Unraveling the Catalytic Promiscuity Mechanism of Triterpene Synthase AaOSC-22030 via Multiscale QM/MM Simulations and Mutagenesis

exhibits remarkable catalytic promiscuity, converting 2,3-oxidosqualene into diverse triterpenoid skeletons. In this study, we employed multiscale molecular dynamics, static QM/MM calculations, and site-directed mutagenesis to elucidate the origins of this diversity. Our analysis identifies residue 728 as a pivotal determinant. We reveal that the substitution of a conserved aromatic residue with a polar serine (S728) creates a relaxed steric environment, permitting broad conformational exploration, while simultaneously serving as a general base to intercept intermediates. Strikingly, the S728Y mutation transformed the promiscuous enzyme into a specific synthase, yielding Dammarendiol II as the exclusive product. Furthermore, the E371A mutant demonstrated that the N369/E371 dyad independently controls the terminal hydration of pentacyclic products. These findings highlight that cyclization and quenching are governed by distinct active site residues, providing a rational strategy for the functional reshaping of triterpene synthases with customized product profiles.