Mechanism of sirtuin inhibition by nicotinamide: altering the NAD(+) cosubstrate specificity of a Sir2 enzyme.
Sir2 enzymes form a unique class of NAD(+)-dependent deacetylases required for diverse biological processes, including transcriptional silencing, regulation of apoptosis, fat mobilization, and lifespan regulation. Sir2 activity is regulated by nicotinamide, a noncompetitive inhibitor that promotes a base-exchange reaction at the expense of deacetylation. To elucidate the mechanism of nicotinamide inhibition, we determined ternary complex structures of Sir2 enzymes containing nicotinamide. The structures show that free nicotinamide binds in a conserved pocket that participates in NAD(+) binding and catalysis. Based on our structures, we engineered a mutant that deacetylates peptides by using nicotinic acid adenine dinucleotide (NAAD) as a cosubstrate and is inhibited by nicotinic acid. The characteristics of the altered specificity enzyme establish that Sir2 enzymes contain a single site that participates in catalysis and nicotinamide regulation and provides additional insights into the Sir2 catalytic mechanism.
Avalos
José L
JL
Howard Hughes Medical Institute, Department of Biophysics and Biophysical Chemistry, School of Medicine, Johns Hopkins University, 725 N. Wolfe Street, Baltimore, Maryland 21205, USA.
Bever
Katherine M
KM
Wolberger
Cynthia
C
D000107:Acetylation; D000595:Amino Acid Sequence; D002384:Catalysis; D015966:Gene Expression Regulation, Fungal; D006655:Histone Deacetylases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009243:NAD / Q000031:analogs & derivatives* / Q000378:metabolism*; D009525:Niacin / Q000494:pharmacology; D009536:Niacinamide / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D056565:Sirtuin 2; D037761:Sirtuins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D014665:Vasodilator Agents / Q000494:pharmacology
D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae; D014665:Vasodilator Agents; D009243:NAD; D009536:Niacinamide; D009525:Niacin; C018348:nicotinic acid adenine dinucleotide; C087778:SIR2 protein, S cerevisiae; D056565:Sirtuin 2; D037761:Sirtuins; D006655:Histone Deacetylases