title: Identification and regulation of the catalytic promiscuity of (-)-γ-lactamase from Microbacterium hydrocarbonoxydans.
abstract: Mhg, a previously reported (-)-γ-lactamase from Microbacterium hydrocarbonoxydans, was identified to have perhydrolase activity by combining structure similarity search with activity assays. Kinetic studies illustrated that perhydrolysis was the native activity owing to lower K m and higher k cat/K m values. Experimental evidence showed that both hydrolysis and perhydrolysis reactions took place at the same active center. Engineering of the putative substrate-binding pocket revealed that Leu233 site played a vital role in the aspects of selective catalysis, soluble protein expression level and optimum temperature shift, etc. The mutants L233A, L233P, and L233T retained (-)-γ-lactamase activity but lost perhydrolase activity, while L233M only kept perhydrolase activity. Substitutions of Leu233 could dramatically influence the state of expressed protein. Computational analysis explicitly explained the relationships between mutations and γ-lactamase activity changes. Our investigations demonstrated that it was an efficient method to identify the enzyme catalytic promiscuity by combining 3D structure alignment with activity validations, and engineering of substrate-binding pocket could serve as a promising way to regulate activities of promiscuous enzymes.
authors.affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, No. 1 West Beichen Road, Chaoyang District, Beijing, 100101, People's Republic of China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Sun
authors.forename: Hongtao
authors.initials: H
authors.lastname: Zhao
authors.forename: Jianjun
authors.initials: J
authors.lastname: Wang
authors.forename: Junge
authors.initials: J
authors.lastname: Zhu
authors.forename: Sheng
authors.initials: S
authors.lastname: Wu
mesh_terms: D039903:Actinobacteria / Q000201:enzymology*; D000581:Amidohydrolases / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D019295:Computational Biology; D004252:DNA Mutational Analysis; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D015202:Protein Engineering
chemical_list: D050505:Mutant Proteins; D000581:Amidohydrolases; C401943:gamma-lactamase