title: Expression, purification, crystallization and preliminary X-ray diffraction analysis of the dihydroorotase domain of human CAD.
abstract: CAD is a 243 kDa eukaryotic multifunctional polypeptide that catalyzes the first three reactions of de novo pyrimidine biosynthesis: glutamine-dependent carbamyl phosphate synthetase, aspartate transcarbamylase and dihydroorotase (DHO). In prokaryotes, these activities are associated with monofunctional proteins, for which crystal structures are available. However, there is no detailed structural information on the full-length CAD protein or any of its functional domains apart from that it associates to form a homohexamer of ∼1.5 MDa. Here, the expression, purification and crystallization of the DHO domain of human CAD are reported. The DHO domain forms homodimers in solution. Crystallization experiments yielded small crystals that were suitable for X-ray diffraction studies. A diffraction data set was collected to 1.75 Å resolution using synchrotron radiation at the SLS, Villigen, Switzerland. The crystals belonged to the orthorhombic space group C222(1), with unit-cell parameters a=82.1, b=159.3, c=61.5 Å. The Matthews coefficient calculation suggested the presence of one protein molecule per asymmetric unit, with a solvent content of 48%.
authors.affiliation: Structural Bases of Genome Integrity Group, Structural Biology and Biocomputing Programme, Spanish National Cancer Research Centre (CNIO), Calle de Melchor Fernández Almagro 3, 28029 Madrid, Spain.
authors.forename: Nada
authors.initials: N
authors.lastname: Lallous
authors.forename: Araceli
authors.initials: A
authors.lastname: Grande-García
authors.forename: Rafael
authors.initials: R
authors.lastname: Molina
authors.forename: Santiago
authors.initials: S
authors.lastname: Ramón-Maiques
mesh_terms: D001221:Aspartate Carbamoyltransferase / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D020134:Catalytic Domain; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003460:Crystallization; D018360:Crystallography, X-Ray; D004080:Dihydroorotase / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D004926:Escherichia coli; D006801:Humans; D008027:Light; D020836:Protein Structure, Quaternary; D012542:Scattering, Radiation
chemical_list: C033626:CAD trifunctional enzyme; D001221:Aspartate Carbamoyltransferase; D004080:Dihydroorotase; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)