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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

Aclacinomycin 10-hydroxylase is a methyltransferase homologue that catalyzes a S-adenosyl-L-methionine (AdoMet)-dependent hydroxylation of the C-10 carbon atom of 15-demethoxy-⑀-rhodomycin, a step in the biosynthesis of the polyketide antibiotic ␤-rhodomycin. SAdenosyl-L-homocysteine is an inhibitor of the enzyme, whereas the AdoMet analogue sinefungin can act as cofactor, indicating that a positive charge is required for catalysis.18 O 2 experiments show that the hydroxyl group is derived from molecular oxygen.

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SnoaB is a cofactor-independent monooxygenase that catalyzes the conversion of 12-deoxynogalonic acid to nogalonic acid in the biosynthesis of the aromatic polyketide nogalamycin in Streptomyces nogalater. In vitro (18)O(2) experiments establish that the oxygen atom incorporated into the substrate is derived from molecular oxygen. The crystal structure of the enzyme was determined in two different space groups to 1.7 and 1.9 A resolution, respectively. The enzyme displays the ferredoxin fold, with the characteristic beta-strand exchange at the dimer interface. The crystal structures reveal a putative catalytic triad involving two asparagine residues, Asn18 and Asn63, and a water molecule, which may play important roles in the enzymatic reaction. Site-directed mutagenesis experiments, replacing the two asparagines individually by alanine, led to a 100-fold drop in enzymatic activity. Replacement of an invariant tryptophan residue in the active site of the enzyme by phenylalanine also resulted in an enzyme variant with about 1% residual activity. Taken together, our findings are most consistent with a carbanion mechanism where the deprotonated substrate reacts with molecular oxygen via one electron transfer and formation of a caged radical.