Cloning, expression, purification, crystallization and X-ray crystallographic analysis of the (S)-3-hydroxybutyryl-CoA dehydrogenase PaaH1 from Ralstonia eutropha H16.

The (S)-3-hydroxybutyryl-CoA dehydrogenase PaaH1 from Ralstonia eutropha (RePaaH1) is an enzyme used in the biosynthesis of n-butanol from acetyl-CoA by the reduction of acetoacetyl-CoA to (S)-3-hydroxybutyryl-CoA. The RePaaH1 protein was crystallized using the hanging-drop vapour-diffusion method in the presence of 1.4 M ammonium sulfate, 0.1 M sodium cacodylate pH 6.0, 0.2 M sodium chloride at 295 K. X-ray diffraction data were collected to a maximum resolution of 2.6 Å on a synchrotron beamline. The crystal belonged to space group P3221, with unit-cell parameters a=b=135.4, c=97.2 Å. With three molecules per asymmetric unit, the crystal volume per unit protein weight (VM) is 2.68 Å3 Da(-1), which corresponds to a solvent content of approximately 54.1%. The structure was solved by the single-wavelength anomalous dispersion method and refinement of the structure is in progress.

Citation

Jieun Kim, Kyung-Jin Kim. Cloning, expression, purification, crystallization and X-ray crystallographic analysis of the (S)-3-hydroxybutyryl-CoA dehydrogenase PaaH1 from Ralstonia eutropha H16. Acta crystallographica. Section F, Structural biology communications. 2014 Jul;70(Pt 7):955-8

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PMID: 25005097

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