CAD is a multifunctional enzyme catalyzing the first three steps of de novo pyrimidine synthesis. The protein sequentially executes three enzymatic activities: carbamoyl-phosphate synthetase (CPSase), aspartate transcarbamylase (ATCase), and dihydroorotase (DHOase) 1. CPSase functions through a two-step mechanism involving glutamine-dependent amidotransferase activity that generates ammonia, followed by ammonium-dependent carbamoyl phosphate synthesis. The endogenously produced carbamoyl phosphate is channeled to ATCase, which catalyzes carbamoyl-L-aspartate formation from L-aspartate and carbamoyl phosphate. Finally, DHOase cyclizes carbamoyl aspartate to dihydroorotate 1. The dihydroorotase domain contains a structurally unusual active site with two zinc ions bridged by a carboxylated lysine and a third zinc coordinating a histidinate ion 1. CAD self-assembles into hexameric complexes of approximately 1.5 MDa. Upregulation of CAD is essential for cell proliferation, making it a potential antitumoral target 1. CAD mutations are associated with developmental and epileptic encephalopathy 50, indicating the clinical significance of proper pyrimidine metabolism for neurological function.