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.     

Production of calcium-mobilizing metabolites by a novel member of the ADP-ribosyl cyclase family expressed in Schistosoma mansoni

ADP-ribosyl cyclases are structurally conserved enzymes that are best known for catalyzing the production of the calcium-mobilizing metabolite, cyclic adenosine diphosphate ribose (cADPR), from nicotinamide adenine dinucleotide (NAD(+)). However, these enzymes also produce adenosine diphosphate ribose (ADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP(+)), both of which have been shown to modulate calcium mobilization in vitro. We have now characterized a new member of the cyclase family from Schistosoma mansoni, a member of the Platyhelminthes phylum. We show that the novel NAD(P)(+) catabolizing enzyme (NACE) expressed by schistosomes is structurally most closely related to the cyclases cloned from Aplysia but also shows significant homology with the mammalian cyclases, CD38 and CD157. NACE expression is developmentally regulated in schistosomes, and the GPI-anchored protein is localized to the outer tegument of the adult schistosome. Importantly, NACE, like all members of the cyclase family, is a multifunctional enzyme and catalyzes NAD(+) glycohydrolase and base-exchange reactions to produce ADPR and NAADP(+). However, despite being competent to generate a cyclic product from NGD(+), a nonphysiologic surrogate substrate, NACE is so far the only enzyme in the cyclase family that is unable to produce significant amounts of cADPR (<0.02% of reaction products) using NAD(+) as the substrate. This suggests that the other calcium-mobilizing metabolites produced by NACE may be more important for calcium signaling in schistosomes. Alternatively, the function of NACE may be to catabolize extracellular NAD(+) to prevent its use by host enzymes that utilize this source of NAD(+) to facilitate immune responses.     

Molecular modeling study for conformational changes of Sirtuin 2 due to substrate and inhibitor binding

Sirtuin is a member of NAD(+)-dependent deacetylase family. The structural details of Sirtuin 2 (SIRT2) complex will be very useful to discover the drug which might have beneficial effects on various diseases like cancer, diabetes, etc. Unfortunately, SIRT2 complex structure is not available yet, hence molecular docking was carried out to dock the substrate (NAD(+) and acetylated lysine) and inhibitor (sirtinol) in the NAD(+) binding site. The suitable binding orientation of substrate and inhibitor in the SIRT2 active site was selected and subjected to 5 ns molecular dynamics simulations to adjust the binding orientation of inhibitor and substrate as well as to identify the conformational changes in the active site. The result provides an insight about 3D SIRT2 structural details as well as the importance of F96 in deacetylation function. In addition, our simulations revealed the displacement of F96 upon substrate and inhibitor binding, inducing an extended conformation of loop3 and changing its interactions with the rest of SIRT2. We believe that our study could be helpful to gain a structural insight of SIRT2 and to design the receptor-based inhibitors.     

Mouse intracellular immunoglobulin M. Structure and identification of a free thiol group

1. The formation of the non-enzymic adduct of NAD(+) and sulphite was investigated. In agreement with others we conclude that the dianion of sulphite adds to NAD(+). 2. The formation of ternary complexes of either lactate dehydrogenase or malate dehydrogenase with NAD(+) and sulphite was investigated. The u.v. spectrum of the NAD-sulphite adduct was the same whether free or enzyme-bound at either pH6 or pH8. This suggests that the free and enzyme-bound adducts have a similar electronic structure. 3. The effect of pH on the concentration of NAD-sulphite bound to both enzymes was measured in a new titration apparatus. Unlike the non-enzymic adduct (where the stability change with pH simply reflects HSO(3) (-)=SO(3) (2-)+H(+)), the enzyme-bound adduct showed a bell-shaped pH-stability curve, which indicated that an enzyme side chain of pK=6.2 must be protonated for the complex to form. Since the adduct does not bind to the enzyme when histidine-195 of lactate dehydrogenase is ethoxycarbonylated we conclude that the protein group involved is histidine-195. 4. The pH-dependence of the formation of a ternary complex of lactate dehydrogenase, NAD(+) and oxalate suggested that an enzyme group is protonated when this complex forms. 5. The rate at which NAD(+) binds to lactate dehydrogenase and malate dehydrogenase was measured by trapping the enzyme-bound NAD(+) by rapid reaction with sulphite. The rate of NAD(+) dissociation from the enzymes was calculated from the bimolecular association kinetic constant and from the equilibrium binding constant and was in both cases much faster than the forward V(max.). No kinetic evidence was found that suggested that there were interactions between protein subunits on binding NAD(+).     

