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, 0.1M Tris-HCl pH 8.5 and 0.2M trimethylamine N-oxide dihydrate at 295K. X-ray diffraction data were collected to a maximum resolution of 1.96Å on a synchrotron beamline. The crystal belonged to space group P21, with unit-cell parameters a = 68.38, b = 105.47, c = 106.91Å, α = γ = 90, β = 106.18°. With four subunits per asymmetric unit, the crystal volume per unit protein weight (V M) is 2.3ųDa^-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.