Expression, purification and preliminary X-ray diffraction analysis of a ketoreductase from a type II polyketide synthase.
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.
Teartasin
Watchrra
W
Molecular Recognition Centre, University of Bristol, School of Medical Sciences, University Walk, Bristol BS8 1TD, England.
Limpkin
Claire
C
Glod
Frank
F
Spencer
James
J
Cox
Russell J
RJ
Simpson
Thomas J
TJ
Crosby
John
J
Crump
Matthew P
MP
Hadfield
Andrea T
AT
D064431:3-Oxoacyl-(Acyl-Carrier-Protein) Reductase; D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D016366:Open Reading Frames; D048630:Polyketide Synthases / Q000737:chemistry*; D011487:Protein Conformation; D048372:Streptomyces coelicolor / Q000235:genetics / Q000378:metabolism; D014961:X-Ray Diffraction / Q000379:methods*
D006639:Histidine; D048630:Polyketide Synthases; D000429:Alcohol Oxidoreductases; D064431:3-Oxoacyl-(Acyl-Carrier-Protein) Reductase