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  • Enhancing the promiscuous phosphotriesterase activity of a thermostable lactonase (GkaP) for the efficient degradation of organophosphate pesticides.

Enhancing the promiscuous phosphotriesterase activity of a thermostable lactonase (GkaP) for the efficient degradation of organophosphate pesticides.

Applied and environmental microbiology (2012-07-17)
Yu Zhang, Jiao An, Wei Ye, Guangyu Yang, Zhi-Gang Qian, Hai-Feng Chen, Li Cui, Yan Feng
ABSTRACT

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.

MATERIALS
Product Number
Brand
Product Description

Supelco
Parathion, PESTANAL®, analytical standard
Supelco
Diazinon solution, 100 μg/mL in acetonitrile, PESTANAL®, analytical standard
Supelco
Diazinon, PESTANAL®, analytical standard
Supelco
Parathion solution, 100 μg/mL in cyclohexane, PESTANAL®, analytical standard
Supelco
Diazinon, certified reference material, TraceCERT®