Catalytic promiscuity potentiated the divergence of new cytochrome P450 enzyme functions in cyanogenic defense metabolism

Abstract Cytochrome P450 monooxygenases (P450s) constitute the largest metabolic enzyme family in plants, responsible for synthesizing hundreds of thousands of specialized metabolites with essential roles in chemical defenses against herbivores and pathogens (Banks et al., 2011; Nelson and Werck-Reichhart, 2011; Wurtzel and Kutchan, 2016). Substrate promiscuity has been documented to play a central role in the evolution of plant specialized metabolic enzymes (Weng et al., 2012; Leong and Last, 2017), however most plant P450s are highly substrate-specific (Verpoorte, 2013). Here, we show the rapid inversion of primary and weak secondary (promiscuous) catalytic activities between two distinct yet evolutionarily linked multifunctional P450s, CYP71A12 and CYP71A13, based on intramolecular epistasis of two amino acid residues under positive selection in CYP71A12. Furthermore, we uncover previously undocumented catalytic activity during the inversion as well as naturally occurring amino acid substitution patterns that could have been present in evolutionary intermediates between the two enzymes. Comparative expression profiling and homology modeling reveal that natural selection acted on the promoter of CYP71A13 and the substrate-recognition elements of CYP71A12 to improve the efficiencies of their promiscuous reactions. The rise in catalytic promiscuity potentiated the divergence of new P450 enzyme functions in cyanogenic defense metabolism. Directed evolution of promiscuous reactions is one of the core technologies underpinning the field of synthetic biology. Our results provide a more complete understanding of how natural selection uses promiscuous reactions to generate new enzymes in nature and chemical diversity in pathogen defense, as well as demonstrate a novel strategy for identifying their molecular origins in highly divergent, related enzymes.