Crystal Structure of a Ternary Complex of DnrK, a Methyltransferase in Daunorubicin Biosynthesis, with Bound Products

Abstract

One of the final steps in the biosynthesis of the widely used anti-tumor drug daunorubicin in Streptomyces peucetius is the methylation of the 4-hydroxyl group of the tetracyclic ring system. This reaction is catalyzed by the S-adenosyl-L-methionine-dependent carminomycin 4-Omethyltransferase DnrK. The crystal structure of the ternary complex of this enzyme with the bound products S-adenosyl-L-homocysteine and 4-methoxy-⑀-rhodomycin T has been determined to a 2.35-Å resolution. DnrK is a homodimer, and the subunit displays the typical fold of small molecule O-methyltransferases. The structure provides insights into the recognition of the anthracycline substrate and also suggests conformational changes as part of the catalytic cycle of the enzyme. The position and orientation of the bound ligands are consistent with an S N 2 mechanism of methyl transfer. Mutagenesis experiments on a putative catalytic base confirm that DnrK most likely acts as an entropic enzyme in that rate enhancement is mainly due to orientational and proximity effects. This contrasts the mechanism of DnrK with that of other O-methyltransferases where acid/base catalysis has been demonstrated to be an essential contribution to rate enhancement.Daunorubicin and doxorubicin are aromatic polyketide antibiotics that exhibit high cytotoxicity and are widely applied in the chemotherapy of a variety of cancers (1, 2). These and related anthracyclines consist of a cyclic polyketide backbone, 7,8,9,10-tetrahydrotetracene-5,12-quinone, glycosylated at position C7 or C10 (Fig. 1). Diversity is generated by variations in the modification of the aglycone moiety and the composition of the attached carbohydrate. Biosynthesis of daunorubicin/doxorubicin starts with the formation of the polyketide backbone catalyzed by a class II polyketide synthase with subsequent cyclization of the polyketide chain (3). These steps lead to the formation of aklavinone, a common intermediate in the synthesis of most anthracyclines. This aglycone is then further modified through a series of steps, i.e. hydroxylation, glycosylation, methylester hydrolysis, decarboxylation, methylation, and, in

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

“…Structural studies of DnrK and RdmB (22,25) have confirmed that both enzymes consist of an N-terminal dimerization domain, an α-helical middle domain, and a C-terminal catalytic domain that binds the cofactor SAM (Fig. 1).…”

“…In addition, DnrK has even been shown to be able to methylate various flavonoids (23). The crystal structure of the ternary complex of DnrK revealed that the mechanism of methylation is consistent with canonical S N 2-type reactions of SAM-dependent methyltransferases, although proximity and orientation effects rather than acid/base catalysis appear to be more critical in the case of DnrK (22).…”

“…1) in the DnrK structure (Fig. 1), which has been noted as the likely explanation for why the enzyme only accepts monoglycosylated substrates (22). The corresponding region in RdmB is rather different and the carbohydrate chain extends from the binding pocket into the bulk solution in the RdmB ternary complex (26), which prompted us to select this region (R3; Fig.…”

“…2) as substrates. The reaction mixtures also included SAM, which is essential as a cosubstrate for DnrK (22) and as a cofactor for RdmB (26), and a reducing agent (DTT) that is required solely by RdmB (26). The reaction with native DnrK resulted in a single main product ( 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). In addition, DnrK has even been shown to be able to methylate various flavonoids (23).…”