Ministry of Education Key Laboratory of Carbohydrate Chemistry and Biotechnology, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, China.
Appl Microbiol Biotechnol. 2018 Aug;102(15):6537-6545. doi: 10.1007/s00253-018-9108-0. Epub 2018 Jun 15.
Methyl parathion hydrolase (MPH) that hydrolyzes a wide range of organophosphorus pesticides can be used to remediate land polluted by the pesticides. Here, the catalytic efficiency of methyl parathion hydrolase from Pseudomonas sp. (WBC-3) was enhanced by searching and engineering a critical site far away from the binding pocket. In the first round, a four-site mutant with a modest increased catalytic efficiency (3.2-fold k/K value of the wild type) was obtained with random mutagenesis. By splitting and re-combining the four substitutions in the mutant, the critical site S277, was identified to show the most significant effects of improving binding affinity and catalytic efficiency. With further site-saturation mutagenesis focused on the residue S277, another two substitutions were discovered to have even more significant decrease in K (40.2 and 47.6 μM) and increased in k/K values (9.5- and 10.3-fold of the wild type) compared to the original four-site mutant (3.0- and 3.2-fold). In the three-dimensional structure, residue S277 is located at a hinge region of a loop, which could act as a "lid" at the substrate entering to the binding pocket. This suggests that substitutions of residue S277 could affect substrate binding via conformational change in substrate entrance region. This work provides a valuable protocol combining random mutagenesis, site-saturation mutagenesis, structural and bioinformatics analyses to obtain mutants with high catalytic efficiency from a screening library of a modest size (3200 strains).
甲基对氧磷水解酶（MPH）能够水解广泛的有机磷农药，可用于修复受农药污染的土地。在这里，通过搜索和工程化远离结合口袋的关键位点，提高了来自假单胞菌（WBC-3）的甲基对氧磷水解酶的催化效率。在第一轮中，通过随机诱变获得了一个催化效率略有提高（野生型 k/K 值的 3.2 倍）的四突变体。通过拆分和重新组合突变体中的四个取代，确定了关键位点 S277，它对提高结合亲和力和催化效率的影响最大。通过进一步针对残基 S277 的定点饱和突变，发现另外两个取代使 K 值（40.2 和 47.6 μM）显著降低，k/K 值（野生型的 9.5-和 10.3 倍）显著提高，与原始四突变体（3.0-和 3.2 倍）相比。在三维结构中，残基 S277 位于环的铰链区域，它可以作为进入结合口袋的底物的“盖子”。这表明取代残基 S277 可以通过底物进入区域的构象变化影响底物结合。这项工作提供了一个有价值的方案，结合随机诱变、定点饱和诱变、结构和生物信息学分析，从一个中等规模（3200 株）的筛选文库中获得高催化效率的突变体。
