Oxidative Maturation and Structural Characterization of Prenylated FMN Binding by UbiD, a Decarboxylase Involved in Bacterial Ubiquinone Biosynthesis

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 -hydroxybenzoic acid-type substrates. Here we show that the 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 of the apoUbiD with reduced prFMN. Furthermore, although the Fdc1 apoenzyme can be readily reconstituted and activated, oxidation to the mature prFMN cofactor stalls at formation of a radical prFMN species in holoUbiD. Further oxidative maturation 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-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.

Phthaloyl‐coenzyme A decarboxylase from Thauera chlorobenzoica: the prenylated flavin‐, K+‐ and Fe2+‐dependent key enzyme of anaerobic phthalate degradation

“…B), and triplicate analyses gave a value of 359 ± 8 kDa (mean value ± standard deviation of triplicate analyses). This result suggests an α 6 ‐ composition and is in line with hexameric UbiD from E. coli (Marshall et al ., ) but differs from the previously reported dimeric compositions of UbiD‐like Fdc1 from yeast or Aspergillus niger (Bhuiya et al ., ; Payne et al ., ).…”

Requirement of a Functional Flavin Mononucleotide Prenyltransferase for the Activity of a Bacterial Decarboxylase in a Heterologous Muconic Acid Pathway in Saccharomyces cerevisiae

“…Sequence comparison of AroY-C iso to other enzymes of this family with an already described prFMN binding capability, Fdc1 from S. cerevisiae and Aspergillus niger and UbiD from E. coli (13,31) using Clustal Omega (18) reveals a relatively low sequence conservation between AroY-C iso and these enzymes (28% identity to E. coli UbiD, 25% identity to A. niger Fdc1, and 23% to S. cerevisiae Fdc1) (see Fig. S2 in the supplemental material).…”

Regioselective para‐Carboxylation of Catechols with a Prenylated Flavin Dependent Decarboxylase

“…However, upon purification of the decarboxylase from the E. coli host, only little enzyme activity could be detected for either of the two enzymes, despite additional co‐expression with the UbiD‐associated prenyltransferase UbiX to provide sufficient prFMN in vivo. Upon in vitro reconstitution with reduced prFMN (Figure 1 a),14a, 15 decarboxylation activity could be detected with 3,4‐dihydroxybenzoic acid (3,4‐DHBA, 1 ) following brief exposure to oxygen to generate the active prFMN iminium form (Figure 1 b). The lack of activity for anaerobically reconstituted protein clearly demonstrates the requirement for oxidative maturation of the prFMN cofactor.…”