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

Anna Jansson,Hanna Koskiniemi,Pekka Mäntsälä,Jarmo Niemi,Gunter Schneider

doi:10.1074/jbc.m407081200

Anna Jansson, Hanna Koskiniemi + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m407081200

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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-O-methyltransferase DnrK. The crystal structure of the ternary complex of this enzyme with the bound products S-adenosyl-L-homocysteine and 4-methoxy-epsilon-rhodomycin T has been determined to a 2.35-angstroms 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 SN2 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.

Highlights

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]
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
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

Summary

Introduction

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]. 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 further modified through a series of steps, i.e. hydroxylation, glycosylation, methylester hydrolysis, decarboxylation, methylation, and, in the case of doxorubicin, oxidation by the action of tailoring enzymes (4 –9). Replacement of a putative catalytic base by site-directed mutagenesis results in little change in catalytic activity This observation suggests that DnrK most likely acts as an entropic enzyme in that rate enhancement is mainly due to orientational and proximity effects. This is different from the mechanism of other related O-methyltransferases where acid/ base catalysis has been demonstrated to be an essential catalytic step [15]

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