Enhanced degradation of chemical warfare agents through molecular engineering of the phosphotriesterase active site
Enhanced degradation of chemical warfare agents through molecular engineering of the phosphotriesterase active site
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DOI：
10.1021/ja0358798
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发表时间：
2003-07-30
影响因子：
15
通讯作者：
Raushel, FM
中科院分区：
文献类型：
--
作者：
Hill, CM;Li, WS;Raushel, FM
细菌磷酸三酯酶已被用作进化用于催化分解有机磷神经毒剂的改良酶的模板。通过对His-254、His-257和Leu-303残基的随机化，构建了活性位点突变的组合库。对突变蛋白质的收集进行了筛选，以了解其对化学战剂梭曼毒性最大的立体异构体的显色类似物的降解能力。突变株H254G/H257W/L303T对目标底物的催化作用比野生型快近3个数量级。在甲基膦酸二异丙酯存在和不存在的情况下解析了X-射线晶体结构。突变的酶被连接到活性部位的另一个二价阳离子上，该阳离子在底物类似物抑制剂结合时被取代。这些研究表明，底物专一性的实质性变化可以通过对初级氨基酸序列进行相对较小的改变来实现。
The bacterial phosphotriesterase has been utilized as a template for the evolution of improved enzymes for the catalytic decomposition of organophosphate nerve agents. A combinatorial library of active site mutants was constructed by randomizing residues His-254, His-257, and Leu-303. The collection of mutant proteins was screened for the ability to hydrolyze a chromogenic analogue of the most toxic stereoisomer of the chemical warfare agent, soman. The mutant H254G/H257W/L303T catalyzed the hydrolysis of the target substrate nearly 3 orders of magnitude faster than the wild-type enzyme. The X-ray crystal structure was solved in the presence and absence of diisopropyl methyl phosphonate. The mutant enzyme was ligated to an additional divalent cation at the active site that was displaced upon the binding of the substrate analogue inhibitor. These studies demonstrate that substantial changes in substrate specificity can be achieved by relatively minor changes to the primary amino acid sequence.