Adaptive landscapes and molecular basis for the functional innovation of an organophosphate hydrolysing enzyme

Abstract

How functional innovation of enzyme, i.e., the acquirement of novel functions, occurs is a fundamental question in molecular evolution. Here, we unveil the evolutionary processes that led to the emergence of methyl parathion hydrolase (MPH), an enzyme that has acquired the ability to degrade the xenobiotic organophosphate (OP), methyl-parathion (Pt-M). Combining ancestral sequence reconstruction (ASR) with biochemical, genetic, and structural analyses, we characterized the adaptive landscape of MPH during its functional transition towards Pt-M activity from a dihydrocoumarin (DHC)-degrading ancestor. Five amino acid substitutions were found to be critical for the conversion between the ancestral DHC activity and the evolved Pt-M activity, accounting for a >600,000-fold functional switch between the two substrates. Characterization of adaptive landscapes of 5 combinatorial mutational space (32 combinations) revealed that the prevalence of epistatic interactions between the residues; consequently, only a fraction (16) of the 120 (5!) possible pathways are actually accessible in the gradually incremental manner. Moreover, multiple adaptive landscapes analysis for four similar OP compounds unveiled molecular basis for the development of high substrate specificity toward Pt-M over other compounds. Our study provides a comprehensive description of the evolutionary and molecular mechanisms that led to the emergence of a novel enzymatic function.