Prenylated flavin mononucleotide explained

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.^[1]

prFMN is flavin prenylated at the N5 and C6 positions resulting in the formation of a fourth non-aromatic ring.^[2]

prFMN was discovered in 2015 at the University of Manchester by David Leys' group.^[3]

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^UbiX) 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^UbiX, 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^Ox

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^ox) cofactor were observed: prFMN^iminium, hydroxylated prFMN^iminium and prFMN^ketimine. Determination of the prFMN isomer that was catalytically relevant involved incubation of AnFdc1^UbiX 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^UbiX with phenylpyruvate revealed a bond between C1’ of prFMN^iminium and a phenylacetaldehyde adduct – a species that can be formed by decarboxylation of α-hydroxycinnamic acid and tautomerization of the α-hydroxystyrene prFMN^iminium adduct.

This observation confirmed that it is the prFMN^iminium that is the catalytically relevant cofactor.

Notes and References

1. Leys . David . Flavin metamorphosis: cofactor transformation through prenylation . Current Opinion in Chemical Biology . December 2018 . 47 . 117–125 . 10.1016/j.cbpa.2018.09.024. 30326424 . free .
2. White . Mark D. . Payne . Karl A. P. . Fisher . Karl . Marshall . Stephen A. . Parker . David . Rattray . Nicholas J. W. . Trivedi . Drupad K. . Goodacre . Royston . Rigby . Stephen E. J. . Scrutton . Nigel S. . Hay . Sam . Leys . David . UbiX is a flavin prenyltransferase required for bacterial ubiquinone biosynthesis . Nature . 17 June 2015 . 522 . 7557 . 502–506 . 10.1038/nature14559. 26083743 . 4988493 . 2015Natur.522..502W .
3. Payne . Karl A. P. . White . Mark D. . Fisher . Karl . Khara . Basile . Bailey . Samuel S. . Parker . David . Rattray . Nicholas J. W. . Trivedi . Drupad K. . Goodacre . Royston . Beveridge . Rebecca . Barran . Perdita . Rigby . Stephen E. J. . Scrutton . Nigel S. . Hay . Sam . Leys . David . New cofactor supports α,β-unsaturated acid decarboxylation via 1,3-dipolar cycloaddition . Nature . 17 June 2015 . 522 . 7557 . 497–501 . 10.1038/nature14560. 26083754 . 4988494 . 2015Natur.522..497P .