Expression, purification, crystallization and preliminary X-ray diffraction analysis of the dihydroorotase domain of human CAD

TY - JOUR

T1 - Expression, purification, crystallization and preliminary X-ray diffraction analysis of the dihydroorotase domain of human CAD

AU - Lallous, Nada

AU - Grande-García, Araceli

AU - Molina, Rafael

AU - Ramón-Maiques, Santiago

PY - 2012/11/1

Y1 - 2012/11/1

N2 - 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%.

AB - 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%.

KW - Aspartate Carbamoyltransferase/biosynthesis

KW - Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/biosynthesis

KW - Catalytic Domain

KW - Chromatography, Affinity

KW - Chromatography, Gel

KW - Crystallization

KW - Crystallography, X-Ray

KW - Dihydroorotase/biosynthesis

KW - Escherichia coli

KW - Humans

KW - Light

KW - Protein Structure, Quaternary

KW - Scattering, Radiation

U2 - 10.1107/S1744309112038857

DO - 10.1107/S1744309112038857

M3 - Journal article

C2 - 23143245

SN - 2053-230X

VL - 68

SP - 1341

EP - 1345

JO - Acta Crystallographica Section F: Structural Biology Communications

JF - Acta Crystallographica Section F: Structural Biology Communications

IS - Pt 11

ER -