Structural Insight into the Core of CAD, the Multifunctional Protein Leading De Novo Pyrimidine Biosynthesis

• Published in: Structure (London) Vol. 25; no. 6; pp. 912 - 923.e5
• Main Authors: Moreno-Morcillo, María, Grande-García, Araceli, Ruiz-Ramos, Alba, del Caño-Ochoa, Francisco, Boskovic, Jasminka, Ramón-Maiques, Santiago
• Format: Journal Article
• Language: English
• Published: United States Elsevier Ltd 06-06-2017
• Subjects: Aspartate Carbamoyltransferase - chemistry; Aspartate Carbamoyltransferase - genetics; Aspartate Carbamoyltransferase - metabolism; aspartate transcarbamoylase; carbamoyl phosphate synthetase; Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) - chemistry; Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) - genetics; Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) - metabolism; Carbamyl Phosphate - chemistry; Carbamyl Phosphate - metabolism; Chaetomium - enzymology; Chaetomium thermophilum; Crystallography, X-Ray; dihydroorotase; Dihydroorotase - chemistry; Dihydroorotase - genetics; Dihydroorotase - metabolism; glutaminase; Humans; Microscopy, Electron; Models, Molecular; multi-angle light scattering; Mutagenesis, Site-Directed; nucleotide metabolism; Protein Domains; Protein Multimerization; Pyrimidines - biosynthesis; single-particle electron microscopy; URA2; X-ray crystallography; X-ray crystallography; carbamoyl phosphate synthetase; multi-angle light scattering; aspartate transcarbamoylase; Chaetomium thermophilum; glutaminase; URA2; nucleotide metabolism; single-particle electron microscopy; dihydroorotase
• ISSN: 0969-2126; 1878-4186
• DOI: 10.1016/j.str.2017.04.012

CAD, the multifunctional protein initiating and controlling de novo biosynthesis of pyrimidines in animals, self-assembles into ∼1.5 MDa hexamers. The structures of the dihydroorotase (DHO) and aspartate transcarbamoylase (ATC) domains of human CAD have been previously determined, but we lack information on how these domains associate and interact with the rest of CAD forming a multienzymatic unit. Here, we prove that a construct covering human DHO and ATC oligomerizes as a dimer of trimers and that this arrangement is conserved in CAD-like from fungi, which holds an inactive DHO-like domain. The crystal structures of the ATC trimer and DHO-like dimer from the fungus Chaetomium thermophilum confirm the similarity with the human CAD homologs. These results demonstrate that, despite being inactive, the fungal DHO-like domain has a conserved structural function. We propose a model that sets the DHO and ATC complex as the central element in the architecture of CAD.
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•The covalently linked DHO and ATC domains of CAD self-assemble as dimers of trimers•The crystal structures of the fungal DHO-like dimer and ATC trimer were determined•The inactive DHO-like domain has a conserved structural role in CAD-like from fungi•A DHO-ATC hexamer is proposed as the central structural element of CAD particles
Moreno-Morcillo et al. provide insights into human CAD, the multifunctional protein initiating and controlling de novo biosynthesis of pyrimidines, by characterizing a construct covering DHO and ATC domains. The authors show that this construct forms hexamers and that this assembly does not depend on whether DHO domain is active. They propose a model with the DHO and ATC domains as the central supporting framework of CAD particles.