caa a22 4500 60550122X CHVBK 20210128100559.0 cr unu---uuuuu 210128e20150901xx s 000 0 eng 10.1007/s00253-015-6503-7 doi (NATIONALLICENCE)springer-10.1007/s00253-015-6503-7 Identification and regulation of the catalytic promiscuity of (−)-γ-lactamase from Microbacterium hydrocarbonoxydans [Elektronische Daten] [Yu Sun, Hongtao Zhao, Jianjun Wang, Junge Zhu, Sheng Wu] 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. Springer-Verlag Berlin Heidelberg, 2015 γ-Lactamase nationallicence Perhydrolase nationallicence Enzyme promiscuity nationallicence Protein engineering nationallicence Microbacterium hydrocarbonoxydans nationallicence Sun Yu State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, No. 1 West Beichen Road, Chaoyang District, 100101, Beijing, People's Republic of China aut Zhao Hongtao Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland aut Wang Jianjun State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, No. 1 West Beichen Road, Chaoyang District, 100101, Beijing, People's Republic of China aut Zhu Junge State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, No. 1 West Beichen Road, Chaoyang District, 100101, Beijing, People's Republic of China aut Wu Sheng State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, No. 1 West Beichen Road, Chaoyang District, 100101, Beijing, People's Republic of China aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/18(2015-09-01), 7559-7568 0175-7598 99:18<7559 2015 99 253 https://doi.org/10.1007/s00253-015-6503-7 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-015-6503-7 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Sun Yu State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, No. 1 West Beichen Road, Chaoyang District, 100101, Beijing, People's Republic of China aut NATIONALLICENCE 700 1- Zhao Hongtao Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland aut NATIONALLICENCE 700 1- Wang Jianjun State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, No. 1 West Beichen Road, Chaoyang District, 100101, Beijing, People's Republic of China aut NATIONALLICENCE 700 1- Zhu Junge State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, No. 1 West Beichen Road, Chaoyang District, 100101, Beijing, People's Republic of China aut NATIONALLICENCE 700 1- Wu Sheng State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, No. 1 West Beichen Road, Chaoyang District, 100101, Beijing, People's Republic of China aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/18(2015-09-01), 7559-7568 0175-7598 99:18<7559 2015 99 253