Structure-based redesign of the bacterial prolidase active-site pocket for efficient enhancement of methyl-parathion hydrolysis

Abstract

The recruitment and rapid evolution of promiscuous catalysis provide insights into the precise mechanisms underlying enzyme repurposing and help understand molecular determinants of the remarkable adaptability of proteins. Starting from an old bacterial prolidase scaffold (PlOPAA), we performed laboratory-based evolution to improve its initially weak methyl-parathion hydrolase activity. Four residues surrounding the active-site pocket were simultaneously mutated, achieving a 10 000-fold increase in methyl-parathion hydrolysis and broadening the substrate specificity against organophosphorus compounds. Structural, kinetic, and computational comparisons of wild-type and the engineered enzyme identified the molecular basis for the notably enhanced catalysis and revealed that the mutations reshaped the substrate pocket to facilitate access to catalytic machinery for methyl-parathion. Our observations provide a new efficient biocatalyst for organophosphrus remediation, and shed light on how enzymes gain new functions in a permissive active-site architecture.