Expression, purification and crystallization of two peroxisomal acyl-CoA oxidases from Arabidopsis thaliana.
Two members of the acyl-CoA oxidase family from Arabidopsis thaliana have been cloned, overexpressed, purified and crystallized. Long-chain-specific acyl-CoA oxidase 1 crystals are characterized by a large variation in diffraction quality and non-isomorphous unit-cell parameters. The best crystals diffract to 2.0 angstrom using synchrotron radiation, have unit-cell parameters a = 85.2, b = 117.0, c = 131.0 angstrom, alpha = beta = gamma = 90 degrees and show P2(1)2(1)2(1) symmetry. There are two polypeptide chains in the asymmetric unit. Short-chain-specific acyl-CoA oxidase 4 crystals are trigonal, space group P3(1)21/P3(2)21, with unit-cell parameters a = b = 198.7, c = 149.6 angstroms. The crystals are most likely to contain six polypeptide chains in the asymmetric unit. Freshly prepared acyl-CoA oxidase 4 crystals diffract to 3.9 angstroms at cryogenic temperature at beamline I711, Max-Lab, but the diffraction quality degenerates after storage for only a few days in the crystallization drop. A selenomethionine-substituted form of the protein was produced and two-wavelength MAD data were collected at beamline BW7A, EMBL Outstation, Hamburg.
Pedersen
Lise
L
Carlsberg Laboratory, Department of Chemistry, Gamle Carlsberg Vej 10, DK-2500 Valby, Denmark.
Henriksen
Anette
A
D042962:Acyl-CoA Oxidase / Q000737:chemistry* / Q000235:genetics; D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray / Q000379:methods*; D017343:Genes, Plant; D008956:Models, Chemical; D010455:Peptides / Q000737:chemistry; D020675:Peroxisomes / Q000201:enzymology*; D010957:Plasmids / Q000378:metabolism; D017356:Synchrotrons; D013696:Temperature; D013997:Time Factors; D014961:X-Ray Diffraction
D029681:Arabidopsis Proteins; D010455:Peptides; C495584:ACX1 protein, Arabidopsis; C495585:ACX4 protein, Arabidopsis; D042962:Acyl-CoA Oxidase