Open AccessExpression, purification, crystallization and preliminary X‐ray diffraction analysis of the dihydroorotase domain of human CAD
Author(s) -
Lallous Nada,
GrandeGarcía Araceli,
Molina Rafael,
RamónMaiques Santiago
Publication year - 2012
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s1744309112038857
Subject(s) - crystallization , orthorhombic crystal system , crystallography , aspartate carbamoyltransferase , resolution (logic) , pyrimidine metabolism , glutamine , chemistry , crystal structure , biology , biochemistry , amino acid , enzyme , gene , escherichia coli , organic chemistry , artificial intelligence , purine , computer science
CAD is a 243 kDa eukaryotic multifunctional polypeptide that catalyzes the first three reactions of de novo pyrimidine biosynthesis: glutamine‐dependent c arbamyl phosphate synthetase, a spartate transcarbamylase and d ihydroorotase (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 C 222 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%.