title: Structure, functional characterization, and evolution of the dihydroorotase domain of human CAD.
abstract: Upregulation of CAD, the multifunctional protein that initiates and controls the de novo biosynthesis of pyrimidines in animals, is essential for cell proliferation. Deciphering the architecture and functioning of CAD is of interest for its potential usage as an antitumoral target. However, there is no detailed structural information about CAD other than that it self-assembles into hexamers of ∼1.5 MDa. Here we report the crystal structure and functional characterization of the dihydroorotase domain of human CAD. Contradicting all assumptions, the structure reveals an active site enclosed by a flexible loop with two Zn²⁺ ions bridged by a carboxylated lysine and a third Zn coordinating a rare histidinate ion. Site-directed mutagenesis and functional assays prove the involvement of the Zn and flexible loop in catalysis. Comparison with homologous bacterial enzymes supports a reclassification of the DHOase family and provides strong evidence against current models of the architecture of CAD.
authors: Structural Bases of Genome Integrity Group, Structural Biology and Biocomputing Programme, Spanish National Cancer Research Centre (CNIO), 28029 Madrid, Spain.
authors: Araceli
authors: A
authors: Grande-García
authors: Structural Bases of Genome Integrity Group, Structural Biology and Biocomputing Programme, Spanish National Cancer Research Centre (CNIO), 28029 Madrid, Spain.
authors: Nada
authors: N
authors: Lallous
authors: Structural Bases of Genome Integrity Group, Structural Biology and Biocomputing Programme, Spanish National Cancer Research Centre (CNIO), 28029 Madrid, Spain.
authors: Celsa
authors: C
authors: Díaz-Tejada
authors: Structural Bases of Genome Integrity Group, Structural Biology and Biocomputing Programme, Spanish National Cancer Research Centre (CNIO), 28029 Madrid, Spain. Electronic address: sramon@cnio.es.
authors: Santiago
authors: S
authors: Ramón-Maiques
mesh_terms: D000595:Amino Acid Sequence; D001221:Aspartate Carbamoyltransferase / Q000737:chemistry*; D001426:Bacterial Proteins / Q000378:metabolism; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000737:chemistry*; D002384:Catalysis; D020134:Catalytic Domain; D002460:Cell Line; D004260:DNA Repair; D004080:Dihydroorotase / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology; D057809:HEK293 Cells; D006639:Histidine / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007477:Ions; D008239:Lysine / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009369:Neoplasms / Q000378:metabolism; D010766:Phosphorylation; D010802:Phylogeny; D011485:Protein Binding; D055503:Protein Multimerization; D017386:Sequence Homology, Amino Acid; D015032:Zinc / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; C033626:CAD trifunctional enzyme; D007477:Ions; D006639:Histidine; D001221:Aspartate Carbamoyltransferase; D004080:Dihydroorotase; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing); D015032:Zinc; D008239:Lysine