domain. ERK binding to the N-terminal noncatalytic domain of MKP3 has been shown to increase (up to 100-fold) the catalytic activity of MKP3 toward small artificial substrates. Here, we address the function of the N-terminal domain of MKP3 in either inter- or intramolecular dephosphorylation of pERK (phosphorylated ERK) and the stoichiometry of the MKP3/pERK Michaelis complex. These are important mechanistic distinctions given the observation that ERK exists in a monomer/dimer equilibrium that is shifted toward the dimer when phosphorylated and given that MKP3 undergoes catalytic activation toward other substrates when bound to ERK. Wild-type and engineered mutants of ERK and MKP3, binding analyses, reaction kinetics, and chemical cross-linking studies were used to demonstrate that the monomer of MKP3 binds to the monomeric form of pERK and that MKP3 within the resulting heterodimer performs intramolecular dephosphorylation of pERK. This study provides the first direct evidence that MKP3 utilizes intramolecular dephosphorylation between a complex consisting of one molecule each of MKP3 and ERK. Catalytic activation and substrate tethering by MKP3 lead to a >or=4000-fold rate enhancement (k(cat)/K(m)) for dephosphorylation of pERK.

Binding, Competitive↗
PubMed2003

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 Brönsted 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.

Amino Acid Substitution↗