Discovery and Characterization of Conserved N-Oxide-Forming Function of FAD-Dependent Monooxygenases for Fungal Prenylated Indole Alkaloid Biosynthesis.

N-oxides are ubiquitous functional groups in natural products, conferring critical biological properties such as enhanced solubility and modulated membrane permeability. However, the biosynthetic pathways for N-oxide formation in complex prenylated indole alkaloids (PIAs), especially the penicimutamides, remain poorly understood. Here, we describe the discovery and biochemical characterization of new activities of FAD-dependent monooxygenase (FMO) PldC, which sequentially catalyzes the indole 2,3-epoxidation-rearrangement of penicimutamide E, and the regioselective N13-oxidation of penicimutamide D to produce penicimutamide D N-oxide. Furthermore, conserved N-oxide-forming activity was confirmed as a new function with another two known FMOs of CtdE and PhqK involved in the biosynthesis of citrinadins and paraherquamides, respectively. These findings provide mechanistic insights into FMO function, revealing a remarkable catalytic plasticity whereby a single enzyme family drives dual oxidative pathways (C-oxidation and N-oxidation) to generate structurally diverse alkaloid metabolites. Collectively, our findings expand the catalytic landscape of FMOs, suggesting N-oxide formation as a conserved functional trait across this enzyme family during fungal PIA biosynthesis. These results also shed new light on the evolutionary trajectories and catalytic design principles of FMOs in the assembly of complex natural products.