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Activity Improvement and Vital Amino Acid Identification on the Marine-Derived Quorum Quenching Enzyme MomL by Protein Engineering
- Wang, Jiayi, Lin, Jing, Zhang, Yunhui, Zhang, Jingjing, Feng, Tao, Li, Hui, Wang, Xianghong, Sun, Qingyang, Zhang, Xiaohua, Wang, Yan
- Marine drugs 2019 v.17 no.5
- Chinese cabbage, Pectobacterium carotovorum subsp. carotovorum, carboxylic ester hydrolases, catalytic activity, enzyme activity, homoserine, lactones, mutants, pathogenicity, polymerase chain reaction, screening, site-directed mutagenesis
- MomL is a marine-derived quorum-quenching (QQ) lactonase which can degrade various N-acyl homoserine lactones (AHLs). Intentional modification of MomL may lead to a highly efficient QQ enzyme with broad application potential. In this study, we used a rapid and efficient method combining error-prone polymerase chain reaction (epPCR), high-throughput screening and site-directed mutagenesis to identify highly active MomL mutants. In this way, we obtained two candidate mutants, MomL<inf>I144V</inf> and MomL<inf>V149A</inf>. These two mutants exhibited enhanced activities and blocked the production of pathogenic factors of Pectobacterium carotovorum subsp. carotovorum (Pcc). Besides, seven amino acids which are vital for MomL enzyme activity were identified. Substitutions of these amino acids (E238G/K205E/L254R) in MomL led to almost complete loss of its QQ activity. We then tested the effect of MomL and its mutants on Pcc-infected Chinese cabbage. The results indicated that MomL and its mutants (MomL<inf>L254R</inf>, MomL<inf>I144V</inf>, MomL<inf>V149A</inf>) significantly decreased the pathogenicity of Pcc. This study provides an efficient method for QQ enzyme modification and gives us new clues for further investigation on the catalytic mechanism of QQ lactonase.