Molecular Evolution Of A Thermostable Phosphotiresterase-Like Lactonase Toward High Degradation Activity For Organophosphate Pesticides

Synthetic organophosphate (OP) compounds have been widely used as agriculturalpesticides since the1950’s. More than100OP pesticides are in use worldwide. Continuousand excessive use of OP compounds has led to the contamination of many terrestrial andaquatic ecosystems. OP compounds are highly toxic due to irreversible inhibitacetylcholinesterase (AChE) and disrupt neurotransmission in the central nervous system forall vertebrates. Currently, decontamination of OP compounds utilizes bleach treatment,alkaline hydrolysis, or incineration. In all cases, the conditions are harsh, and the byproductscan be toxic and corrosive. Therefore, enzymatic degradation of OP compound has receivedconsiderable attention since it provides the possibility of both environmentally friendly and insitu detoxification. The phosphotriesterase (PTE, EC3.1.8.1) within the amidohydrolasesuperfamily can hydrolyze a broad range of OP compounds, including most OP pesticides andCWAs. The phosphotriestrerase has been recognized an ideal candidate for OP detoxification.We are interested in developing more robust phosphotriesterase that can be used asbioremediation tools. By searching bacterial genomic database, we found the locus tagGK1506(GenBank ID:3183579) from Geobacillus kaustophilus HTA426encoding aputative phosphotriesterase (GkaP). The GK1506gene was cloned and heterologousexpressed in Escherichia coli. A systematic study was carried out to reseach its enzymaticproperties, catalytic mechanism and molecular evolution.The recombinant GkaP possessed exceptional biological stability. It was also found toproficiently hydrolyze various lactones, and exhibited promiscuous phosphotriesterase andesterase activities. It shoule be classified into newly emerging phosphotriesterase-lilelactonase (PLL) family. Enzyme promiscuity is a dominant feature for divergent evolution ofnew catalysts. A better understanding of catalytic promiscuity can improve our knowledge ofprotein evolution and ancestry. Therefor, GkaP becomes an ideal model to study enzymepromiscuousty. We investigated the function of hot spots in the active site by site-directedmutagenesis approach. We found that position99in the active site was closely related tosubstrate discrimination. One evolved variant, Y99L, exhibited an11-fold improvement over wild-type in reactivity (kcat/Km) toward the phosphotriesterase substrate ethyl-paraoxon, butshowed a15-fold decrease toward the lactonase substrate δ-decanolactone, with a157-foldinversion of substrate specificity. Structural analysis of Y99L revealed that the mutationcauses a~6.6outward shift of adjacent loop7, which may increase the flexibility of theactive site and facilitate organophosphate substrate accommodation and catalysis. To furtherexplore the catalytic mechanism, the hypothetical proton shuttle pathway was constructed forresidues Asp266, Arg230, and Gly209. Mutation G209D increased both the phosphotriesteraseand lactonase activities by up to10-and3-fold, respectively. Structural analysis of G209Dvariant identified two simultaneous hydrogen bond bridges between a water molecule andArg230and Asp209, which resulted in a conformational fluctuation of the Arg230side chain anda~2shift of the adjacent loop7. Our results demonstrate that a limited mutation in apromiscuous enzyme may lead to alterations in the dynamics and conformational distributionof substrate-binding loop, which benefit for an alternative binding substrate and efficientcatalysis. These findings provide a new clue for understanding the catalytic mechanism of thepromiscuous enzyme.Protein engineering techniques are powerful methods to reshaping enzymatic proficiencyand specificity. We selected GkaP as a template to evolve its ancillary OPs hydrolyticcapability. By combining rational and random mutagenesis strategies, we successfullyobtained several active variants after four rounds of mutation and screening (~10,000colonies). Among these, the best variant26A8C demonstrated232-fold improvement overthe wild-type enzyme in reactivity (kcat/Km) for OP pesticide ethyl-paraoxon. This superiorvariant also exhibited high hydrolytic activities over17-497-fold for several targeted OPpesticides, including parathion, diazinon and chlorpyrifos. Concomitantly, the variant26A8Cshowed a767-fold decrease in lactonase activity, producing specialized for OP rather thanlactone hydrolysis. The variant26A8C accumulated eight mutations: F28I, Y99L, T171S,F228L, N269S, V270G, W271C and G273D. The analysis for the mutagenesis sites in theGkaP structure revealed that the key mutations leading to higher phosphotriesterase activityare located in loops7and8(F228L, N269S, V270G, W271C and G273D), In theamidohydrolase superfamily members in particular, loops7and8are most often involved incontacting the ligands in the active site and determining substrate specificity. These results notonly permit us to obtain further insights into the divergence evolution of plosphotriesterasebut also suggested that laboratory evolution of GkaP may lead to potential biological solutionsfor efficient decontamination of neurotoxic OP compounds.