title: Purification, crystallization and preliminary X-ray diffraction analysis of 3-ketoacyl-CoA thiolase A1887 from Ralstonia eutropha H16.
abstract: The gene product of A1887 from Ralstonia eutropha (ReH16_A1887) has been annotated as a 3-ketoacyl-CoA thiolase, an enzyme that catalyzes the fourth step of β-oxidation degradative pathways by converting 3-ketoacyl-CoA to acyl-CoA. ReH16_A1887 was overexpressed and purified to homogeneity by affinity and size-exclusion chromatography. The degradative thiolase activity of the purified ReH16_A1887 was measured and enzyme-kinetic parameters for the protein were obtained, with Km, Vmax and kcat values of 158 µM, 32 mM min(-1) and 5 × 10(6) s(-1), respectively. The ReH16_A1887 protein was crystallized in 17% PEG 8K, 0.1 M HEPES pH 7.0 at 293 K and a complete data set was collected to 1.4 Å resolution. The crystal belonged to space group P4(3)2(1)2, with unit-cell parameters a = b = 129.52, c = 114.13 Å, α = β = γ = 90°. The asymmetric unit contained two molecules, with a solvent content of 58.9%.
authors.affiliation: School of Life Sciences, KNU Creative BioResearch Group (BK21 Plus Program), Kyungpook National University, Daehak-ro 80, Buk-ku, Daegu 702-701, Republic of Korea.
authors.forename: Jieun
authors.initials: J
authors.lastname: Kim
authors.affiliation: School of Life Sciences, KNU Creative BioResearch Group (BK21 Plus Program), Kyungpook National University, Daehak-ro 80, Buk-ku, Daegu 702-701, Republic of Korea.
authors.forename: Kyung Jin
authors.initials: KJ
authors.lastname: Kim
mesh_terms: D000102:Acetyl-CoA C-Acyltransferase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D000214:Acyl Coenzyme A / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003460:Crystallization; D018360:Crystallography, X-Ray; D020118:Cupriavidus necator / Q000737:chemistry / Q000201:enzymology*; D057075:Enzyme Assays; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D007700:Kinetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D000214:Acyl Coenzyme A; D001426:Bacterial Proteins; D011994:Recombinant Proteins; D000102:Acetyl-CoA C-Acyltransferase
keywords: 3-ketoacyl-CoA thiolase; Ralstonia eutropha; β-oxidation degradative pathway