Oxidative maturation and Structural Characterization of Prenylated-FMN binding by UbiD, a Decarboxylase Involved in Bacterial Ubiquinone Biosynthesis [Protein Structure and Folding]

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 holo-enzyme form, despite the fact active holo-Fdc1 is readily obtained. Formation of holo-UbiD requires reconstitution in vitro of the apo-UbiD with reduced prFMN. Furthermore, while the Fdc1 apo-enzyme can be readily reconstituted and activated, in vitro oxidation to the mature prFMN cofactor stalls at formation of a radical prFMN species in holo-UbiD. 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 holo-UbiD 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 unexpected variation occurs in domain mobility, prFMN binding and maturation by the UbiD enzyme family.

from #AlexandrosSfakianakis via Alexandros G.Sfakianakis on Inoreader http://ift.tt/2iOxt1X
via IFTTT