Prenylated flavin mononucleotide
Prenylated flavin mononucleotide (prFMN) is a cofactor produced by the flavin prenyltransferase UbiX and used by UbiD enzymes in their function as reversible decarboxylases. Hence, prFMN is pivotal for catalysis in the ubiquitous microbial UbiDX 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) 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, 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. PrFMN. 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) cofactor were observed: prFMN, hydroxylated prFMN and prFMN. Determination of the prFMN isomer that was catalytically relevant involved incubation of AnFdc1 with phenylpyruvate (of which a small proportion is α-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 with phenylpyruvate revealed a bond between C1’ of prFMN and a phenylacetaldehyde adduct – a species that can  be formed by decarboxylation of α-hydroxycinnamic acid and tautomerization of the α-hydroxystyrene prFMN adduct. This observation confirmed that it is the prFMN that is the catalytically relevant cofactor.