title: Roles of Conserved Residues of the Glycine Oxidase GoxA in Controlling Activity, Cooperativity, Subunit Composition, and Cysteine Tryptophylquinone Biosynthesis.
abstract: GoxA is a glycine oxidase that possesses a cysteine tryptophylquinone (CTQ) cofactor that is formed by posttranslational modifications that are catalyzed by a modifying enzyme GoxB. It is the second known tryptophylquinone enzyme to function as an oxidase, the other being the lysine ϵ-oxidase, LodA. All other enzymes containing CTQ or tryptophan tryptophylquinone (TTQ) cofactors are dehydrogenases. Kinetic analysis of GoxA revealed allosteric cooperativity for its glycine substrate, but not O2This is the first CTQ- or TTQ-dependent enzyme to exhibit cooperativity. Here, we show that cooperativity and homodimer stabilization are strongly dependent on the presence of Phe-237. Conversion of this residue, which is a Tyr in LodA, to Tyr or Ala eliminates the cooperativity and destabilizes the dimer. These mutations also significantly affect the kcatand Kmvalues for the substrates. On the basis of structural and modeling studies, a mechanism by which Phe-237 exerts this influence is presented. Two active site residues, Asp-547 and His-466, were also examined and shown by site-directed mutagenesis to be critical for CTQ biogenesis. This result is compared with the results of similar studies of mutagenesis of structurally conserved residues of other tryptophylquinone enzymes. These results provide insight into the roles of specific active-site residues in catalysis and CTQ biogenesis, as well as describing an interesting mechanism by which a single residue can dictate whether or not an enzyme exhibits cooperative allosteric behavior toward a substrate.
authors.affiliation: From the Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, Florida 32827.
authors.forename: Esha
authors.initials: E
authors.lastname: Sehanobish
authors.affiliation: From the Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, Florida 32827.
authors.forename: Heather R
authors.initials: HR
authors.lastname: Williamson
authors.affiliation: From the Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, Florida 32827 victor.davidson@ucf.edu.
authors.forename: Victor L
authors.initials: VL
authors.lastname: Davidson
mesh_terms: D000594:Amino Acid Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D002384:Catalysis; D017124:Conserved Sequence; D019281:Dimerization; D004151:Dipeptides / Q000096:biosynthesis* / Q000737:chemistry; D020563:Gammaproteobacteria / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D045563:Indolequinones / Q000096:biosynthesis* / Q000737:chemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D016415:Sequence Alignment
chemical_list: D004151:Dipeptides; D045563:Indolequinones; C447619:cysteine tryptophylquinone; D000594:Amino Acid Oxidoreductases; C115925:glycine oxidase
keywords: cooperativity; enzyme kinetics; enzyme processing; oxidase; protein self-assembly; protein structure-function; quinoprotein