Pairing-Dependent Effects of a Modular UbiX–PhdC–UbiD Cascade in prFMN-Dependent Biocatalysis

UbiD-family decarboxylases, named after the UbiD decarboxylase involved in ubiquinone biosynthesis, require prenylated flavin mononucleotide (prFMN), and efficient formation of the catalytically competent cofactor can limit their functional expression and engineering. Here, we investigated whether the prFMN maturase PhdC could be incorporated as a distinct cofactor-maturation module in a cascade comprising the flavin prenyltransferase UbiX, PhdC, and UbiD. Seven sequence-diverse PhdC homologs were selected and characterized in vitro; EtPhdC and SmPhdC were evaluated as engineered A148E and Q137E variants, respectively. Their effects were examined by whole-cell coexpression with ferulic acid decarboxylase (FDC), the pyrrole-2-carboxylate decarboxylase HudA, the 2,5-furandicarboxylic acid decarboxylase HmfF, and three 3,4-dihydroxybenzoic acid decarboxylase (AroY) homologs. All seven PhdC homologs produced spectral changes at 450 nm consistent with changes associated with prFMN oxidative maturation. However, their effects on whole-cell reaction output were strongly pairing-dependent: PhdC coexpression decreased FDC output, had limited effects on HudA, and increased HmfF output, with RjPhdC giving the highest value. AroY homologs also showed distinct PhdC-dependent responses. Furthermore, PhdC coexpression recovered detectable whole-cell reaction output in selected Glu variants, including KpAroY E289A, E289Q, and E289D, whereas recovery was not universal. These results indicate that cofactor maturation can be treated as an additional design variable in UbiD-family enzyme engineering and highlight PhdC–UbiD functional compatibility as an important consideration for prFMN-dependent biocatalysis.