Hawse William F, Hoff Kevin G, Fatkins David G, Daines Alison, Zubkova Olga V, Schramm Vern L, Zheng Weiping, Wolberger Cynthia
Department of Biophysics and Biophysical Chemistry, Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA.
Structure. 2008 Sep 10;16(9):1368-77. doi: 10.1016/j.str.2008.05.015.
Sirtuin enzymes comprise a unique class of NAD(+)-dependent protein deacetylases. Although structures of many sirtuin complexes have been determined, structural resolution of intermediate chemical steps are needed to understand the deacetylation mechanism. We report crystal structures of the bacterial sirtuin, Sir2Tm, in complex with an S-alkylamidate intermediate, analogous to the naturally occurring O-alkylamidate intermediate, and a Sir2Tm ternary complex containing a dissociated NAD(+) analog and acetylated peptide. The structures and biochemical studies reveal critical roles for the invariant active site histidine in positioning the reaction intermediate, and for a conserved phenylalanine residue in shielding reaction intermediates from base exchange with nicotinamide. The new structural and biochemical studies provide key mechanistic insight into intermediate steps of the Sir2 deacetylation reaction.
沉默调节蛋白（Sirtuin）酶是一类独特的依赖烟酰胺腺嘌呤二核苷酸（NAD⁺）的蛋白质脱乙酰酶。尽管许多沉默调节蛋白复合物的结构已被确定，但仍需要对中间化学步骤进行结构解析，以了解脱乙酰化机制。我们报告了细菌沉默调节蛋白Sir2Tm与一种S-烷基酰胺中间体（类似于天然存在的O-烷基酰胺中间体）以及一个含有解离的NAD⁺类似物和乙酰化肽的Sir2Tm三元复合物的晶体结构。这些结构和生化研究揭示了不变的活性位点组氨酸在定位反应中间体中的关键作用，以及一个保守的苯丙氨酸残基在保护反应中间体免受与烟酰胺进行碱基交换影响方面的关键作用。新的结构和生化研究为Sir2脱乙酰化反应的中间步骤提供了关键的机制见解。
