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

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

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49 Scopus citations

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 arid 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.

Original languageEnglish
Pages (from-to)298-304
Number of pages7
JournalJournal of Bacteriology
Volume181
Issue number1
DOIs
StatePublished - Jan 1999

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