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Purification, Properties, and Characterization of Recombinant Streptomyces sp. Strain C5 DoxA, a Cytochrome P-450 Catalyzing Multiple Steps in Doxorubicin Biosynthesis

1999; American Society for Microbiology; Volume: 181; Issue: 1 Linguagem: Inglês

10.1128/jb.181.1.298-304.1999

1098-5530

Robbie J. Walczak, M L Dickens, Nigel D. Priestley, William R. Strohl,

Metal-Catalyzed Oxygenation Mechanisms

ABSTRACT DoxA is a cytochrome P-450 monooxygenase involved in the late stages of daunorubicin and doxorubicin biosynthesis that has a broad substrate specificity for anthracycline glycone substrates. Recombinant DoxA was purified to homogeneity from Streptomyces lividans transformed with a plasmid containing the Streptomyces sp. strain C5 doxA gene under the control of the strong SnpR-activated snpA promoter. The purified enzyme was a monomeric, soluble protein with an apparent M r of 47,000. Purified DoxA catalyzed the 13-hydroxylation of 13-deoxydaunorubicin, the 13-oxidation of 13-dihydrocarminomycin 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°C. The k cat / K m values for the oxidation of anthracycline substrates by purified DoxA, incubated with appropriate electron-donating components, 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 reaction and that the main anthracycline flux through the late steps of the daunorubicin biosynthetic pathway catalyzed by DoxA is likely directed through the 4- O -methyl series of anthracyclines.