Identification of the NAD(+)-binding fold of glyceraldehyde-3-phosphate dehydrogenase as a novel RNA-binding domain

There is growing evidence that metabolic enzymes may act as multifunctional proteins performing diverse roles in cellular metabolism. Among these functions are the RNA-binding activities of NAD(+)-dependent dehydrogenases. Previously, we have characterized the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an RNA-binding protein with preference to adenine-uracil-rich sequences. In this study, we used GST-GAPDH fusion proteins generated by deletion mutagenesis to search for the RNA binding domain. We established that the N-terminal 43 amino acid residues of GAPDH, which correspond to the first mononucleotide-binding domain of the NAD(+)-binding fold is sufficient to confer RNA-binding. We also provide evidence that this single domain, although it retains most of the RNA-binding activity, loses sequence specificity. Our results suggest a molecular basis for RNA-recognition by NAD(+)-dependent dehydrogenases and (di)nucleotide-binding metabolic enzymes that had been reported to have RNA-binding activity with different specificity. To support this prediction we also identified other members of the family of NAD(+)-dependent dehydrogenases with no previous history of nucleic acid binding as RNA binding proteins in vitro. Based on our findings we propose the addition of the NAD(+)-binding domain to the list of RNA binding domains/motifs.     

Inhibitor binding studies on enoyl reductase reveal conformational changes related to substrate recognition.

Enoyl acyl carrier protein reductase (ENR) is involved in fatty acid biosynthesis. In Escherichia coli this enzyme is the target for the experimental family of antibacterial agents, the diazaborines, and for triclosan, a broad spectrum antimicrobial agent. Biochemical studies have suggested that the mechanism of diazaborine inhibition is dependent on NAD(+) and not NADH, and resistance of Brassica napus ENR to diazaborines is thought to be due to the replacement of a glycine in the active site of the E. coli enzyme by an alanine at position 138 in the plant homologue. We present here an x-ray analysis of crystals of B. napus ENR A138G grown in the presence of either NAD(+) or NADH and the structures of the corresponding ternary complexes with thienodiazaborine obtained either by soaking the drug into the crystals or by co-crystallization of the mutant with NAD(+) and diazaborine. Analysis of the ENR A138G complex with diazaborine and NAD(+) shows that the site of diazaborine binding is remarkably close to that reported for E. coli ENR. However, the structure of the ternary ENR A138G-NAD(+)-diazaborine complex obtained using co-crystallization reveals a previously unobserved conformational change affecting 11 residues that flank the active site and move closer to the nicotinamide moiety making extensive van der Waals contacts with diazaborine. Considerations of the mode of substrate binding suggest that this conformational change may reflect a structure of ENR that is important in catalysis.     

Kinetics of NADH oxidation of NAD+ reduction by mitochondrial complex I]

The kinetics of the NAD: artificial acceptor-oxidoreductase and delta mu H(+)-dependent succinate: NAD(+)-oxidoreductase reactions (reverse electron transfer) reactions catalyzed by the membrane-bound complex I was studied. The values of apparent rate constants of dissociation of complexes of the oxidized and reduced enzyme with NAD+ and NADH were determined. It was shown that the apparent affinity of NADH for the oxidized complex I is by nearly three orders of magnitude as high as that of the reduced one; a reverse correlation is found for NAD+. A kinetic scheme of complex I functioning in the forward and reverse reactions, according to which the free reduced enzyme is not an intermediate of the forward (NADH-oxidase) reaction and the free oxidized enzyme is not an intermediate of the reverse (NAD(+)-reductase) reaction, is proposed.     

The catalytic triad in Drosophila alcohol dehydrogenase: pH, temperature and molecular modelling studies

Drosophila alcohol dehydrogenase belongs to the short chain dehydrogenase/reductase (SDR) family which lack metal ions in their active site. In this family, it appears that the three amino acid residues, Ser138, Tyr151 and Lys155 have a similar function as the catalytic zinc in medium chain dehydrogenases. The present work has been performed in order to obtain information about the function of these residues. To obtain this goal, the pH and temperature dependence of various kinetic coefficients of the alcohol dehydrogenase from Drosophila lebanonensis was studied and three-dimensional models of the ternary enzyme-coenzyme-substrate complexes were created from the X-ray crystal coordinates of the D. lebanonensis ADH complexed with either NAD(+) or the NAD(+)-3-pentanone adduct. The kon velocity for ethanol and the ethanol competitive inhibitor pyrazole increased with pH and was regulated through the ionization of a single group in the binary enzyme-NAD(+) complex, with a DeltaHion value of 74(+/-4) kJ/mol (18(+/-1) kcal/mol). Based on this result and the constructed three-dimensional models of the enzyme, the most likely candidate for this catalytic residue is Ser138. The present kinetic study indicates that the role of Lys155 is to lower the pKa values of both Tyr151 and Ser138 already in the free enzyme. In the binary enzyme-NAD(+) complex, the positive charge of the nicotinamide ring in the coenzyme further lowers the pKa values and generates a strong base in the two negatively charged residues Ser138 and Tyr151. With the OH group of an alcohol close to the Ser138 residue, an alcoholate anion is formed in the ternary enzyme NAD(+) alcohol transition state complex. In the catalytic triad, along with their effect on Ser138, both Lys155 and Tyr151 also appear to bind and orient the oxidized coenzyme.     

