Enhancing the promiscuous phosphotriesterase activity of a thermostable lactonase (GkaP) for the efficient degradation of organophosphate pesticides.
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.
State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, People's Republic of China.
Yu
Y
Zhang
Jiao
J
An
Wei
W
Ye
Guangyu
G
Yang
Zhi-Gang
ZG
Qian
Hai-Feng
HF
Chen
Li
L
Cui
Yan
Y
Feng
D000581:Amidohydrolases / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution; D004390:Chlorpyrifos / Q000378:metabolism; D003976:Diazinon / Q000378:metabolism; D019020:Directed Molecular Evolution; D056506:Geobacillus / Q000201:enzymology*; D007700:Kinetics; D007783:Lactones / Q000378:metabolism; D008958:Models, Molecular; D016296:Mutagenesis; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D010755:Organophosphates / Q000378:metabolism*; D010278:Parathion / Q000378:metabolism; D010575:Pesticides / Q000378:metabolism*; D044345:Phosphoric Triester Hydrolases / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods; D011487:Protein Conformation; D013379:Substrate Specificity
D007783:Lactones; D050505:Mutant Proteins; D010755:Organophosphates; D010575:Pesticides; D010278:Parathion; D044345:Phosphoric Triester Hydrolases; D000581:Amidohydrolases; D004390:Chlorpyrifos; D003976:Diazinon