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首页> 外文期刊>Applied Microbiology >Enhancing the Promiscuous Phosphotriesterase Activity of a Thermostable Lactonase (GkaP) for the Efficient Degradation of Organophosphate Pesticides
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Enhancing the Promiscuous Phosphotriesterase Activity of a Thermostable Lactonase (GkaP) for the Efficient Degradation of Organophosphate Pesticides

机译：增强热稳定的Lactonase（GkaP）的混杂磷酸三酯酶活性，以有效降解有机磷酸酯农药。

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The phosphotriesterase-like lactonase (PLL) enzymes in the amidohydrolase superfamily hydrolyze various lactones and exhibit latent phosphotriesterase activities. These enzymes serve as attractive templates for in vitro evolution of neurotoxic organophosphates (OPs) with hydrolytic capabilities that can be used as bioremediation tools. Here, a thermostable PLL from Geobacillus kaustophilus HTA426 (GkaP) was targeted for joint laboratory evolution with the aim of enhancing its catalytic efficiency against OP pesticides. By a combination of site saturation mutagenesis and whole-gene error-prone PCR approaches, several improved variants were isolated. The most active variant, 26A8C, accumulated eight amino acid substitutions and demonstrated a 232-fold improvement over the wild-type enzyme in reactivity ( k _(cat)/ K_(m) ) for the OP pesticide ethyl -paraoxon. Concomitantly, this variant showed a 767-fold decrease in lactonase activity with δ-decanolactone, imparting a specificity switch of 1.8 × 10~(5)-fold. 26A8C also exhibited high hydrolytic activities (19- to 497-fold) for several OP pesticides, including parathion, diazinon, and chlorpyrifos. Analysis of the mutagenesis sites on the GkaP structure revealed that most mutations are located in loop 8, which determines substrate specificity in the amidohydrolase superfamily. Molecular dynamics simulation shed light on why 26A8C lost its native lactonase activity and improved the promiscuous phosphotriesterase activity. These results permit us to obtain further insights into the divergent evolution of promiscuous enzymes and suggest that laboratory evolution of GkaP may lead to potential biological solutions for the efficient decontamination of neurotoxic OP compounds.
机译：酰胺水解酶超家族中的磷酸三酯酶样内酯酶（PLL）酶水解各种内酯，并表现出潜在的磷酸三酯酶活性。这些酶可作为具有体外水解能力的神经毒性有机磷酸酯（OPs）体外进化的诱人模板，可用作生物修复工具。在这里，来自kaustophilus嗜热芽孢杆菌HTA426（GkaP）的热稳定PLL被用于联合实验室开发，目的是增强其对OP农药的催化效率。通过位点饱和诱变和全基因易错PCR方法的组合，分离了几个改进的变体。最活跃的变体26A8C累积了8个氨基酸取代，并证明OP农药乙基对氧磷的反应性（k_（cat）/ K_（m））比野生型酶高232倍。同时，该变体显示出与δ-癸醇内酯的内酯酶活性降低了767倍，特异性转换为1.8×10〜（5）倍。 26A8C还对几种对硫磷农药，对硫磷，二嗪农和毒死rif具有高水解活性（19到497倍）。对GkaP结构上诱变位点的分析表明，大多数突变位于环8中，这决定了酰胺水解酶超家族中的底物特异性。分子动力学模拟揭示了为何26A8C失去其天然内酯酶活性并改善了混杂的磷酸三酯酶活性。这些结果使我们能够进一步了解混杂酶的不同进化，并表明GkaP的实验室进化可能会导致潜在的生物解决方案，以对神经毒性OP化合物进行有效净化。

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