title: Tuning the substrate selectivity of meta-cleavage product hydrolase by domain swapping.
abstract: meta-Cleavage product (MCP) hydrolases can catalyze relatively low reactive carbon-carbon bond hydrolysis of products, which are derived from the meta-cleavage of catechols. The strict substrate selectivity of MCP hydrolases attracts an interest to understand the determinants of substrate specificity. Compared with conventional site-directed mutagenesis, domain swapping is an effective strategy to explore substrate specificity due to the large-scale reorganization of three-dimensional structure. In the present study, the hybrid MCP hydrolases BphDLidA and MfphALidD were constructed by exchanging the lid domain of two parental enzymes MfphA and BphD. The residues Gly130/Ala196 (MfphA) and Gly136/Ala211 (BphD) were selected as crossover points according to structural disruption score analysis and molecular dynamics simulations. It was shown that the hybrid enzymes exhibited similar substrate selectivity with the parent enzyme providing the lid domain. Docking studies suggested that the lid domain may play a key role in determining substrate specificity by reshaping the active pocket and modulating the orientation of the substrate.
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: E
authors.initials: E
authors.lastname: Shen
authors.forename: Chunlei
authors.initials: C
authors.lastname: Kong
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: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D006867:Hydrolases / Q000235:genetics* / Q000378:metabolism*; D060847:Metabolic Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D006867:Hydrolases