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

Acta Crystallogr D Biol Crystallogr. 2004 Jun;60(Pt 6):1137-8. doi: 10.1107/S0907444904007905. Epub 2004 May 21.

Abstract

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 P3(2)21, with unit-cell parameters a = b = 103.9, c = 123.1 angstroms. The crystals diffract to 2.5 angstroms resolution. A complete data set has been collected and structure solution and refinement is under way.

MeSH terms

  • 3-Oxoacyl-(Acyl-Carrier-Protein) Reductase
  • Alcohol Oxidoreductases / chemistry*
  • Alcohol Oxidoreductases / genetics
  • Alcohol Oxidoreductases / metabolism
  • Cloning, Molecular
  • Electrophoresis, Polyacrylamide Gel
  • Histidine / chemistry
  • Hydrogen-Ion Concentration
  • Open Reading Frames
  • Polyketide Synthases / chemistry*
  • Protein Conformation
  • Streptomyces coelicolor / genetics
  • Streptomyces coelicolor / metabolism
  • X-Ray Diffraction / methods*

Substances

  • Histidine
  • Polyketide Synthases
  • Alcohol Oxidoreductases
  • 3-Oxoacyl-(Acyl-Carrier-Protein) Reductase