title: Evidence for catalytic intermediates involved in generating the chromopyrrolic acid scaffold of rebeccamycin by RebO and RebD.
abstract: We provide the first experimental evidence for intermediates being involved in catalysis by RebD in generating the chromopyrrolic acid (CPA) scaffold of rebeccamycin. In the presence of its substrates (indole pyruvate imine - IPAI - and H2O2 both produced by the flavoprotein oxidase RebO that oxidizes tryptophan), RebD reacts as a peroxidase forming two IPAI radicals that recombine as a C-C bond in the CPA. When catalase is included to remove H2O2, CPA can still be formed because the IPAI rapidly reduces RebD, which reacts with O2, utilizing oxidase-peroxidase chemistry to produce CPA. Reduced RebD can also react with H2O2 forming Cpd II directly, which can oxidize IPAI. Stopped-flow spectrophotometric studies demonstrated that during the reaction of RebO and RebD with Trp and oxygen, a species with a red-shifted Soret band at 424.5 nm appeared. This species can react with either guaiacol or ABTS to form ferric RebD, suggesting that it is Cpd II of RebD involved in the formation of CPA. In summary, the studies reveal new and unusual aspects peroxidase and peroxygenase chemistry used by RebD in catalyzing carbon-carbon oxidative coupling reactions that are involved in biosynthesis of indolocarbazoles.
authors.affiliation: Department of Biological Chemistry, Medical School, University of Michigan, Ann Arbor, MI 48109-0600, USA.
authors.forename: Tatyana
authors.initials: T
authors.lastname: Spolitak
authors.affiliation: Department of Biological Chemistry, Medical School, University of Michigan, Ann Arbor, MI 48109-0600, USA. Electronic address: dballou@umich.edu.
authors.forename: David P
authors.initials: DP
authors.lastname: Ballou
mesh_terms: D000594:Amino Acid Oxidoreductases / Q000737:chemistry*; D002227:Carbazoles / Q000737:chemistry*; D002384:Catalysis; D029968:Escherichia coli Proteins / Q000737:chemistry*; D006861:Hydrogen Peroxide / Q000737:chemistry; D007211:Indoles / Q000737:chemistry; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry; D010544:Peroxidases / Q000737:chemistry*; D011758:Pyrroles / Q000138:chemical synthesis / Q000737:chemistry*; D014364:Tryptophan / Q000737:chemistry
chemical_list: D002227:Carbazoles; D029968:Escherichia coli Proteins; D007211:Indoles; D011758:Pyrroles; C541708:chromopyrrolic acid; C000598991:indole-3-pyruvic acid imine; D014364:Tryptophan; C053339:rebeccamycin; D006861:Hydrogen Peroxide; D010544:Peroxidases; D000594:Amino Acid Oxidoreductases; C507411:RebO protein, E coli; D010100:Oxygen
keywords: Chromopyrrolic acid; Cpd II; Indolocarbazoles; Peroxidase; RebD; Rebeccamycin