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Investigating the catalytic mechanism of the meta-cleavage product hydrolases

University of British Columbia

The 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. The catalytic mechanism of the MCP hydrolases is poorly defined and particularly interesting due to a requisite substrate ketonization that precedes hydrolysis. To resolve the catalytic mechanism of the MCP hydrolases, two enzymes were studied: tetrameric BphDLB400 from Burkholderia xenovorans LB400 and dimeric DxnB2 from Sphingomonas witichii RW1. Both efficiently hydrolyze 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) to produce 2-hydroxypenta-2,4-dienoic acid (HPD) and benzoic acid. A series of experiments established that BphDLB400 uses an histidine-independent nucleophilic mechanism of catalysis and is half-site reactive. Benzoylation of Ser112 was demonstrated by LC ESI/MS/MS analyses and a pre-steady-state kinetic burst of HPD formation indicated the reactivity. While acylation during HOPDA hydrolysis by BphDLB400 occurred on a similar timescale for the WT and H265Q variant, esterase activity was abrogated in the histidine variant. Thus, alternative mechanisms of nucleophile activation are employed for C-C and C-O bond cleavage. A covalent mechanism of catalysis was inferred for DxnB2, however, the turnover of HOPDA was 1:1 with respect to enzyme concentration. A solvent kinetic isotope effect suggested that a proton transfer, and therefore, substrate ketonization determines the rate of acylation in the MCP hydrolases. Substrate ketonization, and therefore acylation, can be indirectly observed as consumption of ESred, an intermediate named for its bathochromically-shifted absorption spectrum. A proton transfer to ESred allowed the assignment of this species to an enzyme-bound HOPDA dianion. An extended Brønsted analysis revealed a linear correlation between substrate basicity and the rate constant determined for the ketonization reaction. Finally, the MCP hydrolase P-subsite, which contacts the MCP dienoate moiety, was definitively linked to substrate ketonization. In DxnB2 Asn43 and Arg180 variants, ESred formation was found to limit this proton transfer reaction. A substrate-assisted nucleophilic mechanism of catalysis has been proposed for the MCP hydrolases. Therein, the electron-rich dienoate moiety substitutes for the His-Asp pair as the general base for nucleophile activation. Overall, definition of the chemical mechanism of the MCP hydrolases has implications for environmental bioremediation strategies and the rational design of therapeutics.

Text

2013-09-19

CC0 1.0 Universal

http://hdl.handle.net/2429/45105

Biochemistry and Molecular Biology

University of British Columbia

UBCV

http://creativecommons.org/publicdomain/zero/1.0/