title: Improved efficiency of a novel methyl parathion hydrolase using consensus approach.
abstract: A methyl parathion hydrolase (MPH) gene, bjmpd, was cloned from a newly isolated MP-degrading bacterial strain, Burkholderia jiangsuensis MP-1Tand 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 BjMPHT64Nalso 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.
authors.affiliation: State Key Laboratory of Bioreactor Engineering and Shanghai Collaborative Innovation Center for Biomanufacturing, East China University of Science and Technology, Shanghai 200237, China; The National Engineering Research Center for Miniaturized Detection Systems, Xi'an 710069, China.
authors.forename: Xu-Yun
authors.initials: XY
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Bioreactor Engineering and Shanghai Collaborative Innovation Center for Biomanufacturing, East China University of Science and Technology, Shanghai 200237, China.
authors.forename: Fei-Fei
authors.initials: FF
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Bioreactor Engineering and Shanghai Collaborative Innovation Center for Biomanufacturing, East China University of Science and Technology, Shanghai 200237, China. Electronic address: chunxiuli@ecust.edu.cn.
authors.forename: Chun-Xiu
authors.initials: CX
authors.lastname: Li
authors.affiliation: State Key Laboratory of Bioreactor Engineering and Shanghai Collaborative Innovation Center for Biomanufacturing, East China University of Science and Technology, Shanghai 200237, China.
authors.forename: Xiao-Jing
authors.initials: XJ
authors.lastname: Luo
authors.affiliation: State Key Laboratory of Bioreactor Engineering and Shanghai Collaborative Innovation Center for Biomanufacturing, East China University of Science and Technology, Shanghai 200237, China.
authors.forename: Qi
authors.initials: Q
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Bioreactor Engineering and Shanghai Collaborative Innovation Center for Biomanufacturing, East China University of Science and Technology, Shanghai 200237, China.
authors.forename: Yun-Peng
authors.initials: YP
authors.lastname: Bai
authors.affiliation: State Key Laboratory of Bioreactor Engineering and Shanghai Collaborative Innovation Center for Biomanufacturing, East China University of Science and Technology, Shanghai 200237, China. Electronic address: jianhexu@ecust.edu.cn.
authors.forename: Jian-He
authors.initials: JH
authors.lastname: Xu
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001673:Biodegradation, Environmental; D019117:Burkholderia / Q000201:enzymology / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D016384:Consensus Sequence; D017124:Conserved Sequence; D019020:Directed Molecular Evolution; D004795:Enzyme Stability; D005798:Genes, Bacterial; D006860:Hydrogen Bonding; D007306:Insecticides / Q000378:metabolism; D007700:Kinetics; D008743:Methyl Parathion / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D040681:Structural Homology, Protein
chemical_list: D001426:Bacterial Proteins; D007306:Insecticides; D011994:Recombinant Proteins; D008743:Methyl Parathion; C495424:methyl parathion hydrolase, Pseudomonas; D010744:Phosphoric Monoester Hydrolases
keywords: Biodegradation; Catalytic efficiency; Consensus approach; Methyl parathion hydrolase; Organophosphorus pesticides; Site-directed mutagenesis