Prenylated flavin mononucleotide

Prenylated flavin mononucleotide (prFMN) is a cofactor produced by the flavin prenyltransferase UbiX and utilised by UbiD enzymes in their function as reversible decarboxylases. Hence, prFMN is pivotal for catalysis in the ubiquitous microbial UbiDX system[1]. prFMN is flavin prenylated at the N5 and C6 positions resulting in the formation of a fourth non-aromatic ring (Figure 1)[2] prFMN was discovered in 2015 in the University of Manchester by David Leys’ group[2][3].

Figure 1. Structure of FMN and prFMN^iminium. The leftmost structure shows FMN (Flavin mononucleotide), the rightmost structure shows prFMN^iminium (prenylated flavin mononucleotide), Note the presence of the quaternary carbon (C3’) in prFMN^iminium indicative of cofactor prenylation.

Prenylated flavin mononucleotide

Identifiers
3D model (JSmol)	Interactive image
PubChem CID	102515050
InChI InChI=1S/C22H31N4O9P/c1-10-7-12-16-15(11(10)2)22(3,4)5-6-25(16)17-19(23-21(31)24-20(17)30)26(12)8-13(27)18(29)14(28)9-35-36(32,33)34/h7,13-14,18,27-29H,5-6,8-9H2,1-4H3,(H2,32,33,34)(H2,23,24,30,31)/p-2/t13-,14+,18-/m0/s1 Key: GLNFBGKHGHVPRL-IYOUNJFTSA-L
SMILES CC1=CC2=C3C(=C1C)C(CCN3C4=C(N2C[C@@H]([C@@H]([C@@H](COP(=O)([O-])[O-])O)O)O)NC(=O)NC4=O)(C)C
Properties
Chemical formula	C22H29N4O9P^2
Molar mass	524.466 g·mol^−1
Except where otherwise noted, data are given for materials in their standard state (at 25 °C [77 °F], 100 kPa).
Infobox references

Two studies in 2015 characterised UbiX as a flavin prenyltransferase, supplying prFMN to UbiD/Fdc1 which utilises the cofactor to catalyse a reversible decarboxylation reaction[2][3]. 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 (Figure 2). 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.

Figure 2. prFMN^iminium in the active site of AnFDC1. PDB file: 4ZA4.

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 tautomerisation of the α-hydroxystyrene prFMN^iminium adduct.

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

References

1. Leys, David (December 2018). "Flavin metamorphosis: cofactor transformation through prenylation". Current Opinion in Chemical Biology. 47: 117–125. doi:10.1016/j.cbpa.2018.09.024. PMID 30326424.
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 (17 June 2015). "UbiX is a flavin prenyltransferase required for bacterial ubiquinone biosynthesis". Nature. 522 (7557): 502–506. Bibcode:2015Natur.522..502W. doi:10.1038/nature14559. PMC 4988493. PMID 26083743.
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 (17 June 2015). "New cofactor supports α,β-unsaturated acid decarboxylation via 1,3-dipolar cycloaddition". Nature. 522 (7557): 497–501. Bibcode:2015Natur.522..497P. doi:10.1038/nature14560. PMC 4988494. PMID 26083754.