title: Deacylation Mechanism by SIRT2 Revealed in the 1'-SH-2'-O-Myristoyl Intermediate Structure.
abstract: 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.
authors.affiliation: School of Biomedical Sciences, University of Hong Kong, Hong Kong, China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Synthetic Chemistry and Department of Chemistry, University of Hong Kong, Hong Kong, China.
authors.forename: Yi Man Eva
authors.initials: YME
authors.lastname: Fung
authors.affiliation: School of Biomedical Sciences, University of Hong Kong, Hong Kong, China; Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
authors.forename: Weizhe
authors.initials: W
authors.lastname: Zhang
authors.affiliation: College of Pharmacy, Engineering Research Center for the Development and Application of Ethnic Medicine and TCM Ministry of Education, Guizhou Medical University, Guizhou 550004, China.
authors.forename: Bin
authors.initials: B
authors.lastname: He
authors.affiliation: School of Biomedical Sciences, University of Hong Kong, Hong Kong, China.
authors.forename: Matthew Wai Heng
authors.initials: MWH
authors.lastname: Chung
authors.affiliation: School of Biomedical Sciences, University of Hong Kong, Hong Kong, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Jin
authors.affiliation: Department of Chemistry and Chemical Biology, Howard Hughes Medical Institute, Cornell University, Ithaca, NY 14853, USA.
authors.forename: Jing
authors.initials: J
authors.lastname: Hu
authors.affiliation: Department of Chemistry and Chemical Biology, Howard Hughes Medical Institute, Cornell University, Ithaca, NY 14853, USA. Electronic address: hl379@cornell.edu.
authors.forename: Hening
authors.initials: H
authors.lastname: Lin
authors.affiliation: School of Biomedical Sciences, University of Hong Kong, Hong Kong, China. Electronic address: qhao@hku.hk.
authors.forename: Quan
authors.initials: Q
authors.lastname: Hao
mesh_terms: D000215:Acylation; D055162:Biocatalysis; D018360:Crystallography, X-Ray; D005229:Fatty Acids, Monounsaturated / Q000737:chemistry*; D006801:Humans; D009243:NAD / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000032:analysis / Q000138:chemical synthesis / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D056565:Sirtuin 2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D005229:Fatty Acids, Monounsaturated; D010455:Peptides; D011994:Recombinant Proteins; D009243:NAD; C055311:carbanicotinamide adenine dinucleotide; D056565:Sirtuin 2; C054211:9-tetradecenoic acid
keywords: crystallography; enzyme; post-translational modifications; reactive intermediates; sirtuins