title: Identification of a monooxygenase from Streptomyces coelicolor A3(2) involved in biosynthesis of actinorhodin: purification and characterization of the recombinant enzyme.
abstract: The oxidation of phenols to quinones is an important reaction in the oxidative tailoring of many aromatic polyketides from bacterial and fungal systems. Sequence similarity between ActVA-Orf6 protein from the actinorhodin biosynthetic cluster and the previously characterized TcmH protein that is involved in tetracenomycin biosynthesis suggested that ActVA-Orf6 might catalyze this transformation as a step in actinorhodin biosynthesis. To investigate the role of ActVA-Orf6 in this oxidation, we have expressed the actVA-Orf6 gene in Escherichia coli and purified and characterized the recombinant protein. ActVA-Orf6 was shown to catalyze the monooxygenation of the tetracenomycin intermediate TcmF1 to TcmD3, strongly suggesting that it catalyzes oxidation of a similar intermediate in actinorhodin biosynthesis. The monooxygenase obeys simple reaction kinetics and has a Km of 4.8 +/- 0.9 microM, close to the figure reported for the homologous enzyme TcmH. The enzyme contains no prosthetic groups and requires only molecular oxygen to catalyze the oxidation. Site-directed mutagenesis was used to investigate the role of histidine residues thought to be important in the reaction; mutants lacking His-52 displayed much-reduced activity, consistent with the proposed mechanistic hypothesis that this histidine acts as a general base during catalysis.
authors.affiliation: Department of Biochemistry and Cambridge Centre for Molecular Recognition, University of Cambridge, United Kingdom.
authors.forename: S G
authors.initials: SG
authors.lastname: Kendrew
authors.forename: D A
authors.initials: DA
authors.lastname: Hopwood
authors.forename: E N
authors.initials: EN
authors.lastname: Marsh
mesh_terms: D000880:Anthraquinones / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000378:metabolism*; D002384:Catalysis; D004926:Escherichia coli; D015870:Gene Expression; D006639:Histidine; D007700:Kinetics; D005810:Multigene Family; D009154:Mutation; D009279:Naphthacenes / Q000378:metabolism; D016366:Open Reading Frames; D010105:Oxygenases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D000095622:Benzoisochromanequinones
chemical_list: D000880:Anthraquinones; D000900:Anti-Bacterial Agents; D006639:Histidine; D009279:Naphthacenes; D010105:Oxygenases; D011993:Recombinant Fusion Proteins; C083904:tetracenomycin F1; C013390:actinorhodin; D000095622:Benzoisochromanequinones