Organisation and sequence determination of glutamine-dependent carbamoyl phosphate synthetase II in Toxoplasma gondii

Carbamoyl phosphate synthetase II encodes the first enzymic step of de novo pyrimidine biosynthesis. Carbamoyl phosphate synthetase II is essential for Toxoplasma gondii replication and virulence. In this study, we characterised the primary structure of a 28kb gene encoding Toxoplasma gondii carbamoyl phosphate synthetase II. The carbamoyl phosphate synthetase II gene was interrupted by 36 introns. The predicted protein encoded by the 37 carbamoyl phosphate synthetase II exons was a 1,687 amino acid polypeptide with an N-terminal glutamine amidotransferase domain fused with C-terminal carbamoyl phosphate synthetase domains. This bifunctional organisation of carbamoyl phosphate synthetase II is unique, so far, to protozoan parasites from the phylum Apicomplexa (Plasmodium, Babesia, Toxoplasma) or zoomastigina (Trypanosoma, Leishmania). Apicomplexan parasites possessed the largest carbamoyl phosphate synthetase II enzymes due to insertions in the glutamine amidotransferase and carbamoyl phosphate synthetase domains that were not present in the corresponding gene segments from bacteria, plants, fungi and mammals. The C-terminal allosteric regulatory domain, the carbamoyl phosphate synthetase linker domain and the oligomerisation domain were also distinct from the corresponding domains in other species. The novel C-terminal regulatory domain may explain the lack of activation of Toxoplasma gondii carbamoyl phosphate synthetase II by the allosteric effector 5-phosphoribosyl 1-pyrophosphate. Toxoplasma gondii growth in vitro was markedly inhibited by the glutamine antagonist acivicin, an inhibitor of glutamine amidotransferase activity typically associated with carbamoyl phosphate synthetase II, guanosine monophosphate synthetase, or CTP synthetase.     

Control of pyrimidine biosynthesis in the perfused liver. Feedback inhibition of glutamine-dependent carbamoyl phosphate synthetase.

The site of feedback inhibition of the biosynthesis of pyrimidine nucleotides de novo was investigated in the isolated perfused rat liver. Hepatic uridine phosphate contents were specifically depleted by use of D-galactosamine. The effective activities of enzymes involved in the synthetic pathway were deduced from the rats of incorporation of labeled precursors into the acid-soluble uracil nucleotide pool and into some intermediates of the pathway. The labeling of hepatic urea was also monitored. When the uridine phosphate contents were less than 20% of controls, the incorporation of [14-C]-bicarbonate was stimulated about 20-fold. Label from [U-14C]oxaloacetate used as permeable precursor of intrace-lular aspartate was introduced into the uridylates to the same extent in normal and UTP-depleted livers. Similar results were obtained with labeled carbamoyl phosphate although the uptake of this compound by the liver was rather low. The lack of labeling of urea from exogenous carbamoyl phosphate does not indicate a free exchange of extra- and intramitochondrial carbamoyl phosphate. [ureido-14C]Ureidosuccinate produced in normal and D-galactosamine-treated livers almost identical labeling patterns of dihydroorotate, orotate and orotidine 5'-phosphate. The steady state concentrations of these intermediates were all below 15 nmol/g liver wet weight.     

Effects of dimethyl sulfoxide and glycerol on catalytic and regulatory properties of glutamine-dependent carbamoyl phosphate synthase from rat liver and dual effects of uridine triphosphate.

The cryoprotectants dimethyl sulfoxide and glycerol markedly affected the activity of glutamine-dependent carbamoyl phosphate synthase (EC 2.7.2.9) of rat liver, the first enzyme of de novo pyrimidine biosynthesis. The apparent K_m for MgATP^2? decreased with increases in the solvent concentrations, from 7.0 mm without the solvents to 0.1 and 0.8 mm in the presence of 25% ($\text{v}\text{v}$) dimethyl sulfoxide and 30% ($\text{w}\text{w}$) glycerol, respectively. The apparent K_m for bicarbonate also decreased with these solvents, while that for glutamine was not significantly affected. The response in the maximal velocity to the solvents was biphasic; the value of V increased 1.39- and 1.18-fold with 7.5% dimethyl sulfoxide and 10% glycerol, respectively, but then decreased as the concentrations of the solvents were further increased. The extents of inhibition by 25% dimethyl sulfoxide and 30% glycerol were 63 and 44%, respectively. The effects of 5-phosphoribosyl 1-pyrophosphate and MgUTP, allosteric effectors, were greatly modified by these solvents. Kinetic parameters as affected by the effectors in the presence of various concentrations of the solvents are described. A notable observation was that MgUTP, a potent inhibitor under ordinary conditions, stimulated the activity in the presence of high concentrations of dimethyl sulfoxide; in the presence of 30% dimethyl sulfoxide and 1 mm MgATP^2?, 0.5 to 2.0 mm MgUTP enhanced the enzymatic activity about twofold. MgUTP at higher concentrations was inhibitory. The dimethyl sulfoxide-dependent dual effects of MgUTP indicate the possible existence of at least two MgUTP-binding sites on the enzyme molecule.     

