Purification, properties, and characterization of recombinant Streptomycessp. strain C5 DoxA, a cytochrome P-450 catalyzing multiple steps in doxorubicin biosynthesis

DoxA is a cytochrome P-450 monooxygenase involved in the late stages of dau norubicin and doxorubicin biosynthesis that has a broad substrate specifici ty for anthracycline glycone substrates. Recombinant DoxA was purified to h omogeneity from Streptomyces lividans transformed with a plasmid containing the Streptomyces sp, strain C5 doxA gene under the control of the strong S npR-activated snpA promoter. The purified enzyme was a monomeric, soluble p rotein with an apparent M-r of 47,000. Purified DoxA catalyzed the 13-hydro xylation of 13-deoxydaunorubicin, the 13-oxidation of 13-dihydrocarminomyci n and 13-dihydrodaunorubicin, and the 14-hydroxylation of daunorubicin. The pH optimum for heme activation was pH 7.5, and the temperature optimum was 30 degrees C. The k(cat)/K-m values for the oxidation of anthracycline sub strates by purified DoxA, incubated with appropriate electron-donating comp onents, were as follows: for 13-deoxydaunorubicin, 22,000 M-1 s(-1); for 13 -dihydrodaunorubicin, 14,000 M-1 s(-1); for 13-dihydrocarminomycin, 280 M-1 s(-1); and for daunorubicin, 130 M-1 s(-1). Our results indicate that the conversion of daunorubicin to doxorubicin by this enzyme is not a favored r eaction and that the main anthracycline flux through the late seeps of the daunorubicin biosynthetic pathway catalyzed by DoxA is likely directed thro ugh the 4-O-methyl series of anthracyclines.