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Expression, purification and preliminary X-ray diffraction analysis of a ketoreductase from a type II polyketide synthase

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Polyketide metabolites produced by bacteria and other organisms include antibiotics, anticancer and antifungal compounds. In type II polyketide synthesis, three enzymes are sufficient to form a polyketide product of the requisite chain length, although the fidelity of the first cyclization is variable. Addition of ketoreductase (KR) to this system results in the formation of a product with correct cyclization and reduction. This paper reports the cloning of the Streptomyces coelicolor actIII ORF5 gene that codes for the ketoreductase. The 261-amino-acid protein has been overexpressed with a 20-residue His tag, purified by affinity chromatography and crystallized in space group P3221, with unit-cell parameters a = b = 103.9, c = 123.1 Å. The crystals diffract to 2.5 Å resolution. A complete data set has been collected and structure solution and refinement is under way.

Keywords: ketoreductase; polyketide synthesis; short-chain dehydrogenase/reductase

Document Type: Research Article

DOI: http://dx.doi.org/10.1107/S0907444904007905

Publication date: June 1, 2004



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