Role of the four conserved histidine residues in the amidotransferase domain of carbamoyl phosphate synthetase

Carbamoyl phosphate synthetase from Escherichia coli catalyzes the formation of carbamoyl phosphate from ATP, bicarbonate, and glutamine. The amidotransferase activity of this enzyme is catalyzed by the smaller of the two subunits of the heterodimeric protein. The roles of four conserved histidine residues within this subunit were probed by site-directed mutagenesis to asparagine. The catalytic activities of the H272N and H341N mutants are not significantly different than that of the wild-type enzyme. The H353N mutant is unable to utilize glutamine as a nitrogen source in the synthetase reaction or the partial glutaminase reaction. However, binding to the glutamine active site is not impaired in the H353N enzyme since glutamine is found to activate the partial ATPase reaction by 40% with a Kd of 54 microM. The H312N mutant has a Michaelis constant for glutamine that is 2 orders of magnitude larger than the wild-type value, but the maximal rate of glutamine hydrolysis is unchanged. These results are consistent with His-353 functioning as a general acid/base catalyst for proton transfers while His-312 serves a critical role for the binding of glutamine to the active site.     

Mutational analysis of ATP-grasp residues in the two ATP sites of Saccharomyces cerevisiae carbamoyl phosphate synthetase

The ATP-grasp fold is found in enzymes that catalyze the formation of an amide bond and occurs twice in carbamoyl phosphate synthetase. We have used site-directed mutagenesis to further define the relationship of these ATP folds to the ATP-grasp family and to probe for distinctions between the two ATP sites. Mutations at D265 and D810 severely diminished activity, consistent with consensus ATP-grasp roles of facilitating the transfer of the gamma-phosphate group of ATP. H262N was inactive whereas H807N, the corresponding mutation in the second ATP domain, exhibited robust activity, suggesting that these residues were not involved in the ATP-grasp function common to both domains. Mutations at I316 were somewhat catalytically impaired and were structurally unstable, consistent with a consensus role of interaction with the adenine and/or ribose moiety of ATP. L229G was too unstable to be purified and characterized. S228A showed essentially wild-type behavior.     

Immunoferritin location of carbamoyl phosphate synthetase in rat liver.

Experiments were carried out to locate carbamoyl phosphate synthetase (CPS) in rat liver by direct immunoferritin labeling. By using Epon sections treated with sodium methoxide, homogenates or mitochondrial and mitoplast fractions, carbamoyl phosphate synthetase was found homogeneously distributed in the mitochondrial matrix. Immunoferritin was detected with high resolution which permits the identification of individual molecules. Measurements were made of the number of ferritin particles per square micron of mitochondrial surface, providing a novel and independent assessment of the carbamoyl phosphate synthetase concentration.     

Inhibition of carbamoyl phosphate synthetase-I by dietary dehydroepiandrosterone.

Dehydroepiandrosterone (DHEA), administered per os, serves to prevent or retard the development of a variety of genetic and induced disorders in mice and rats. This treatment also results in the development of hepatomegaly, a change of liver color from pink to mahogany, peroxisome proliferation in hepatocytes and alterations in hepatocyte mitochondria morphology and respiration. We used one- and two-dimensional polyacrylamide gel electrophoresis (PAGE) to identify changes in the relative levels of liver proteins produced by DHEA treatment of rodents. In mouse liver, there were apparent increases in the levels of 26 proteins and decreases in the levels of 7 proteins. Of the induced proteins the most prominent had Mr approximately 72 K; this protein was identified in a previous study as enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase. Another protein of Mr approximately 28 K, of unknown nature, also was induced markedly by DHEA treatment of mice and rats. A protein of Mr approximately 160 K, which was identified as carbamoyl phosphate synthetase-I (CPS-I), was decreased markedly by DHEA action. This enzyme, which comprises approx. 15-20% of mitochondrial matrix protein, is involved in the entry and rate-limiting step of the urea cycle. The specific activity of CPS-I also was significantly decreased by DHEA, but serum urea levels were normal. To determine whether steroids other than DHEA also induced similar changes, mice were treated with various steroids for 14 days and, thereafter, liver proteins were evaluated by SDS-PAGE: estradiol-17 beta and isoandrosterone induced both the approximately 72 and approximately 28 kDa proteins, testosterone and androsterone induced the 28 kDa protein only, but etiocholanolone, pregnenolone and progesterone were without effect. The findings of this study serve to demonstrate that: (i) hepatic protein levels are affected by DHEA treatment of mice and rats; (ii) liver CPS-I activity is decreased significantly by DHEA treatment, but serum urea levels remain within the normal range; and (iii) sex steroids and some of their precursors, when administered per os, also alter liver protein levels.     

