title: Identification of an acyl-enzyme intermediate in a meta-cleavage product hydrolase reveals the versatility of the catalytic triad.
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 enzyme and proposed herein to be a carbanion, was not observed during hydrolysis by H265Q. In the newly proposed mechanism, the carbanion abstracts a proton from Ser112, thereby completing tautomerization and generating a serinate for nucleophilic attack on the C6-carbonyl. Finally, quantification of an observed pre-steady-state kinetic burst suggests that BphD is a half-site reactive enzyme. While the updated catalytic mechanism shares features with the serine proteases, MCP hydrolase-specific chemistry highlights the versatility of the Ser-His-Asp triad.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver BC V6T 1Z3, Canada.
authors.forename: Antonio C
authors.initials: AC
authors.lastname: Ruzzini
authors.forename: Subhangi
authors.initials: S
authors.lastname: Ghosh
authors.forename: Geoff P
authors.initials: GP
authors.lastname: Horsman
authors.forename: Leonard J
authors.initials: LJ
authors.lastname: Foster
authors.forename: Jeffrey T
authors.initials: JT
authors.lastname: Bolin
authors.forename: Lindsay D
authors.initials: LD
authors.lastname: Eltis
mesh_terms: D000215:Acylation; D055162:Biocatalysis; D006860:Hydrogen Bonding; D006867:Hydrolases / Q000737:chemistry*; D006868:Hydrolysis; D008958:Models, Molecular
chemical_list: D006867:Hydrolases