Purification, properties, and characterization of recombinant Streptomycessp. strain C5 DoxA, a cytochrome P-450 catalyzing multiple steps in doxorubicin biosynthesis
Rj. Walczak et al., Purification, properties, and characterization of recombinant Streptomycessp. strain C5 DoxA, a cytochrome P-450 catalyzing multiple steps in doxorubicin biosynthesis, J BACT, 181(1), 1999, pp. 298-304
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.