title: Catalytic role for arginine 188 in the C-C hydrolase catalytic mechanism for Escherichia coli MhpC and Burkholderia xenovorans LB400 BphD.
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.
authors.affiliation: Department of Chemistry, University of Warwick, Coventry CV4 7AL, UK.
authors.forename: Chen
authors.initials: C
authors.lastname: Li
authors.forename: Jian-Jun
authors.initials: JJ
authors.lastname: Li
authors.forename: Mark G
authors.initials: MG
authors.lastname: Montgomery
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Wood
authors.forename: Timothy D H
authors.initials: TD
authors.lastname: Bugg
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001120:Arginine / Q000737:chemistry; D001483:Base Sequence; D019117:Burkholderia / Q000201:enzymology* / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D018360:Crystallography, X-Ray; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006867:Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D001120:Arginine; D006867:Hydrolases; C497337:MhpC protein, E coli; C062907:2,6-dioxo-6-phenylhexa-3-enoate hydrolase