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- Title
Purification, crystallization and preliminary X-ray diffraction analysis of 3-ketoacyl-CoA thiolase A1887 from Ralstonia eutropha H16.
- Authors
Kim, Jieun; Kim, Kyung-Jin
- 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 µ M, 32 m M min−1 and 5 × 106 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 P43212, 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%.
- Subjects
THIOLASES; RALSTONIA eutropha; CRYSTALLIZATION; X-ray diffraction; HOMOGENEITY; CHROMATOGRAPHIC analysis
- Publication
Acta Crystallographica: Section F, Structural Biology Communications, 2015, Vol 71, Issue 6, p758
- ISSN
2053-230X
- Publication type
Article
- DOI
10.1107/S2053230X15007888