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 K m, Vmax and kcat values of 158 μM, 32 mM 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%.