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Applied Microbiology and Biotechnology

, Volume 99, Issue 18, pp 7559–7568 | Cite as

Identification and regulation of the catalytic promiscuity of (−)-γ-lactamase from Microbacterium hydrocarbonoxydans

  • Yu Sun
  • Hongtao Zhao
  • Jianjun Wang
  • Junge Zhu
  • Sheng WuEmail author
Biotechnologically relevant enzymes and proteins

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.

Keywords

γ-Lactamase Perhydrolase Enzyme promiscuity Protein engineering Microbacterium hydrocarbonoxydans 

Notes

Acknowledgments

We thank the financial support from the State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences.

Supplementary material

253_2015_6503_MOESM1_ESM.pdf (369 kb)
ESM 1 (PDF 368 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Yu Sun
    • 1
    • 2
  • Hongtao Zhao
    • 3
  • Jianjun Wang
    • 1
  • Junge Zhu
    • 1
  • Sheng Wu
    • 1
    Email author
  1. 1.State Key Laboratory of Microbial Resources, Institute of MicrobiologyChinese Academy of SciencesBeijingPeople’s Republic of China
  2. 2.University of Chinese Academy of SciencesBeijingPeople’s Republic of China
  3. 3.Department of BiochemistryUniversity of ZurichZurichSwitzerland

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