Identification of an acyl-enzyme intermediate in a meta-cleavage product hydrolase reveals the versatility of the catalytic triad

Journal of the American Chemical Society
Antonio C RuzziniLindsay D Eltis

Abstract

Meta-cleavage product (MCP) hydrolases are members of the α/β-hydrolase superfamily that utilize a Ser-His-Asp triad to catalyze the hydrolysis of a C-C bond. BphD, the MCP hydrolase from the biphenyl degradation pathway, hydrolyzes 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) to 2-hydroxypenta-2,4-dienoic acid (HPD) and benzoate. A 1.6 Å resolution crystal structure of BphD H265Q incubated with HOPDA revealed that the enzyme's catalytic serine was benzoylated. The acyl-enzyme is stabilized by hydrogen bonding from the amide backbone of 'oxyanion hole' residues, consistent with formation of a tetrahedral oxyanion during nucleophilic attack by Ser112. Chemical quench and mass spectrometry studies substantiated the formation and decay of a Ser112-benzoyl species in wild-type BphD on a time scale consistent with turnover and incorporation of a single equivalent of (18)O into the benzoate produced during hydrolysis in H(2)(18)O. Rapid-scanning kinetic studies indicated that the catalytic histidine contributes to the rate of acylation by only an order of magnitude, but affects the rate of deacylation by over 5 orders of magnitude. The orange-colored catalytic intermediate, ES(red), previously detected in the wild-type enzym...Continue Reading

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Citations

Oct 31, 2012·Chemical Communications : Chem Comm·Adam G NewmanCraig A Townsend
Jun 16, 2018·Chembiochem : a European Journal of Chemical Biology·Sven NerdingerVictor Snieckus
Apr 17, 2014·The Journal of Biological Chemistry·Yueru SunZhihong Guo
Jul 12, 2017·Acta Crystallographica. Section F, Structural Biology Communications·Qi QiHong Lv
Dec 8, 2019·Biochemical and Biophysical Research Communications·Hujian ZhouXiao-Jian Hu

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