title: The key role of a non-active-site residue Met148 on the catalytic efficiency of meta-cleavage product hydrolase BphD.
abstract: meta-Cleavage product (MCP) hydrolases (EC 3.7.1.9) can catalyze a specific C-C bond fission during the microbial aerobic degradation of aromatics. The previous studies on structure-function relationship of MCP hydrolases mainly focus on the active site residues by site-directed mutagenesis. However, the information about the role of the non-active-site residues is still unclear. In this study, a non-active-site residue Met148 of MCP hydrolase BphD was selected as the mutagenesis site according to the sequence alignments, structure superimpose and the tunnel analysis, which underwent the saturation mutagenesis resulting 19 mutants. The catalytic efficiencies of the mutants on 6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) were all decreased compared with the wild-type one except for the M148D mutant. Especially, the M148P mutant exhibited 290-fold lower k cat/K m than that of the wild-type BphD. Transient kinetic analyses of M148P showed the reciprocal relaxation time corresponded to C-C bond cleavage and product release steps (9.6 s(-1)) was 4.08-fold lower than BphD WT (39.2 s(-1)). Tunnel cluster analysis of BphD WT, M148P and M148W demonstrated that only the bulky Trp148 could block tunnel T2 in the BphD WT, but it exhibited slight effects on the catalytic efficiency (0.94-fold of BphD WT). Therefore, product release was not the main reason for the efficiency decrease of M148P. On the other hand, molecular dynamics simulations on the BphD WT and BphD M148P in complex with HOPDA indicated that the dramatic decrease of the catalytic efficiencies of BphD M148P should be due to the unproductive binding of HOPDA. The study demonstrated the catalytic efficiency of MCP hydrolase can be engineered by modification of non-active site residue.
authors.affiliation: Key Laboratory of Industrial Ecology and Environmental Engineering (MOE), School of Environmental Science and Technology, Dalian University of Technology, Dalian, 116024, China.
authors.forename: Hao
authors.initials: H
authors.lastname: Zhou
authors.forename: Yuanyuan
authors.initials: Y
authors.lastname: Qu
authors.forename: Chunlei
authors.initials: C
authors.lastname: Kong
authors.forename: E
authors.initials: E
authors.lastname: Shen
authors.forename: Jingwei
authors.initials: J
authors.lastname: Wang
authors.forename: Xuwang
authors.initials: X
authors.lastname: Zhang
authors.forename: Qiao
authors.initials: Q
authors.lastname: Ma
authors.forename: Jiti
authors.initials: J
authors.lastname: Zhou
mesh_terms: D004252:DNA Mutational Analysis; D005231:Fatty Acids, Unsaturated / Q000378:metabolism; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense
chemical_list: D005231:Fatty Acids, Unsaturated; D050505:Mutant Proteins; C018971:2-hydroxy-6-oxo-6-phenyl-2,4-hexadienoic acid; D006867:Hydrolases; C062907:2,6-dioxo-6-phenylhexa-3-enoate hydrolase