Switching a newly discovered lactonase into an efficient and thermostable phosphotriesterase by simple double mutations His250Ile/Ile263Trp

Abstract

OPHC2 is a thermostable organophosphate (OP) hydrolase in the β-lactamase superfamily. OPs are highly toxic synthetic chemicals with no natural analogs. How did OPHC2 acquire phosphotriesterase (PTE) activity remained unclear. In this study, an OPHC2 analogue, PoOPH was discovered from Pseudomonas oleovorans exhibiting high lactonase and esterase activities and latent PTE activity. Sequence analysis revealed conserved His250 and Ile263 and site-directed mutagenesis at these crucial residues enhanced PTE activity. The best variant PoOPHM2 carrying H250I/I263W mutations displayed 6,962- and 106-fold improvements in catalytic efficiency for methyl-parathion and ethyl-paraoxon degradation, whereas the original lactonase and esterase activities decreased dramatically. A 1.4 × 10(7) -fold of specificity inversion was achieved by only two residue substitutions. Significantly, thermostability of the variants was not compromised. Crystal structure of PoOPHM2 was determined at 2.25 Å resolution and docking studies suggested that the two residues in the binding pocket determine substrate recognition. Lastly, new organophosphorus hydrolases (OPHs) were discovered using simple double mutations. Among them, PpOPHM2 from Pseudomonas putida emerged as a new promising OPH with very high activity (41.0 U mg(-1) ) toward methyl-parathion. Our results offer a first scrutiny to PTE activity evolution of OPHs in β-lactamase superfamily and provide efficient and robust enzymes for OP detoxification.

Marked enhancement of Acinetobacter sp. organophosphorus hydrolase activity by a single residue substitution Ile211Ala

“…PoOPH shares 98.5 % sequence identity with OPHC2. By simple double mutations His250Ile/Ile263Trp, PoOPH was switched into an efficient OPH, displaying 6962-fold improvement in catalytic efficiency toward MP (Luo et al 2014). The study proves the emergence of efficient and robust enzymes for OP detoxification by OPH activity evolution in the β-lactamase superfamily.…”

“…as template with the following primers: 5′-GGGTTTCATATGATGCTAAAAAATAGAC-3′ (forward) and 5′-CCCAAGCTTATCTTTAAAATG GATCGGA-3′ (reverse) (Luo et al 2014). The PCR fragment was subsequently cloned into expression vector pET-28a(+) to generate the recombinant plasmid pET28a-AbOPH.…”

“…In recent years, clarifying the evolutionary pathway (Bar-Rogovsky et al 2013;Luo et al 2014) and structure-activity relationship (Dong et al 2005;Gotthard et al 2013) of organophosphorus hydrolase (OPH) has been extensively studied. Phosphotriesterase (PTE) from Brevundimonas diminuta was the best-characterized and potentially important OPH, which exhibited near diffusion-limit reaction rate (10 ) to ethyl-paraoxon (Omburo et al 1992).…”

“…In parallel with MPH, a newly identified OPH, OPHC2, isolated from Pseudomonas pseudoalcaligenes, is a thermostable OPH (T m = 97.8 ± 3.2 °C) (Gotthard et al 2013) in β-lactamase superfamily, which shares 46 % sequence identity with MPH. PoOPH, a newly discovered lactonase from Pseudomonas oleovorans, exhibited high lactonase and esterase activities, but low OPH activity (Luo et al 2014). PoOPH shares 98.5 % sequence identity with OPHC2.…”

Review on Bioremediation of Methyl Parathion Contaminated Agricultural Soil by Microorganisms

“…A methyl parathion hydrolase (MPH) gene, bjmpd, was cloned from a newly isolated MPdegrading bacterial strain, Burkholderia jiangsuensis MP-1T and heterologously expressed in Escherichia coli BL21 (DE3) [52].Although the amino acid sequence of the bjmpd-encoded enzyme, named BjMPH, differed from that of MPH from Pseudomonas sp. WBC-3 (PsMPH) in only three residues, Ser132, Val247 and Ala267, a significantly higher specific activity towards MP was exhibited by BjMPH than PsMPH.…”