Cloning, expression, purification, crystallization and X-ray crystallographic analysis of (S)-3-hydroxybutyryl-CoA...

(S)-3-Hydroxybutyryl-CoA dehydrogenase from Clostridium butyricum (CbHBD) is an enzyme that catalyzes the second step in the biosynthesis of n-butanol from acetyl-CoA by the reduction of acetoacetyl-CoA to 3-hydroxybutyryl-CoA. The CbHBD protein was crystallized using the hanging-drop vapour-diffusion method in the presence of 2 M ammonium sulfate, 0.1 M CAPS pH 10.5, 0.2 M lithium sulfate at 295 K. X-ray diffraction data were collected to a maximum resolution of 2.3 Å on a synchrotron beamline. The crystal belonged to space group R3, with unit-cell parameters a = b = 148.5, c = 201.6 Å. With four molecules per asymmetric unit, the crystal volume per unit protein weight (VM) is 3.52 Å(3) Da(-1), which corresponds to a solvent content of approximately 65.04%. The structure was solved by the molecular-replacement method and refinement of the structure is in progress.