Hoff Kevin G, Avalos José L, Sens Kristin, Wolberger Cynthia
Department of Biophysics and Biophysical Chemistry, Howard Hughes Medical Institute, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205, USA.
Structure. 2006 Aug;14(8):1231-40. doi: 10.1016/j.str.2006.06.006.
Sirtuin proteins comprise a unique class of NAD+-dependent protein deacetylases. Although several structures of sirtuins have been determined, the mechanism by which NAD+ cleavage occurs has remained unclear. We report the structures of ternary complexes containing NAD+ and acetylated peptide bound to the bacterial sirtuin Sir2Tm and to a catalytic mutant (Sir2Tm(H116Y)). NAD+ in these structures binds in a conformation different from that seen in previous structures, exposing the alpha face of the nicotinamide ribose to the carbonyl oxygen of the acetyl lysine substrate. The NAD+ conformation is identical in both structures, suggesting that proper coenzyme orientation is not dependent on contacts with the catalytic histidine. We also present the structure of Sir2Tm(H116A) bound to deacteylated peptide and 3'-O-acetyl ADP ribose. Taken together, these structures suggest a mechanism for nicotinamide cleavage in which an invariant phenylalanine plays a central role in promoting formation of the O-alkylamidate reaction intermediate and preventing nicotinamide exchange.
沉默调节蛋白构成了一类独特的烟酰胺腺嘌呤二核苷酸（NAD⁺）依赖性蛋白质脱乙酰酶。尽管已经确定了几种沉默调节蛋白的结构，但NAD⁺裂解发生的机制仍不清楚。我们报道了含有与细菌沉默调节蛋白Sir2Tm和催化突变体（Sir2Tm(H116Y)）结合的NAD⁺和乙酰化肽的三元复合物的结构。这些结构中的NAD⁺以与先前结构中不同的构象结合，将烟酰胺核糖的α面暴露于乙酰赖氨酸底物的羰基氧。两种结构中的NAD⁺构象相同，表明适当的辅酶取向不依赖于与催化组氨酸的接触。我们还展示了与去乙酰化肽和3'-O-乙酰基ADP核糖结合的Sir2Tm(H116A)的结构。综合这些结构，提出了一种烟酰胺裂解机制，其中一个不变的苯丙氨酸在促进O-烷基酰胺反应中间体的形成和防止烟酰胺交换中起核心作用。
