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.

Essential role for the SANT domain in the functioning of multiple chromatin remodeling enzymes.

The SANT domain is a novel motif found in a number of eukaryotic transcriptional regulatory proteins that was identified based on its homology to the DNA binding domain of c-myb. Here we show that the SANT domain is essential for the in vivo functions of yeast Swi3p, Ada2p, and Rsc8p, subunits of three distinct chromatin remodeling complexes. We also find that the Ada2p SANT domain is essential for histone acetyltransferase activity of native, Gcn5p-containing HAT complexes. Furthermore, kinetic analyses indicate that an intact SANT domain is required for an Ada2p-dependent enhancement of histone tail binding and enzymatic catalysis by Gcn5p. Our results are consistent with a general role for SANT domains in functional interactions with histone N-terminal tails.

Modulating acetyl-CoA binding in the GCN5 family of histone acetyltransferases.

The histone acetyltransferase (HAT) GCN5 is the founding member for a family of chromatin remodeling enzymes. GCN5 is the catalytic subunit of a large multi-subunit complex that functions in the regulation of gene activation via acetylation of lysine residues within the N-terminal tails of core histone proteins. Using acetyl-CoA as a co-substrate, the high affinity binding of acetyl-CoA is a critical first step in the reaction. Here, we examine the biochemical and biological importance of a conserved hydroxyl-bearing residue in signature motif A. Interestingly, one major exception is the Saccharomyces cerevisiae GCN5, where an alanine (Ala(190)) is located in the corresponding position. In related GCN5 family structures, a hydroxyl-containing side chain residue is hydrogen-bonded to the alpha-phosphate oxygen of CoA. We demonstrate that this key hydrogen bond contributes approximately 10-fold to the binding affinity of GCN5 HATs for acetyl-CoA. Human p300/CBP-associating factor, human GCN5, and tetrahymena GCN5 displayed dissociation constants (K(d)) for acetyl-CoA of 0.64 +/- 0.12, 0.56 +/- 0.15, and 0.62 +/- 0.17 microm, respectively. In contrast, S. cerevisiae GCN5 displayed a K(d) of 8.5 microm. When Ala(190) was replaced with threonine, the A190T derivative yielded a K(d) value of 0.56 +/- 0.1 microm for acetyl-CoA, completely restoring the higher affinity binding seen with the GCN5 homologs that naturally harbor a threonine at this position. Detailed kinetic analyses revealed that the A190T derivative was otherwise catalytically indistinguishable from wild type GCN5. We also demonstrate that the A190T allele rescued the slow growth phenotype and the defect in HO transcription caused by a deletion of GCN5. Furthermore, the A190T allele supported wild type levels of transcriptionally targeted and global histone H3 acetylation. In each case, the A190T derivative behaved similarly to wild type GCN5, suggesting that the efficacy of HAT activity by GCN5 is not limited by the availability of nuclear acetyl-CoA pools.

Noncysteinyl coordination to the [4Fe-4S]2+ cluster of the DNA repair adenine glycosylase MutY introduced via site-directed mutagenesis. Structural characterization of an unusual histidinyl-coordinated cluster.

The Escherichia coli DNA repair enzyme MutY plays an important role in the recognition and repair of 7,8-dihydro-8-oxo-2'-deoxyguanosine-2'-deoxyadenosine (OG*A) mismatches in DNA. MutY prevents DNA mutations caused by the misincorporation of A opposite OG by catalyzing the deglycosylation of the aberrant adenine. MutY is representative of a unique subfamily of DNA repair enzymes that also contain a [4Fe-4S]2+ cluster, which has been implicated in substrate recognition. Previously, we have used site-directed mutagenesis to individually replace the cysteine ligands to the [4Fe-4S]2+ cluster of E. coli MutY with serine, histidine, or alanine. These experiments suggested that histidine coordination to the iron-sulfur cluster may be accommodated in MutY at position 199. Purification and enzymatic analysis of C199H and C199S forms indicated that these forms behave nearly identical to the WT enzyme. Furthermore, introduction of the C199H mutation in a truncated form of MutY (C199HT) allowed for crystallization and structural characterization of the modified [4Fe-4S] cluster coordination. The C199HT structure showed that histidine coordinated to the iron cluster although comparison to the structure of the WT truncated enzyme indicated that the occupancy of iron at the modified position had been reduced to 60%. Electron paramagnetic resonance (EPR) spectroscopy on samples of C199HT indicates that a significant percentage (15-30%) of iron clusters were of the [3Fe-4S]1+ form. Oxidation of the C199HT enzyme with ferricyanide increases the amount of the 3Fe cluster by approximately 2-fold. Detailed kinetic analysis on samples containing a mixture of [3Fe-4S]1+ and [4Fe-4S]2+ forms indicated that the reactivity of the [3Fe-4S]1+ C199HT enzyme does not differ significantly from that of the WT truncated enzyme. The relative resistance of the [4Fe-4S]2+ cluster toward oxidation, as well as the retention of activity of the [3Fe-4S]1+ form, may be an important aspect of the role of MutY in repair of DNA damage resulting from oxidative stress.