Effect of carbamoyl phosphate on nitrogenase in Anabaena cylindrica Lemm.

Carbamoyl phosphate inhibited acetylene reduction by whole cells and cell-free extracts of Anabaena cylindrica. Higher levels of both endogenous carbamoyl phosphate and carbamoyl phosphate synthase activity were present in NH4+-grown cells (in which acetylene reduction was absent) than in N2-grown cells (in which acetylene reduction was present). However, inhibition of acetylene reduction was observed also with cyanate, the main initial decomposition product under the conditions used. It is concluded that carbamoyl phosphate or one of its metabolites may act as a physiological regulator of both nitrogenase activity and synthesis, but caution must be used in interpreting effects observed several hours after the addition of carbamoyl phosphate, because the effects may be due to cyanate.     

The regulation of carbamoyl phosphate synthase activity in rat liver mitochondria.

The rate at which isolated rat liver mitochondria synthesized citrulline with NH4C1 as nitrogen source was markedly dependent on the protein content of the diet. 2. Citrulline synthesis was not rate-limited by substrate concentration, substrate transport or ornithine transcarbamoylase activity under the conditions used. 3. The intramitochondrial content of an activator of carbamoyl phosphate synthase, assumed to be N-acetyl-glutamate, varied markedly with dietary protein content. The variation in the concentration of this activator was sufficient to account for the observed variation in the rates of citrulline synthesis if this synthesis were rate-limited by the activity of carbamoyl phosphate synthase. 4. The rates of urea formation from NH4Cl as nitrogen source in isolated liver cells showed variations in response to diet that closely paralleled the variations in the rates of citrulline synthesis observed in isolated mitochondria. 5. These results are consistent with the postulate that when NH4Cl plus ornithine are present in an excess, the rate of urea synthesis is regulated at the level of carbamoyl phosphate synthase activity.     

Simultaneous separation by high-performance liquid chromatography of carbamoyl aspartate, carbamoyl phosphate and dihydroorotic acid

Leflunomide is an immunomodulatory drug which acts by inhibiting dihydroorotic acid dehydrogenase, the fourth enzyme of pyrimidine biosynthesis. We modified our high-performance liquid chromatography method to demonstrate that the principal metabolite in mitogen-stimulated human T-lymphocytes incubated with leflunomide was not dihydroorotic acid, but carbamoyl aspartate. Identification involved preparation of [¹⁴C]carbamoyl aspartate from [¹⁴C]aspartic acid and mammalian aspartate transcarbamoylase. Accumulation of carbamoyl aspartate indicates that under these conditions the equilibrium constant for dihydroorotase favours the reverse reaction. This HPLC method, enabling simultaneous separation of the first four intermediates in the de novo pyrimidine pathway may be of use in a variety of experimental situations.     

Mechanism of allosteric modulation of Escherichia coli carbamoyl phosphate synthetase probed by site-directed mutagenesis of ornithine site residues

The role of residues of the ornithine activator site is probed by mutagenesis in Escherichia coli carbamoyl phosphate synthetase (CPS). Mutations E783A, E783L, E892A and E892L abolish ornithine binding, E783D and T1042V decrease 2-3 orders of magnitude and E892D decreased 10-fold apparent affinity for ornithine. None of the mutations inactivates CPS. E783 mutations hamper carbamate phosphorylation and increase K(+) and MgATP requirements, possibly by perturbing the K(+)-loop near the carbamate phosphorylation site. Mutation E892A activates the enzyme similarly to ornithine, possibly by altering the position of K891 at the opening of the tunnel that delivers the carbamate to its phosphorylation site. T1042V also influences modulation by IMP and UMP, supporting signal transmission from the nucleotide effector to the ornithine site mediated by a hydrogen bond network involving T1042. Ornithine activation of CPS may be mediated by K(+)-loop and tunnel gating changes.     

Characterization of pyrimidine-repressible and arginine-repressible carbamyl phosphate synthetases from Bacillus subtilis.

