Purification, properties, and characterization of recombinant Streptomyces sp. 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 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 degreesC. 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 x s-1; for 13-dihydrodaunorubicin, 14,000 M-1 x s-1; for 13-dihydrocarminomycin, 280 M-1 x s-1; and for daunorubicin, 130 M-1 x 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.
Walczak
R J
RJ
Department of Microbiology, Ohio State University, Columbus, Ohio 43210, USA.
Dickens
M L
ML
Priestley
N D
ND
Strohl
W R
WR
D018943:Anthracyclines / Q000737:chemistry / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004317:Doxorubicin / Q000096:biosynthesis* / Q000737:chemistry / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D007700:Kinetics; D008970:Molecular Weight; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
D018943:Anthracyclines; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D004317:Doxorubicin; D003577:Cytochrome P-450 Enzyme System