Characterization\nof the N5-dimethylallyl-FMN Intermediate\nin the Biosynthesis of Prenylated-FMN Catalyzed by UbiX

Prenylated-FMN (prFMN) is the cofactor used by the UbiD-like\nfamily\nof decarboxylases that catalyzes the decarboxylation of various aromatic\nand unsaturated carboxylic acids. prFMN is synthesized from reduced\nFMN and dimethylallyl phosphate (DMAP) by a specialized prenyl transferase,\nUbiX. UbiX catalyzes the sequential formation of two bonds, the first\nbetween N5 of the flavin and C1 of DMAP, and the second between C6\nof the flavin and C3 of DMAP. We have examined the reaction of UbiX\nwith both FMN and riboflavin. Although UbiX converts FMN to prFMN,\nwe show that significant amounts of the N5-dimethylallyl-FMN intermediate\nare released from the enzyme during catalysis. With riboflavin as\nthe substrate, UbiX catalyzes only a partial reaction, resulting in\nonly N5-dimethylallyl-riboflavin being formed. Purification of the\nN5-dimethylallyl-FMN adduct allowed its structure to be verified by <sup>1</sup>H NMR spectroscopy and its reactivity to be investigated.\nSurprisingly, whereas reduced prFMN oxidizes in seconds to form the\nstable prFMN semiquinone radical when exposed to air, N5-dimethylallyl-FMN\noxidizes much more slowly over several hours; in this case, oxidation\nis accompanied by spontaneous hydrolysis to regenerate FMN. These\nstudies highlight the important contribution that cyclization of the\nprenyl-derived ring of prFMN makes to the cofactor’s biological\nactivity.