# Structure and Mechanism of PhdC, a Prenylated‐Flavin Maturase

## TL;DR

This paper reveals the structure and function of PhdC, an enzyme that helps activate a key cofactor for certain enzymes.

## Contribution

The study provides structural and mechanistic insights into PhdC, a novel prFMN maturase enzyme.

## Key findings

- PhdC is a distant member of the class I HpaC-like flavin reductases and catalyzes prFMN radical to prFMNiminium.
- Co-expression of PhdC or its homologue YclD activates UbiD, similar to the effect of LpdD.
- Conserved Glu residues in PhdC suggest a proton-abstraction coupled oxidation mechanism.

## Abstract

Prenylated flavin mononucleotide (prFMN) is a modified flavin cofactor required by the UbiD family of (de)carboxylase enzymes. While the reduced prFMNH2 form is produced by the flavin prenyltransferase UbiX, the corresponding two‐electron oxidized prFMNiminium form is required to support UbiD catalysis. Thus, oxidative maturation of prFMNH2 is required, which can be catalyzed by UbiD. However, heterologous (over)expression of UbiDs frequently leads to the accumulation of the stable but non‐active one‐electron oxidized purple prFMNradical species. A dedicated prFMN maturase enzyme (PhdC) from Mycolicibacterium fortuitum was recently identified as capable of catalyzing the oxidative maturation of prFMNradical to prFMNiminium, thereby enabling an effective supply of active cofactor to the associated phenazine‐1‐carboxylate (de)carboxylase PhdA. We report the crystal structure of PhdC in complex with flavin, revealing it is a distant member of the class I HpaC‐like family of short‐chain dimeric flavin reductases and demonstrate catalytic conversion of the prFMNradical species to prFMNiminium in the presence of oxygen or ferricyanide. Co‐expression of PhdC or a distant homologue from Priestia megaterium (YclD) with the canonical UbiD from
Escherichia coli
leads to activation of the latter, similar in effect to co‐expression with the prFMNH2‐binding chaperone LpdD. Conserved Glu residues in the PhdC active site suggest catalysis occurs through C1′ proton‐abstraction coupled oxidation. This study thus provides both structural and mechanistic insight into the function of PhdC, adding to the expanding repertoire of prFMN‐binding proteins associated with the widespread UbiDX system.