Structure and Mechanism of the Prenylated-FMN Maturase, PhdC

Prenylated-FMN-dependent (UbiD-like) enzymes catalyze (de)carboxylation reactions on a wide range of unreactive aromatic and heterocyclic compounds, making them attractive as biocatalysts. The oxidative maturation of prFMN, which is synthesized as its inactive, reduced form, is an essential yet poorly understood step. Incorrect cofactor oxidation prevents many UbiD-like enzymes from being produced as active holoenzymes. Here we report the structure of the recently identified prFMN maturase, PhdC, which catalyzes the oxidation of the spontaneously formed prFMN semiquinone radical to the active cofactor. The enzyme is a homodimer that possesses a split barrel fold seen in other flavin-dependent oxidases. prFMN is bound in a shallow cleft at the dimer interface, which leaves one face of the cofactor partially exposed to solvent. The structure reveals that E134 is positioned to act as the general base required to deprotonate the substrate, a step that deuterium kinetic isotope effect measurements indicate is rate-determining. U.V.–visible spectral changes indicate that on binding the substrate PhdC promotes formation of the prFMN semiquinone radical anion. Based on these experiments we propose a mechanism for the oxidative maturation of prFMN catalyzed by PhdC.