Cloning, expression, purification, crystallization and X-ray crystallographic analysis of PhaA from Ralstonia eutropha.
Polyhydroxybutyrate (PHB) is a biopolymer that is in the spotlight because of its broad applications in bioplastics, fine chemicals, implant biomaterials and biofuels. PhaA from Ralstonia eutropha (RePhaA) is the first enzyme in the PHB biosynthetic pathway and catalyzes the condensation reaction of two acetyl-CoA molecules to give acetoacetyl-CoA. RePhaA was crystallized using the hanging-drop vapour-diffusion method in the presence of 20% polyethylene glycol monomethyl ether 2K, 0.1 M Tris-HCl pH 8.5 and 0.2 M trimethylamine N-oxide dihydrate at 295 K. X-ray diffraction data were collected to a maximum resolution of 1.96 Å on a synchrotron beamline. The crystal belonged to space group P2₁, with unit-cell parameters a=68.38, b=105.47, c=106.91 Å, α=γ=90, β=106.18°. With four subunits per asymmetric unit, the crystal volume per unit protein weight (VM) is 2.3 Å3 Da(-1), which corresponds to a solvent content of approximately 46.2%. The structure was solved by the molecular-replacement method and refinement of the structure is in progress.
School of Life Sciences, KNU Creative BioResearch Group, Kyungpook National University, Daehak-ro 80, Buk-ku, Daegu 702-701, Republic of Korea.
Eun-Jung
EJ
Kim
School of Life Sciences, KNU Creative BioResearch Group, Kyungpook National University, Daehak-ro 80, Buk-ku, Daegu 702-701, Republic of Korea.
Kyung-Jin
KJ
Kim
D000217:Acyltransferases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D003460:Crystallization; D018360:Crystallography, X-Ray; D020118:Cupriavidus necator / Q000201:enzymology* / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data
D001426:Bacterial Proteins; D000217:Acyltransferases; C077225:poly(3-hydroxyalkanoic acid) synthase
PhaA; Ralstonia eutropha; polyhydroxyalkanoate; thiolase