Abstract:
:A methyl parathion hydrolase (MPH) gene, bjmpd, was cloned from a newly isolated MP-degrading bacterial strain, Burkholderia jiangsuensis MP-1T and heterologously expressed in Escherichia coli BL21 (DE3). Although the amino acid sequence of the bjmpd-encoded enzyme, named BjMPH, differed from that of MPH from Pseudomonas sp. WBC-3 (PsMPH) in only three residues, Ser132, Val247 and Ala267, a significantly higher specific activity towards MP was exhibited by BjMPH than PsMPH. Among them, Ala267 was identified as a key site affecting the catalytic efficiency, and it was rather conservative (Ala or Ser) in homologous proteins, suggesting that a simple substitution of the residue in conservative site with another conservative residue based on the consensus sequence approach might possibly enhance the catalytic efficiency of the MP-degrading enzyme. Inspired by such an observation, we identified a new mutant, BjMPHT64N, exhibiting 3.78-fold higher catalytic efficiency (kcat/KM) towards MP than its wild-type, reaching 4.20×106M-1s-1. The mutant BjMPHT64N also displayed enhanced reactivities (kcat/KM) towards other organophosphorus pesticides. Homology-modelling analysis indicates that enhanced polar contacts of the 64th residue in this mutant may contribute to stabilizing the structure of the enzyme and promote the interactions between enzyme and substrate. This study generated an efficient MP-degrading enzyme, and provides useful information for enhancing the catalytic efficiency of MPHs via conservative residue substitution based on the consensus approach.
journal_name
Enzyme Microb Technoljournal_title
Enzyme and microbial technologyauthors
Liu XY,Chen FF,Li CX,Luo XJ,Chen Q,Bai YP,Xu JHdoi
10.1016/j.enzmictec.2016.07.005subject
Has Abstractpub_date
2016-11-01 00:00:00pages
11-17eissn
0141-0229issn
1879-0909pii
S0141-0229(16)30134-Xjournal_volume
93-94pub_type
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