Abstract
Aclacinomycin methylesterase (RdmC) is one of the tailoring enzymes that modify the aklavinone skeleton in the biosynthesis of anthracyclines in Streptomyces species. The crystal structures of this enzyme from Streptomyces purpurascens in complex with the product analogues 10-decarboxymethylaclacinomycin T and 10-decarboxymethylaclacinomycin A were determined to nominal resolutions of 1.45 and 1.95 A, respectively. RdmC is built up of two domains. The larger alpha/beta domain shows the common alpha/beta hydrolase fold, whereas the smaller domain is alpha-helical. The active site and substrate binding pocket are located at the interface between the two domains. Decarboxymethylaclacinomycin T and decarboxymethylaclacinomycin A bind close to the catalytic triad (Ser102-His276-Asp248) in a hydrophobic pocket, with the sugar moieties located at the surface of the enzyme. The binding of the ligands is dominated by hydrophobic interactions, and specificity appears to be controlled mainly by the shape of the binding pocket rather than through specific hydrogen bonds. Mechanistic key features consistent with the structure of complexes of RdmC with product analogues are Ser102 acting as nucleophile and transition state stabilization by an oxyanion hole formed by the backbone amides of residues Gly32 and Met103.
Highlights
Aclacinomycin methylesterase (RdmC) is one of the tailoring enzymes that modify the aklavinone skeleton in the biosynthesis of anthracyclines in Streptomyces species
The crystal structures of this enzyme from Streptomyces purpurascens in complex with the product analogues 10-decarboxymethylaclacinomycin T and 10decarboxymethylaclacinomycin A were determined to nominal resolutions of 1.45 and 1.95 Å, respectively
Decarboxymethylaclacinomycin T and decarboxymethylaclacinomycin A bind close to the catalytic triad (Ser102-His276-Asp248) in a hydrophobic pocket, with the sugar moieties located at the surface of the enzyme
Summary
Aclacinomycin methylesterase (RdmC) is one of the tailoring enzymes that modify the aklavinone skeleton in the biosynthesis of anthracyclines in Streptomyces species. 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]. Modeling of the substrate complex based on these structures suggests that the catalytic serine residue is perfectly located to act as nucleophile during hydrolysis and that the negative charge developing in the transition state and the tetrahedral intermediate could be stabilized by an oxyanion hole consisting of the main chain nitrogens of residues Gly and Met103
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