Catalytic role for Arginine 188 in the C-C hydrolase catalytic mechanism for Escherichia coli MhpC and Burkholderia xenovorans LB400 BphD


Li, C., Li, J.J., Montgomery, M.G., Wood, S.P. and Bugg, T.D.H. (2006) Catalytic role for Arginine 188 in the C-C hydrolase catalytic mechanism for Escherichia coli MhpC and Burkholderia xenovorans LB400 BphD. Biochemistry, 45, (41), 12470-12479. (doi:10.1021/bi061253t).

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Original Publication URL: http://dx.doi.org/10.1021/bi061253t

Description/Abstract

The /-hydrolase superfamily, comprised mainly of esterase and lipase enzymes, contains a family of bacterial C-C hydrolases, including MhpC and BphD which catalyze the hydrolytic C-C cleavage of meta-ring fission intermediates on the Escherichia coli phenylpropionic acid pathway and Burkholderia xenovorans LB400 biphenyl degradation pathway, respectively. Five active site amino acid residues (Arg-188, Asn-109, Phe-173, Cys-261, and Trp-264) were identified from sequence alignments that are conserved in C-C hydrolases, but not in enzymes of different function. Replacement of Arg-188 in MhpC with Gln and Lys led to 200- and 40-fold decreases, respectively, in kcat; the same replacements for Arg-190 of BphD led to 400- and 700-fold decreases, respectively, in kcat. Pre-steady-state kinetic analysis of the R188Q MhpC mutant revealed that the first step of the reaction, keto-enol tautomerization, had become rate-limiting, indicating that Arg-188 has a catalytic role in ketonization of the dienol substrate, which we propose is via substrate destabilization. Mutation of nearby residues Phe-173 and Trp-264 to Gly gave 4-10-fold reductions in kcat but 10-20-fold increases in Km, indicating that these residues are primarily involved in substrate binding. The X-ray structure of a succinate-H263A MhpC complex shows concerted movements in the positions of both Phe-173 and Trp-264 that line the approach to Arg-188. Mutation of Asn-109 to Ala and His yielded 200- and 350-fold reductions, respectively, in kcat and pre-steady-state kinetic behavior similar to that of a previous S110A mutant, indicating a role for Asn-109 is positioning the active site loop containing Ser-110. The catalytic role of Arg-188 is rationalized by a hydrogen bond network close to the C-1 carboxylate of the substrate, which positions the substrate and promotes substrate ketonization, probably via destabilization of the bound substrate.

Item Type: Article
ISSNs: 0006-2960 (print)
Related URLs:
Subjects: Q Science > Q Science (General)
R Medicine > R Medicine (General)
Divisions: University Structure - Pre August 2011 > School of Biological Sciences
ePrint ID: 55878
Date Deposited: 06 Aug 2008
Last Modified: 27 Mar 2014 18:38
URI: http://eprints.soton.ac.uk/id/eprint/55878

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