Identification of a monooxygenase from Streptomyces coelicolor A3(2) involved in biosynthesis of actinorhodin: purification and characterization of the recombinant enzyme.
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.
Kendrew
S G
SG
Department of Biochemistry and Cambridge Centre for Molecular Recognition, University of Cambridge, United Kingdom.
Hopwood
D A
DA
Marsh
E N
EN
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
D000880:Anthraquinones; D000900:Anti-Bacterial Agents; D006639:Histidine; D009279:Naphthacenes; D010105:Oxygenases; D011993:Recombinant Fusion Proteins; C083904:tetracenomycin F1; C013390:actinorhodin; D000095622:Benzoisochromanequinones