Abstract
The activity of the reversible decarboxylase enzyme Fdc1 is dependent on prenylated FMN (prFMN), a recently discovered cofactor. The oxidized prFMN supports a 1,3-dipolar cycloaddition mechanism that underpins reversible decarboxylation. Fdc1 is a distinct member of the UbiD family of enzymes, with the canonical UbiD catalyzing the (de)carboxylation of para-hydroxybenzoic acid-type substrates. Here we show that the Escherichia coli UbiD enzyme, which is implicated in ubiquinone biosynthesis, cannot be isolated in an active holoenzyme form despite the fact active holoFdc1 is readily obtained. Formation of holoUbiD requires reconstitution in vitro of the apoUbiD with reduced prFMN. Furthermore, although the Fdc1 apoenzyme can be readily reconstituted and activated, in vitro oxidation to the mature prFMN cofactor stalls at formation of a radical prFMN species in holoUbiD. Further oxidative maturation in vitro occurs only at alkaline pH, suggesting a proton-coupled electron transfer precedes formation of the fully oxidized prFMN. Crystal structures of holoUbiD reveal a relatively open active site potentially occluded from solvent through domain motion. The presence of a prFMN sulfite-adduct in one of the UbiD crystal structures confirms oxidative maturation does occur at ambient pH on a slow time scale. Activity could not be detected for a range of putative para-hydroxybenzoic acid substrates tested. However, the lack of an obvious hydrophobic binding pocket for the octaprenyl tail of the proposed ubiquinone precursor substrate does suggest UbiD might act on a non-prenylated precursor. Our data reveals an unexpected variation occurs in domain mobility, prFMN binding, and maturation by the UbiD enzyme family.
Highlights
Research Council (BBSRC) Grant BB/K017802/1 and a BBSRC iCASE studentship to S
The widespread UbiD family of enzymes catalyzes the reversible decarboxylation of ␣,-unsaturated acids, a chemical feat that is accomplished by the use of the recently discovered cofactor prenylated FMN4 [6]
In the case of the fungal enzyme Fdc1, the N5-alkyl linkage from the UbiX prenylated FMN (prFMN) product is oxidized to the corresponding N5AC iminium species, generating an azomethine ylide species required for activity (Fig. 1A)
Summary
The activity of the reversible decarboxylase enzyme Fdc is dependent on prenylated FMN (prFMN), a recently discovered cofactor. The widespread UbiD family of enzymes catalyzes the reversible decarboxylation of ␣,-unsaturated acids, a chemical feat that is accomplished by the use of the recently discovered cofactor prenylated FMN (prFMN)4 [6] The latter is synthesized from FMNH2 and dimethylallyl monophosphate (DMAP) by the non-canonical terpene cyclase UbiX, producing a highly modified FMN with linkages via the N5 and C6 positions to the DMAP dimethylallyl moiety [7]. It is proposed that Fdc and by extension other UbiD enzymes make use of a 1,3 dipolar cycloaddition reaction between prFMN and the ␣, -unsaturated acid substrate to generate a transient pyrrolidine substrate cofactor adduct (Fig. 1B, species 6) [6] The latter can readily undergo decarboxylation concomitant with pyrrolidine ring opening. Fdc belongs to a distinct branch of the UbiD family of enzymes
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call