Cosubstrate-induced dynamics of D-3-hydroxybutyrate dehydrogenase from Pseudomonas putida

D-3-Hydroxybutyrate dehydrogenase from Pseudomonas putida belongs to the family of short-chain dehydrogenases/reductases. We have determined X-ray structures of the D-3-hydroxybutyrate dehydrogenase from Pseudomonas putida, which was recombinantly expressed in Escherichia coli, in three different crystal forms to resolutions between 1.9 and 2.1 A. The so-called substrate-binding loop (residues 187-210) was partially disordered in several subunits, in both the presence and absence of NAD(+). However, in two subunits, this loop was completely defined in an open conformation in the apoenzyme and in a closed conformation in the complex structure with NAD(+). Structural comparisons indicated that the loop moves as a rigid body by about 46 degrees . However, the two small alpha-helices (alphaFG1 and alphaFG2) of the loop also re-orientated slightly during the conformational change. Probably, the interactions of Val185, Thr187 and Leu189 with the cosubstrate induced the conformational change. A model of the binding mode of the substrate D-3-hydroxybutyrate indicated that the loop in the closed conformation, as a result of NAD(+) binding, is positioned competent for catalysis. Gln193 is the only residue of the substrate-binding loop that interacts directly with the substrate. A translation, libration and screw (TLS) analysis of the rigid body movement of the loop in the crystal showed significant librational displacements, describing the coordinated movement of the substrate-binding loop in the crystal. NAD(+) binding increased the flexibility of the substrate-binding loop and shifted the equilibrium between the open and closed forms towards the closed form. The finding that all NAD(+) -bound subunits are present in the closed form and all NAD(+) -free subunits in the open form indicates that the loop closure is induced by cosubstrate binding alone. This mechanism may contribute to the sequential binding of cosubstrate followed by substrate.     

Glycerol dehydrogenase. structure, specificity, and mechanism of a family III polyol dehydrogenase

BACKGROUND: Bacillus stearothermophilus glycerol dehydrogenase (GlyDH) (glycerol:NAD(+) 2-oxidoreductase, EC 1.1.1.6) catalyzes the oxidation of glycerol to dihydroxyacetone (1,3-dihydroxypropanone) with concomitant reduction of NAD(+) to NADH. Analysis of the sequence of this enzyme indicates that it is a member of the so-called iron-containing alcohol dehydrogenase family. Despite this sequence similarity, GlyDH shows a strict dependence on zinc for activity. On the basis of this, we propose to rename this group the family III metal-dependent polyol dehydrogenases. To date, no structural data have been reported for any enzyme in this group. RESULTS: The crystal structure of B. stearothermophilus glycerol dehydrogenase has been determined at 1.7 A resolution to provide structural insights into the mechanistic features of this family. The enzyme has 370 amino acid residues, has a molecular mass of 39.5 kDa, and is a homooctamer in solution. CONCLUSIONS: Analysis of the crystal structures of the free enzyme and of the binary complexes with NAD(+) and glycerol show that the active site of GlyDH lies in the cleft between the enzyme's two domains, with the catalytic zinc ion playing a role in stabilizing an alkoxide intermediate. In addition, the specificity of this enzyme for a range of diols can be understood, as both hydroxyls of the glycerol form ligands to the enzyme-bound Zn(2+) ion at the active site. The structure further reveals a previously unsuspected similarity to dehydroquinate synthase, an enzyme whose more complex chemistry shares a common chemical step with that catalyzed by glycerol dehydrogenase, providing a striking example of divergent evolution. Finally, the structure suggests that the NAD(+) binding domain of GlyDH may be related to that of the classical Rossmann fold by switching the sequence order of the two mononucleotide binding folds that make up this domain.     

A shared binding site for NAD+ and coenzyme A in an acetaldehyde dehydrogenase involved in bacterial degradation of aromatic compounds

The meta-cleavage pathway for catechol is a central pathway for the bacterial dissimilation of a wide variety of aromatic compounds, including phenols, methylphenols, naphthalenes, and biphenyls. The last enzyme of the pathway is a bifunctional aldolase/dehydrogenase that converts 4-hydroxy-2-ketovalerate to pyruvate and acetyl-CoA via acetaldehyde. The structure of the NAD (+)/CoASH-dependent aldehyde dehydrogenase subunit is similar to that of glyceraldehyde-3-phosphate dehydrogenase, with a Rossmann fold-based NAD (+) binding site observed in the NAD (+)-enzyme complex [Manjasetty, B. A., et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 6992-6997]. However, the location of the CoASH binding site was not determined. In this study, hydrogen-deuterium exchange experiments, coupled with peptic digest and mass spectrometry, were used to examine cofactor binding. The pattern of hydrogen-deuterium exchange in the presence of CoASH was almost identical to that observed with NAD (+), consistent with the two cofactors sharing a binding site. This is further supported by the observations that either CoASH or NAD (+) is able to elute the enzyme from an NAD (+) affinity column and that preincubation of the enzyme with NAD (+) protects against inactivation by CoASH. Consistent with these data, models of the CoASH complex generated using AUTODOCK showed that the docked conformation of CoASH can fully occupy the cavity containing the enzyme active site, superimposing with the NAD (+) cofactor observed in the X-ray crystal structure. Although CoASH binding Rossmann folds have been described previously, this is the first reported example of a Rossmann fold that can alternately bind CoASH or NAD (+) cofactors required for enzymatic catalysis.