Dihydroorotase from the Hyperthermophile Aquifiex aeolicus Is Activated by Stoichiometric Association with Aspartate Transcarbamoylase and Forms a One-Pot Reactor for Pyrimidine Biosynthesis

Abstract

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 Å 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 Å 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.

Bioenergetics-based modeling of Plasmodium falciparum metabolism reveals its essential genes, nutritional requirements, and thermodynamic bottlenecks

“…The presence of substrate channeling between ethanolamine phosphate and PE in the Kennedy pathway and between carbamoyl phosphate and (S)-dihydroorotate in the pyrimidine biosynthetic pathway turns them thermodynamically feasible. Hypotheses on substrate channeling through these pathways have been suggested in other cell types based on the metabolite concentration levels and thermodynamic studies [42, 54, 56]. We demonstrate that network thermodynamics can provide a framework for integrating and testing hypotheses on enzyme mechanisms and their function in metabolic networks [14, 16].…”

“…The malarial enzyme DHOase shares some characteristics with both types I (e.g., mammals) and II (e.g., Escherichia coli ) enzymes [53]. Previous experimental [54] and computational [55] studies in DHOases of type II have hypothesized that channeling exists from carbamoyl phosphate to (S)-dihydroorotate through the enzymes E.C. 2.1.3.2 and E.C.…”

Characterization of the Dihydroorotase from Methanococcus jannaschii

“…The latter form dimers and frequently associate with ATCase to form a stoichiometric 1:1 complex e.g. in Aa [24] and Ba [25]. It is assumed [3] that the stereochemistry of the Aa complex [24] is adopted by the other DHOases that associate with ATCase.…”

“…in Aa [24] and Ba [25]. It is assumed [3] that the stereochemistry of the Aa complex [24] is adopted by the other DHOases that associate with ATCase. In contrast, the Mj enzyme is a monomer in solution.…”

“…For instance, A. aeolicus DHOase has one Zn whether complexed (pdb id: 3d6n) [24] or uncomplexed (pdb id: 1xrf and 1xrt) [27] with ATCase. The S. aureus enzyme (pdb id: 3gri) also has one Zn center bridged to the conserved Asp via a water molecule.…”