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 o xidative tailoring of many aromatic polyketides from bacterial and fun gal systems. Sequence similarity between ActVA-Orf6 protein from the a ctino-rhodin biosynthetic cluster and the previously characterized Tcm H protein that is involved in tetracenomycin biosynthesis suggested th at ActVA-Orf6 might catalyze this transformation as a step in actinorh odin biosynthesis. To investigate the role of ActVA-Orf6 in this oxida tion, we have expressed the actVA-Orf6 gene in Escherichia coil and pu rified and characterized the recombinant protein. ActVA-Orf6 was shown to catalyze the monooxygenation of the tetracenomycin intermediate Tc mF1 to TcmD3, strongly suggesting that it catalyzes oxidation of a sim ilar intermediate in actinorhodin biosynthesis. The monooxygenase obey s simple reaction kinetics and has a K-m of 4.8 +/- 0.9 mu M, close to the figure reported for the homologous enzyme TcmH. The enzyme contai ns 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; mu tants lacking His-52 displayed much-reduced activity, consistent with the proposed mechanistic hypothesis that this histidine acts as a gene ral base during catalysis.