Prenylated flavin mononucleotide (prFMN) is a cofactor biosynthesized by the flavin prenyltransferase UbiX and used by UbiD enzymes for reversible decarboxylation reactions. Hence, prFMN is pivotal for catalysis in the ubiquitous microbial UbiD/X system.
prFMN is flavin prenylated at the N5 and C6 positions resulting in the formation of a fourth non-aromatic ring.
prFMN was discovered in 2015 at the University of Manchester by David Leys' group.
Two studies in 2015 characterized UbiX as a flavin prenyltransferase, supplying prFMN to UbiD/Fdc1 which utilises the cofactor to catalyse a reversible decarboxylation reaction. Ferulic acid decarboxylase (Fdc1) from A. niger co-expressed in E. coli with UbiX from E. coli (AnFdc1<sup>UbiX</sup>) once purified had clear spectral differences to singly expressed AnFdc1, and was capable of in vitro decarboxylation of a range of aromatic carboxylic acids. The atomic resolution of the crystal structure of AnFdc1<sup>UbiX</sup>, allowed elucidation of the structure of the modified FMN cofactor classified as prFMN. The crystal structure revealed an isopentenyl-adduct to the N5-C6 of FMN, with the modifications branched nature and the position of the covalent linkages with flavin suggesting prenylation.
UbiD activation by UbiX/prFMN was found to be dependent on oxygen suggesting that the reduced prFMN product of UbiX is oxidised to the catalytically relevant form. Several variations of the oxidised prFMN (prFMN<sup>ox</sup>) cofactor were observed: prFMN<sup>iminium</sup>, hydroxylated prFMN<sup>iminium</sup> and prFMN<sup>ketimine</sup>. Determination of the prFMN isomer that was catalytically relevant involved incubation of AnFdc1<sup>UbiX</sup> with phenylpyruvate (of which a small proportion is ñ-hydroxycinnamic acid which closely resembles cinnamic acid - a model substrate). Incubation with phenylpyruvate lead to an altered UV-Vis spectrum and reversible enzyme inhibition. The crystal structure of AnFdc1<sup>UbiX</sup> with phenylpyruvate revealed a bond between C1â of prFMN<sup>iminium</sup> and a phenylacetaldehyde adduct â a species that can be formed by decarboxylation of ñ-hydroxycinnamic acid and tautomerization of the ñ-hydroxystyrene prFMN<sup>iminium</sup> adduct.
This observation confirmed that it is the prFMN<sup>iminium</sup> that is the catalytically relevant cofactor.