In vivo and in vitro bioconversion of epsilon-rhodomycinone glycoside to doxorubicin: functions of DauP, DauK, and DoxA

Abstract

We recently determined the function of the gene product of Streptomyces sp. strain C5 doxA, a cytochrome P-450-like protein, to be daunorubicin C-14 hydroxylase (M. L. Dickens and W. R. Strohl, J. Bacteriol. 178: [3389][3390][3391][3392][3393][3394][3395] 1996). In the present study, we show that DoxA also catalyzes the hydroxylation of 13-deoxycarminomycin and 13-deoxydaunorubicin to 13-dihydrocarminomycin and 13-dihydrodaunorubicin, respectively, as well as oxidizing the 13-dihydro-anthracyclines to their respective 13-keto forms. The Streptomyces sp. strain C5 dauP gene product also was shown unequivocally to remove the carbomethoxy group of the -rhodomycinone-glycoside (rhodomycin D) to form 10-carboxy-13-deoxycarminomycin. Additionally, Streptomyces sp. strain C5 DauK was found to methylate the anthracyclines rhodomycin D, 10-carboxy-13-deoxycarminomycin, and 13-deoxy-carminomycin, at the 4-hydroxyl position, indicating a broader substrate specificity than was previously known. The products of Streptomyces sp. strain C5 doxA, dauK, and dauP were sufficient and necessary to confer on Streptomyces lividans TK24 the ability to convert rhodomycin D, the first glycoside in daunorubicin and doxorubicin biosynthesis, to doxorubicin. Daunorubicin (daunomycin [11,17]) and doxorubicin (14-hydroxydaunorubicin; adriamycin [2]) ( Fig. 1) are clinically important anthracycline chemotherapeutic agents (2, 11, 17), which are synthesized by a type II polyketide synthase (23,(42)(43)(44). The aglycone is formed by condensing nine extender units derived from malonyl coenzyme A onto a propionyl moiety to make a C 21 polyketide intermediate (21,23,(42)(43)(44)(45)50), which is reduced at C-9 from the carboxy terminus (43, 44), cyclized, and aromatized to form aklanonic acid (18,19,23,40,(42)(43)(44). Aklanonic acid is converted in four steps to ε-rhodomycinone (3,7,15,16,30,40,42,44), an intermediate that is accumulated in large quantities by most daunorubicin-producing strains (27,42,44,46). It is postulated that ε-rhodomycinone is glycosylated by condensation with TDP-daunosamine (3,8,23,38,42,44) to form the first glycoside intermediate, here named rhodomycin D, which is then converted by a series of reactions to daunorubicin and doxorubicin. Previously, however, the order of the reactions and the enzymes catalyzing some of the steps in the conversion of rhodomycin D to doxorubicin were not known. We recently showed that the doxA gene of Streptomyces sp. strain C5 encoded a cytochrome P-450 enzyme that was capable of conferring on Streptomyces lividans TK24 the ability to convert daunorubicin to doxorubicin (14). In the present work, we describe the enzymatic activities from Streptomyces sp. strain C5 that are responsible for the conversion of rhodomycin D to doxorubicin both in vivo and in vitro (Fig. 1). MATERIALS AND METHODSBacterial strains, plasmids, media, and genetic manipulations. Streptomyces sp. strain C5, originally obtained from the Frederick Cancer Research Center (33) and previously described in detail (3), was...

Divergent evolution of an atypical S -adenosyl- l -methionine–dependent monooxygenase involved in anthracycline biosynthesis

“…1) (28). This is in contrast to the daunorubicin pathway, where the reaction order of the two enzymes DnrP and DnrK is more relaxed, as DnrK is able to methylate at least five biosynthetic intermediates (21).…”

“…1) have been described as unstable and undergo spontaneous 10-decarboxylation on the rhodomycin and daunorubicin pathways, respectively (21,29). However, indirect evidence has suggested that the decarboxylation might also be enzymatically catalyzed by DnrP (21).…”

“…1) in one of the final steps in the biosynthesis of the antitumor drug daunorubicin (2; Fig. 1) in Streptomyces peucetius (20,21). The enzyme possesses rather relaxed substrate specificity in regard to modifications in the polyaromatic anthracycline ring system, but it is quite specific with respect to the length of the carbohydrate chain at C-7, accepting only monoglycosides (22).…”

Crystal Structure of Aclacinomycin Methylesterase with Bound Product Analogues

“…RdmC shows about 50% sequence identity to related enzymes from other Streptomyces species, for instance DnrP from Streptomyces peucetius and DauP from Streptomyces sp strain C5 (17,18). These enzymes catalyze similar reactions, hydrolyzing 10-carbomethoxy-13-deoxycarminomycin to 10-carboxy-13-deoxycarminomycin (12,17). They all contain the same sequence motif G-X-S-X-G, typical for a serine hydrolase active site (18).…”

“…DnrP from Streptomyces peucetius and DauP from Streptomyces sp. C5 exhibit sequence identities to RdmC of 51 and 53%, respectively (12,17). DnrP and DauP have both been proposed to act as 10-carbomethoxy-13-deoxycarminomycin esterases on anthracycline substrates removing the carboxymethyl side chain at the C-10 position of, for example, the rhodomycin D aglycones (DauP) (18).…”