甲基对硫磷水解酶(MPH)基因,bjmpd,从新分离的MP降解细菌菌株中克隆,江苏伯克氏菌MP-1T并在大肠杆菌BL21(DE3)中异源表达。虽然bjmpd编码酶的氨基酸序列,叫BjMPH,与假单胞菌的MPH不同。WBC-3(PsMPH)只有三个残基,Ser132,Val247和Ala267,BjMPH对MP的比活性明显高于PsMPH。其中,Ala267被确定为影响催化效率的关键位点,在同源蛋白中相当保守(Ala或Ser),这表明,基于共有序列方法,用另一个保守残基简单地取代保守位点中的残基可能会增强MP降解酶的催化效率。受到这种观察的启发,我们发现了一个新的突变体,BjMPHT64N,对MP的催化效率(kcat/KM)比其野生型高3.78倍,达到4.20×106M-1s-1。突变体BjMPHT64N还表现出对其他有机磷农药的增强的反应性(kcat/KM)。同源性建模分析表明,该突变体中第64个残基的极性接触增强可能有助于稳定酶的结构并促进酶与底物之间的相互作用。这项研究产生了一种高效的MP降解酶,并为基于共识方法通过保守残基取代提高MPHs的催化效率提供了有用的信息。
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.