Ferulic Acid Decarboxylase Controls Oxidative Maturation of the Prenylated Flavin Mononucleotide Cofactor.

Prenylated flavin mononucleotide (prFMN) is a recently discovered modified flavin cofactor containing an additional nonaromatic ring, connected to the N5 and C6 atoms. This cofactor underpins reversible decarboxylation catalyzed by members of the widespread UbiD enzyme family and is produced by the flavin prenyltransferase UbiX. Oxidative maturation of the UbiX product prFMNH to the corresponding oxidized prFMN is required for ferulic acid decarboxylase (Fdc1; a UbiD-type enzyme) activity. However, it is unclear what role the Fdc1 enzyme plays in this process. Here, we demonstrate that, in the absence of Fdc1, prFMNH oxidation by O proceeds via a transient semiquinone prFMN species and culminates in a remarkably stable prFMN-hydroperoxide species. Neither forms of prFMN are able to support Fdc1 activity. Instead, enzyme activation using O-mediated oxidation requires prFMNH binding prior to oxygen exposure, confirming that UbiD enzymes play a role in O-mediated oxidative maturation. In marked contrast, alternative oxidants such as potassium ferricyanide support prFMN formation both in solution and in Fdc1.