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Journal of Bacteriology, January 1999, p. 298-304, Vol. 181, No. 1
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

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

Robbie J. Walczak,1 Michael L. Dickens,1,dagger Nigel D. Priestley,2 and William R. Strohl1,*

Department of Microbiology1 and College of Pharmacy,2 Ohio State University, Columbus, Ohio 43210

Received 27 July 1998/Accepted 16 October 1998

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 Mr 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 kcat/Km 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.

* Corresponding author. Present address: Merck Research Laboratories, P.O. Box 2000, R80Y-215, Rahway, NJ 07065. Phone: (732) 594-8433. Fax: (732) 594-1399. E-mail: william_strohl{at}merck.com.

dagger Present address: Merck Research Laboratories, P.O. Box 2000, RY810, Rahway, NJ 07065.

Journal of Bacteriology, January 1999, p. 298-304, Vol. 181, No. 1
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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