Wang Yi, Fung Yi Man Eva, Zhang Weizhe, He Bin, Chung Matthew Wai Heng, Jin Jing, Hu Jing, Lin Hening, Hao Quan
School of Biomedical Sciences, University of Hong Kong, Hong Kong, China.
State Key Laboratory of Synthetic Chemistry and Department of Chemistry, University of Hong Kong, Hong Kong, China.
Cell Chem Biol. 2017 Mar 16;24(3):339-345. doi: 10.1016/j.chembiol.2017.02.007. Epub 2017 Mar 9.
Sirtuins are NAD-dependent deacylases. Previous studies have established two important enzymatic intermediates in sirtuin-catalyzed deacylation, an alkylamidate intermediate I, which is then converted to a bicyclic intermediate II. However, how intermediate II is converted to products is unknown. Based on potent SIRT2-specific inhibitors we developed, here we report crystal structures of SIRT2 in complexes with a thiomyristoyl lysine peptide-based inhibitor and carba-NAD or NAD. Interestingly, by soaking crystals with NAD, we capture a distinct covalent catalytic intermediate (III) that is different from the previously established intermediates I and II. In this intermediate, the covalent bond between the S and the myristoyl carbonyl carbon is broken, and we believe this intermediate III to be the decomposition product of II en route to form the end products. MALDI-TOF data further support the intermediate III formation. This is the first time such an intermediate has been captured by X-ray crystallography and provides more mechanistic insights into sirtuin-catalyzed reactions.
沉默调节蛋白是依赖烟酰胺腺嘌呤二核苷酸（NAD）的去酰基酶。以往的研究已经确定了沉默调节蛋白催化去酰化过程中的两个重要酶促中间体，即烷基酰胺中间体I，随后它会转化为双环中间体II。然而，中间体II如何转化为产物尚不清楚。基于我们开发的强效SIRT2特异性抑制剂，在此我们报告了SIRT2与硫代肉豆蔻酰赖氨酸肽基抑制剂以及碳环NAD或NAD形成复合物的晶体结构。有趣的是，通过用NAD浸泡晶体，我们捕获了一种与先前确定的中间体I和II不同的独特共价催化中间体（III）。在这个中间体中，S与肉豆蔻酰羰基碳之间的共价键断裂，我们认为这个中间体III是II在形成终产物过程中的分解产物。基质辅助激光解吸电离飞行时间（MALDI-TOF）数据进一步支持了中间体III的形成。这是首次通过X射线晶体学捕获到这样的中间体，并为沉默调节蛋白催化的反应提供了更多的作用机制见解。
