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Cloning, expression, purification, crystallization and X‐ray crystallographic analysis of ( S )‐3‐hydroxybutyryl‐CoA dehydrogenase from Clostridium butyricum
Author(s) -
Kim EunJung,
Kim KyungJin
Publication year - 2014
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 37
ISSN - 2053-230X
DOI - 10.1107/s2053230x14004348
Subject(s) - ammonium sulfate , lithium sulfate , clostridium butyricum , crystallization , crystallography , chemistry , crystal (programming language) , crystal structure , protein crystallization , stereochemistry , biochemistry , chromatography , organic chemistry , fermentation , computer science , programming language , ion , ionic bonding
( S )‐3‐Hydroxybutyryl‐CoA dehydrogenase from Clostridium butyricum ( Cb HBD) 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 Cb HBD 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 R 3, 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 ( V M ) 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.