Multifunctional switch mediates the catalytic activity and product ratio in fungal CYP512W2

Abstract

Eukaryotic cytochrome P450 (CYP) monooxygenases serve as indispensable biocatalysts for the oxidative modification of bioactive compounds, yet their complicated membrane-anchored nature and structure–function relationships pose significant engineering challenges. Here, we design a computer-aided strategy and report an elegant evolutionary paradigm for fungal CYP512W2 by identifying the critical sites (I108 and I364) as “multifunctional switches” that govern both catalytic activity and product ratio during the biosynthesis of bioactive triterpenoids (GA-Y and GA-Jb). By combining computational mutational scanning, MD simulations, QM/MM calculations, and in vivo assays, we demonstrate that I108 substitutions modulate the catalytic outcomes via four aspects: (1) reshaping the pocket microenvironment, (2) enabling substrate conformational adaptation, (3) altering pre-reaction state populations, and (4) decreasing the energy barriers in catalysis. Remarkably, I108S and I108G enhanced the GA-Y production by 2.5- and 2.9-fold, respectively, while I108N reversed the product selectivity (79.2% for GA-Y in I108N vs. 23.6% in wild-type). Structural analyses revealed that, unlike most mutations that precisely adjust substrate conformations through steric and polar interactions, the I108N mutation disfavors GA-Jb formation by weakening GA-Y binding. This work identifies the key residues that serve as programmable nodes for functional divergence and provides a rational blueprint for the single-residue engineering of CYP regiospecificity to access diverse oxidized compounds.