Sir2 enzymes are broadly conserved from bacteria to humans and have been implicated to play roles in gene silencing, DNA repair, genome stability, longevity, metabolism, and cell physiology. These enzymes bind NAD ؉ and acetyllysine within protein targets and generate lysine, 2-O-acetyl-ADP-ribose, and nicotinamide products. To provide structural insights into the chemistry catalyzed by Sir2 proteins we report the high-resolution ternary structure of yeast Hst2 (homologue of Sir two 2) with an acetyllysine histone H4 peptide and a nonhydrolyzable NAD ؉ analogue, carba-NAD ؉ , as well as an analogous ternary complex with a reaction intermediate analog formed immediately after nicotinamide hydrolysis, ADP-ribose. The ternary complex with carba-NAD ؉ reveals that the nicotinamide group makes stabilizing interactions within a binding pocket harboring conserved Sir2 residues. Moreover, an asparagine residue, N116, strictly conserved within Sir2 proteins and shown to be essential for nicotinamide exchange, is in position to stabilize the oxocarbenium intermediate that has been proposed to proceed the hydrolysis of nicotinamide. A comparison of this structure with the ADP-ribose ternary complex and a previously reported ternary complex with the 2-O-acetyl-ADP-ribose reaction product reveals that the ribose ring of the cofactor and the highly conserved ␤1-␣2 loop of the protein undergo significant structural rearrangements to facilitate the ordered NAD ؉ reactions of nicotinamide cleavage and ADP-ribose transfer to acetate. Together, these studies provide insights into the chemistry of NAD ؉ cleavage and acetylation by Sir2 proteins and have implications for the design of Sir2-specific regulatory molecules. T he Sir2 (silent information regulator 2) or sirtuin family of deacetylases requires NAD ϩ as a cofactor to hydrolyze the acetyl moiety of an acetyllysine within protein targets to regulate diverse biological functions, including gene silencing, genome stability, longevity, metabolism, and cell physiology (for reviews, see refs. 1 and 2). The mechanism for Sir2 activity has been extensively studied at both structural and enzymatic levels. Structural studies reveal that the Sir2 proteins contain a structurally conserved elongated core domain containing a large Rossmann fold at one end, a structurally more variable zincbinding motif at the opposite end, and a series of loops connecting these regions and forming a cleft in the central region of the core domain (3-6). The acetyllysine and NAD ϩ cosubstrates bind to opposite sides of the cleft and highlight the region of the core domain containing the highest degree of sequence conservation within the Sir2 proteins, implying a conserved catalytic mechanism (3, 7). Biochemical and structural studies reveal that the deacetylation of acetyllysine is coupled to the hydrolysis of NAD ϩ to nicotinamide and 2Ј-O-acetyl-ADP-ribose (8, 9).A detailed structural understanding of how the Sir2 proteins mediate nicotinamide cleavage and ADP-ribose transfer to acetate has been hampered...

Hsf1p, the heat-shock transcription factor from Saccharomyces cerevisiae, has a low level of constitutive transcriptional activity and is kept in this state through negative regulation. In an effort to understand this negative regulation, we developed a novel genetic selection that detects altered expression from the HSP26 promoter. Using this reporter strain, we identified mutations and dosage compensators in the Ras/ cAMP signaling pathway that decrease cAMP levels and increase expression from the HSP26 promoter. In yeast, low cAMP levels reduce the catalytic activity of the cAMP-dependent kinase PKA. Previous studies had proposed that the stress response transcription factors Msn2p/4p, but not Hsf1p, are repressed by PKA. However, we found that reduction or elimination of PKA activity strongly derepresses transcription of the small heat-shock genes HSP26 and HSP12, even in the absence of MSN2/4. In a strain deleted for MSN2/4 and the PKA catalytic subunits, expression of HSP12 and HSP26 depends on HSF1 expression. Our findings indicate that Hsf1p functions downstream of PKA and suggest that PKA might be involved in negative regulation of Hsf1p activity. These results represent a major change in our understanding of how PKA signaling influences the heat-shock response and heat-shock protein expression.