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Purification, crystallization and preliminary X‐ray diffraction analysis of 3‐ketoacyl‐CoA thiolase A1887 from Ralstonia eutropha H16
Author(s) -
Kim Jieun,
Kim KyungJin
Publication year - 2015
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/s2053230x15007888
Subject(s) - thiolase , ralstonia , enzyme , crystallization , chemistry , enzyme kinetics , size exclusion chromatography , stereochemistry , biochemistry , active site , organic chemistry , dehydrogenase
The gene product of A1887 from Ralstonia eutropha ( Re H16_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. Re H16_A1887 was overexpressed and purified to homogeneity by affinity and size‐exclusion chromatography. The degradative thiolase activity of the purified Re H16_A1887 was measured and enzyme‐kinetic parameters for the protein were obtained, with K m , V max and k cat values of 158 µ M , 32 m M min −1 and 5 × 10 6 s −1 , respectively. The Re H16_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 P 4 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%.