Dihydroorotase from the hyperthermophile Aquifex aeolicus is activated by stoichiometric association with aspartate transcarbamoylase and forms a one-pot reactor for pyrimidine biosynthesis.

In prokaryotes, the first three enzymes in pyrimidine biosynthesis, carbamoyl phosphate synthetase (CPS), aspartate transcarbamoylase (ATC), and dihydroorotase (DHO), are commonly expressed separately and either function independently (Escherichia coli) or associate into multifunctional complexes (Aquifex aeolicus). In mammals the enzymes are expressed as a single polypeptide chain (CAD) in the order CPS-DHO-ATC and associate into a hexamer. This study presents the three-dimensional structure of the noncovalent hexamer of DHO and ATC from the hyperthermophile A. aeolicus at 2.3 A resolution. It is the first structure of any multienzyme complex in pyrimidine biosynthesis and is a possible model for the core of mammalian CAD. The structure has citrate, a near isosteric analogue of carbamoyl aspartate, bound to the active sites of both enzymes. Three active site loops that are intrinsically disordered in the free, inactive DHO are ordered in the complex. The reorganization also changes the peptide bond between Asp153, a ligand of the single zinc atom in DHO, and Gly154, to the rare cis conformation. In the crystal structure, six DHO and six ATC chains form a hollow dodecamer, in which the 12 active sites face an internal reaction chamber that is approximately 60 A in diameter and connected to the cytosol by narrow tunnels. The entrances and the interior of the chamber are both electropositive, which suggests that the architecture of this nanoreactor modifies the kinetics of the bisynthase, not only by steric channeling but also by preferential escape of the product, dihydroorotase, which is less negatively charged than its precursors, carbamoyl phosphate, aspartate, or carbamoyl aspartate.

Protein kinase A phosphorylation of the multifunctional protein CAD antagonizes activation by the MAP kinase cascade.

The flux through the de novo pyrimidine biosynthetic pathway is controlled by the multifunctional protein CAD, which catalyzes the first three steps. The cell cycle dependent regulation of pyrimidine biosynthesis is a consequence of sequential phosphorylation of CAD Thr456 and Ser1406 by the MAP kinase and PKA cascades, respectively. Coordinated regulation of the pathway requires precise timing of the two phosphorylation events. These studies show that phosphorylation of purified CAD by PKA antagonizes MAP kinase phosphorylation, and vice versa. Similar results were observed in vivo. Forskolin activation of PKA in BHK-21 cells resulted in a 8.5 fold increase in Ser1406 phosphorylation and severely curtailed the MAP kinase mediated phosphorylation of CAD Thr456. Moreover, the relative activity of MAP kinase and PKA was found to determine the extent of Thr456 phosphorylation. Transfectants expressing elevated levels of MAP kinase resulted in a 11-fold increase in Thr456 phosphorylation, whereas transfectants that overexpress PKA reduced Thr456 phosphorylation 5-fold. While phosphorylation of one site by one kinase may induce conformational changes that interfere with phosphorylation by the other, the observation that both MAP kinase and PKA form stable complexes with CAD suggest that the mutual antagonism is the result of steric interference by the bound kinases. The reciprocal antagonism of CAD phosphorylation by MAP kinase and PKA provides an elegant mechanism to coordinate the cell cycle-dependent regulation of pyrimidine biosynthesis ensuring that signals for up- and down-regulation of the pathway do not conflict.

The crystal structure of a novel, latent dihydroorotase from Aquifex aeolicus at 1.7A resolution.

Dihydroorotases (EC 3.5.2.3) catalyze the reversible cyclization of carbamoyl aspartate to form dihydroorotate in de novo pyrimidine biosynthesis. The X-ray structures of Aquifex aeolicus dihydroorotase in two space groups, C222(1) and C2, were determined at a resolution of 1.7A. These are the first structures of a type I dihydroorotase, a class of molecules that includes the dihydroorotase domain of mammalian CAD. The type I enzymes are more ancient and larger, at 45 kDa, than the type II enzymes exemplified by the 38 kDa Escherichia coli dihydroorotase. Both dihydroorotases are members of the metallo-dependent hydrolase superfamily, whose members have a distorted "TIM barrel" domain containing the active site. However, A.aeolicus dihydroorotase has a second, composite domain, which the E.coli enzyme lacks and has only one of the two zinc atoms present in the E.coli enzyme. A.aeolicus dihydroorotase is unique in exhibiting significant activity only when complexed with aspartate transcarbamoylase, whereas the E.coli dihydroorotase and the CAD dihydroorotase domain are active as free proteins. The latency of A.aeolicus dihydroorotase can be related to two differences between its structure and that of E.coli dihydroorotase: (1) the monoclinic structure has a novel cysteine ligand to the zinc that blocks the active site and possibly functions as a "cysteine switch"; and (2) active site residues that bind the substrate in E.coli dihydroorotase are located in disordered loops in both crystal structures of A.aeolicus dihydroorotase and may function as a disorder-to-order "entropy switch".