Article info Vol. 6  No. 2   pp.  46 ~ 49
Title Purification, crystallization and X-ray crystallographic analysis of enoyl-CoA hydratase/isomerase-family protein from Cupriavidus necator H16
Authors Hogyun Seo and Kyung-Jin Kim*
Institutions School of Life Sciences, KNU Creative BioResearch Group, Kyungpook National University, Daehak-ro 80, Buk-ku, Daegu 41566, Korea *Correspondence: kkim@knu.ac.kr
Abstract H16_B0756 from Cupriavidus necator H16 is a putative enoyl-coenzyme A (CoA) hydratase/isomerase-family protein. Enoyl-CoA hydratase (ECH) in β-oxidation might provide hydroxyalkanoate monomers for PHA production. However, β-oxidation pathway and ECH family enzymes in C. necator have not been fully characterized. To identify the function of H16_B0756, the protein was overexpressed and purified to homogeneity by affinity and size-exclusion chromatography. The H16_B0756 protein was crystallized using hanging-drop vapor-diffusion method in the presence of 30% polyethylene glycol 550 monomethyl ether, 0.1 M sodium chloride and 0.1 M bicine, pH 9.0 at 295 K. X-ray diffraction data were collected to a maximum resolution of 2.0 Å. The crystal belonged to space group P3, with unit cell parameters a = b = 132.94 Å, c = 44.16, α = β = 90.0°, γ = 120.0°. With two molecules per asymmetric unit, the crystal volume per unit protein mass was 1.94 Å3 Da-1, which correspond to a solvent content of approximately 36.80%.