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Catalytic role for Arginine 188 in the C-C hydrolase catalytic mechanism for Escherichia coli MhpC and Burkholderia xenovorans LB400 BphD
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Li, Chen, Li, Jian-Jun, Montgomery, Mark G., Wood, Stephen P. and Bugg, Tim (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). pp. 12470-12479. doi:10.1021/bi061253t ISSN 0006-2960.
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Official URL: http://dx.doi.org/10.1021/bi061253t
Abstract
The alpha/beta-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 k(cat); the same replacements for Arg-190 of BphD led to 400- and 700-fold decreases, respectively, in k(cat). 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 k(cat) but 10-20-fold increases in K-m, 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 k(cat) 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: | Journal Article | ||||
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Subjects: | Q Science > QD Chemistry | ||||
Journal or Publication Title: | BIOCHEMISTRY | ||||
Publisher: | AMER CHEMICAL SOC | ||||
ISSN: | 0006-2960 | ||||
Official Date: | 17 October 2006 | ||||
Dates: |
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Volume: | 45 | ||||
Number: | 41 | ||||
Number of Pages: | 10 | ||||
Page Range: | pp. 12470-12479 | ||||
DOI: | 10.1021/bi061253t | ||||
Publication Status: | Published |
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