title: 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 ofpara-hydroxybenzoic acid-type substrates. Here we show that theEscherichia coliUbiD 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 reconstitutionin vitroof the apoUbiD with reduced prFMN. Furthermore, although the Fdc1 apoenzyme can be readily reconstituted and activated,in vitrooxidation to the mature prFMN cofactor stalls at formation of a radical prFMN species in holoUbiD. Further oxidative maturationin vitrooccurs 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 putativepara-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.
authors.affiliation: From the Manchester Institute of Biotechnology, University of Manchester, 131 Princess Street Manchester, M1 7DN, United Kingdom and.
authors.forename: Stephen A
authors.initials: SA
authors.lastname: Marshall
authors.affiliation: From the Manchester Institute of Biotechnology, University of Manchester, 131 Princess Street Manchester, M1 7DN, United Kingdom and.
authors.forename: Karl
authors.initials: K
authors.lastname: Fisher
authors.affiliation: From the Manchester Institute of Biotechnology, University of Manchester, 131 Princess Street Manchester, M1 7DN, United Kingdom and.
authors.forename: Aisling
authors.initials: A
authors.lastname: Ní Cheallaigh
authors.affiliation: From the Manchester Institute of Biotechnology, University of Manchester, 131 Princess Street Manchester, M1 7DN, United Kingdom and.
authors.forename: Mark D
authors.initials: MD
authors.lastname: White
authors.affiliation: From the Manchester Institute of Biotechnology, University of Manchester, 131 Princess Street Manchester, M1 7DN, United Kingdom and.
authors.forename: Karl A P
authors.initials: KA
authors.lastname: Payne
authors.affiliation: Innovation/Biodomain, Shell International Exploration and Production, Westhollow Technology Center, Houston, Texas 77082-3101.
authors.forename: D A
authors.initials: DA
authors.lastname: Parker
authors.affiliation: From the Manchester Institute of Biotechnology, University of Manchester, 131 Princess Street Manchester, M1 7DN, United Kingdom and.
authors.forename: Stephen E J
authors.initials: SE
authors.lastname: Rigby
authors.affiliation: From the Manchester Institute of Biotechnology, University of Manchester, 131 Princess Street Manchester, M1 7DN, United Kingdom and david.leys@manchester.ac.uk.
authors.forename: David
authors.initials: D
authors.lastname: Leys
mesh_terms: D001665:Binding Sites; D002262:Carboxy-Lyases / Q000737:chemistry / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism*; D005486:Flavin Mononucleotide / Q000378:metabolism*; D008958:Models, Molecular; D010084:Oxidation-Reduction; D054876:Prenylation; D000072417:Protein Domains; D014451:Ubiquinone / Q000737:chemistry / Q000378:metabolism*
chemical_list: D014451:Ubiquinone; D005486:Flavin Mononucleotide; C415986:3-octaprenyl-4-hydroxybenzoate carboxy-lyase; D002262:Carboxy-Lyases
keywords: crystal structure; decarboxylase; electron paramagnetic resonance (EPR); enzyme catalysis; flavin; ubiquinone