CAD: A Multifunctional Protein Leading De Novo Pyrimidine Biosynthesis

Abstract

Pyrimidines are essential precursors for DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) synthesis, protein glycosylation and lipid synthesis. In resting cells, pyrimidines are largely obtained through salvage pathways, but in proliferating cells, particularly in tumours, the synthesis of pyrimidines de novo is indispensable to fuel the high demand of nucleic acids and other cellular components. In animals, the de novo pathway is initiated and controlled by CAD, a ∼240‐kDa multifunctional protein with four different enzymatic domains: glutaminase (GLN), carbamoyl phosphate synthetase (CPS), dihydroorotase (DHO) and aspartate transcarbamoylase (ATC). In contrast, in bacteria, archaeans and plants, GLN, CPS, DHO and ATC are distinct monofunctional proteins. The structures of a number of these enzymes from bacteria and archaea are known, but until recently, there was no structural information about CAD other than that it self‐assembles into ∼1.5‐megaDa hexamers.

Key Concepts

  • De novo synthesis of pyrimidine nucleotides is essential for cell growth and proliferation.
  • In animals, the multifunctional protein CAD catalyses the first three reactions of de novo pyrimidine synthesis.
  • CAD is a 243‐kDa polypeptide with four enzymatic domains [glutaminase (GLN), carbamoyl phosphate synthetase (CPS), dihydroorotase (DHO) and aspartate transcarbamoylase (ATC)] that oligomerises into 1.5‐megaDa hexamers.
  • In bacteria, GLN, CPS, DHO and ATC are individual proteins for which structural information is available.
  • The crystal structures of the DHO and ATC domains of human CAD were recently reported.
  • The GLN and CPS domains of CAD are expected to be similar to the Escherichia coli CPS and human mitochondrial CPS1 crystal structures.
  • A model of CAD is proposed that sets the DHO and ATC domains as the central framework of the hexameric particles.

Keywords: nucleotide metabolism; multifunctional protein; glutaminase; carbamoyl phosphate synthetase; dihydroorotase; aspartate transcarbamoylase; URA2; protein structure; X‐ray crystallography

Figure 1. De novo synthesis of pyrimidines and organisation of the enzymes initiating the pathway. (a) Overview of the de novo pathway for the biosynthesis of pyrimidines. The enzymes catalysing each step in the pathway are as follows: (1) glutaminase‐dependent carbamoyl phosphate synthetase (GLN‐CPS); (2) aspartate transcarbamoylase (ATC); (3) dihydroorotase (DHO); (4) dihydroorotate dehydrogenase; (5) orotate phosphoribosyltransferase; (6) OMP descarboxylase; (7) UMP kinase and (8) nucleotide diphosphate kinase. (b) In animals, a single polypeptide named CAD contains the GLN (brown), CPS (orange), DHO (green) and ATC (purple) activities fused as distinct functional domains. A different CPS (CPS1) present in the mitochondria and fused to an inactive GLN domain (light brown) makes CP for the synthesis of arginine. Fungi have a CAD‐like protein with an inactive DHO‐like domain (light green), whereas in bacteria, archaeans and plants, the GLN, CPS, DHO and ATC activities are encoded as distinct monofunctional proteins. The allosteric activators and inhibitors for the different proteins are shown in blue and red, respectively. The phosphorylation sites in CAD are also indicated.
Figure 2. CP is made in a multistep reaction catalysed by GLN and CPS. (a) CP synthesis from glutamine, bicarbonate and two ATP molecules. (b) Cartoon representation of the GLN‐CPS dimer, exemplified by human CPS1 (PDB code: 5DOU). GLN is shown in brown, and CPS is shown with the N‐ and C‐halves coloured in yellow and orange, respectively. The regulatory region of CPS is shown in green with a molecule of NAG represented in ball and stick. The tunnel running through the interior of the protein is indicated with a blue dashed line. (c) Cartoon representation of different protein regions.
Figure 3. The DHO domain of CAD forms dimers. (a) Cartoon representation of the DHO domain of human CAD in two perpendicular views (PDB code: 4C6L). Each subunit has a molecule of the inhibitor FOA (fluoroorotic acid) and three Zn2+ ions bound at the active site. (b) Scheme of the reversible reaction catalysed by DHO. The Zn2+ ions are represented as spheres.
Figure 4. The ATC domain of CAD forms a catalytic trimer. (a) Cartoon representation of the human ATC trimer bound to PALA (PDB code: 5G1N). Each subunit is depicted in a different colour, and PALA is shown in ball and stick. (b) Ribbon representation of the human ATC subunit with the N‐ and C‐domains coloured in blue and green, respectively. A molecule of PALA, shown in yellow ball and stick, binds at a cleft between both domains. The structure of the subunit in the absence of PALA (coloured grey) is superimposed to show the conformational changes. (c) View of the active site. The interactions of the inhibitor with the active site residues are indicated with dashed lines.
Figure 5. A possible model for the organisation of CAD and comparison with bacterial ATC and DHO complexes. (a) Proposed model for the architecture of CAD hexamers. Two ATC trimers (purple) and three DHO dimers (green) form a central hexamer, which is surrounded by three GLN‐CPS dimers (in brown and wheat colours). The three‐ and twofold symmetry axes are indicated. The linkers connecting the different domains in one of the CAD subunits are represented by dashed lines. The red stars indicate the binding sites for the allosteric effectors. (b) Representation of the complex between DHO and ATC in the bacteria Aquifex aeolicus. (c) Representation of the E. coli ATC holoenzyme in the T and R states. The regulatory dimers are shown in grey with red arrows indicating the binding site for the allosteric effectors.
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Moreno‐Morcillo, María, and Ramón‐Maiques, Santiago(Jul 2017) CAD: A Multifunctional Protein Leading De Novo Pyrimidine Biosynthesis. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0027193]