The number and properties of carbamyl phosphate synthetases in Bacillus subtilis have been uncertain because of conflicting genetic results and instability of the enzyme in extracts. The discovery of a previously unrecognized requirement of B. subtilis carbamyl phosphate synthetases for a high concentration of potassium ions for activity and stability permitted unequivocal demonstration that this bacterium elaborates two carbamyl phosphate synthetases. Carbamyl phosphate synthetase A was shown to be repressed by arginine, to have a molecular weight of about 200,000, and to be coded for by a gene that maps near argC4. This isozyme was insensitive to metabolites of the arginine and pyrimidine biosynthetic pathways. Carbamyl phosphate synthetase P was found to be repressed by uracil, to have a molecular weight of 90,000 to 100,000, and to be coded for by a gene that maps near the other pyr genes. This isozyme was activated by phosphoridine nucleotides. Other kinetic properties of the two isozymes were compared. Bacillus thus resembles eucaryotic microbes in producing two carbamyl phosphate synthetases, rather than the enteric bacteria, which produce a single carbamyl phosphate synthetase.     

Arginine-specific carbamoyl phosphate metabolism in mitochondria of Neurospora crassa. Channeling and control by arginine

Citrulline is synthesized in mitochondria of Neurospora crassa from ornithine and carbamoyl phosphate. In mycelia grown in minimal medium, carbamoyl phosphate limits citrulline (and arginine) synthesis. Addition of arginine to such cultures reduces the availability of intramitochondrial ornithine, and ornithine then limits citrulline synthesis. We have found that for some time after addition of excess arginine, carbamoyl phosphate synthesis continued. Very little of this carbamoyl phosphate escaped the mitochondrion to be used in the pyrimidine pathway in the nucleus. Instead, mitochondrial carbamoyl phosphate accumulated over 40-fold and turned over rapidly. This was true in ornithine- or ornithine carbamoyltransferase-deficient mutants and in normal mycelia during feedback inhibition of ornithine synthesis. The data suggest that the rate of carbamoyl phosphate synthesis is dependent to a large extent upon the specific activity of the slowly and incompletely repressible synthetic enzyme, carbamoyl-phosphate synthetase A. In keeping with this conclusion, we found that when carbamoyl-phosphate synthetase A was repressed 2-10-fold by growth of mycelia in arginine, carbamoyl phosphate was still synthesized in excess of that used for residual citrulline synthesis. Again, only a small fraction of the excess carbamoyl phosphate could be accounted for by diversion to the pyrimidine pathway. The continued synthesis and turnover of carbamoyl phosphate in mitochondria of arginine-grown cells may allow rapid resumption of citrulline formation after external arginine disappears and no longer exerts negative control on ornithine biosynthesis.     

The binding of inosine monophosphate to Escherichia coli carbamoyl phosphate synthetase.

Carbamoyl phosphate synthetase (CPS) from Escherichia coli catalyzes the formation of carbamoyl phosphate, which is subsequently employed in both the pyrimidine and arginine biosynthetic pathways. The reaction mechanism is known to proceed through at least three highly reactive intermediates: ammonia, carboxyphosphate, and carbamate. In keeping with the fact that the product of CPS is utilized in two competing metabolic pathways, the enzyme is highly regulated by a variety of effector molecules including potassium and ornithine, which function as activators, and UMP, which acts as an inhibitor. IMP is also known to bind to CPS but the actual effect of this ligand on the activity of the enzyme is dependent upon both temperature and assay conditions. Here we describe the three-dimensional architecture of CPS with bound IMP determined and refined to 2.1 A resolution. The nucleotide is situated at the C-terminal portion of a five-stranded parallel beta-sheet in the allosteric domain formed by Ser(937) to Lys(1073). Those amino acid side chains responsible for anchoring the nucleotide to the polypeptide chain include Lys(954), Thr(974), Thr(977), Lys(993), Asn(1015), and Thr(1017). A series of hydrogen bonds connect the IMP-binding pocket to the active site of the large subunit known to function in the phosphorylation of the unstable intermediate, carbamate. This structural analysis reveals, for the first time, the detailed manner in which CPS accommodates nucleotide monophosphate effector molecules within the allosteric domain.     

Glutamine utilization in both the arginine-specific and pyrimidine-specific carbamoyl phosphate synthase enzymes of eurospora crassa.

Summary As glutamine-dependent carbamoyl phosphate synthetase (CPS) activity in some organisms is composed of a glutaminase and an ammonium-dependent CPS, CPS^- mutants in Neurospora crassa were examined for glutamine- and ammonium-dependent CPS activities. No evidence was found that the genetic location of these two functions were separable. This is discussed with reference to the close genetic proximity of the CPS_pyr and aspartate carbamoyl-transferase (ACT) structural gene (pyr-3) and the arg-2 gene which appears to specify a subunit responsible for glutamine utilisation in CPS_arg.Supported by Science Research Council Grant B/RG/2981