Distinct Active-Site Architectures Drive Divergent Catalysis for Convergent Spirooxindole Biosynthesis

Abstract Prenylated indole alkaloids containing a spirooxindole scaffold exhibit diverse biological activities. Here, we report that the flavin-dependent monooxygenase PldC catalyzes the conversion of (+)-premalbrancheamide to 3S-hydroxyindolenine, which undergoes spontaneous rearrangement to form 3R-spirooxindole. In contrast, the homologous enzyme PhqK directly furnishes the 3R-spirooxindole from the same substrate. Structural modeling and site-directed mutagenesis reveal that divergent active-site architectures dictate substrate binding orientation, directing epoxide opening to control the product outcome. These findings highlight how distinct active site architectures drive divergent catalysis to achieve convergent spirooxindole biosynthesis.