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.     

Site of Synthesis of the Enzymes of the Pyrimidine Biosynthetic Pathway in Oat (Avena sativa L.) Leaves

Heat-bleached oat (Avena sativa L. cv Porter) leaves lacking 70S chloroplast ribosomes have been used to demonstrate that four chloroplast-localized enzymes of pyrimidine nucleotide biosynthesis: aspartate carbamoyl-transferase, dihydroorotase, orotidine phosphoribosyl-transferase, and orotidine-5′-phosphate decarboxylase, are synthesized on cytoplasmic ribosomes. Two other chloroplast enzymes, carbamoyl phosphate synthetase, involved in both pyrimidine and arginine biosynthesis, and ornithine carbamoyltransferase, an enzyme of arginine biosynthesis, were also shown to be made on 80S ribosomes.     

Carbamoyl-phosphate synthetases from Neurospora crassa. Immunological relatedness of the enzymes from Neurospora, bacteria, yeast, and mammals

Neurospora crassa contains two carbamoyl-phosphate synthetases: a mitochondrial enzyme (CPS-A) which supplies carbamoyl phosphate for arginine biosynthesis, and a nuclear enzyme whose product is used for the synthesis of pyrimidines. We have prepared antiserum against a highly purified preparation of the large subunit of CPS-A and have used the antiserum to demonstrate that the large subunit is, like most mitochondrially localized proteins, initially synthesized as a higher molecular weight precursor. The CPS-A antiserum cross-reacts with the nuclear enzyme, allowing us to identify the product of the complex N. crassa pyr-3 genetic locus as a protein with a subunit molecular weight of 180,000. Finally, we have found that the CPS-A antiserum also cross-reacts with carbamoyl-phosphate synthetases from bacteria, yeast, and mammals. The immunological relatedness of carbamoyl-phosphate synthetases from such diverse species suggests that the protein sequences required for carbamoyl phosphate production have been highly conserved during the course of evolution.     

Nucleotide sequence of the pyrimidine specific carbamoyl phosphate synthetase, a part of the yeast multifunctional protein encoded by the URA2 gene

Summary Yeast URA2 encodes a multifunctional carbamoyl phosphate synthetase-aspartate transcarbamylase of 220,000 molecular weight. We determined the nucleotide sequence of the 5 proximal part of the gene which is responsible for the glutamine amide transfer function of the carbamoyl phosphate synthetase activity. Alignment of the enzyme sequence derived from URA2 with sequences from Escherichia coli carA carB and yeast arginine-specific CPA1 CPA2 indicates that monofunctional and bifunctional carbamoyl phosphate synthetases are probably homologous. The URA2-derived enzyme organization is NH₂-carbamoyl phosphate synthetase-aspartate transcarbamylase-CO₂H.     

Toxoplasma gondii lacks the enzymes required for de novo arginine biosynthesis and arginine starvation triggers cyst formation

Two separate carbamoyl phosphate synthetase activities are required for the de novo synthesis of pyrimidines and arginine in most eukaryotes. Toxoplasma gondii is novel in possessing a single carbamoyl phosphate synthetase II gene that corresponds to a glutamine-dependent form required for pyrimidine biosynthesis. We therefore examined arginine acquisition in T. gondii to determine whether the single carbamoyl phosphate synthetase II activity could provide both pyrimidine and arginine biosynthesis. We found that arginine deprivation efficiently blocks the replication of intracellular T. gondii, yet has little effect on long-term parasite viability. Addition of citrulline, but not ornithine, rescues the growth defect observed in the absence of exogenous arginine. This rescue with citrulline is ablated when parasites are cultured in a human citrullinemia fibroblast cell line that is deficient in argininosuccinate synthetase activity. These results reveal the absence of genes and activities of the arginine biosynthetic pathway and demonstrate that T. gondii is an arginine auxotroph. Arginine starvation was also found to efficiently trigger differentiation of replicative tachyzoites into bradyzoites contained within stable cyst-like structures. These same parasites expressing bradyzoite antigens can be efficiently switched back to rapidly proliferating tachyzoites several weeks after arginine starvation. We hypothesise that the absence of gene activities that are essential for the biosynthesis of arginine from carbamoyl phosphate confers a selective advantage by increasing bradyzoite switching during the host response to T. gondii infection. These findings are consistent with a model of host-parasite evolution that allowed host control of bradyzoite induction by trading off virulence for increased transmission.     

The sole Serine/Threonine protein kinase and its cognate phosphatase from Aquifex aeolicus targets Pyrimidine biosynthesis

Serine/Threonine kinases participate in complex, interacting signaling pathways in eukaryotes, prokaryotes, and archae. While most organisms contain many different kinases, the extreme hyperthermophile, Aquifex aeolicus encodes a single hypothetical Ser/Thr kinase. A gene homologous to eukaryotic protein phosphatases overlaps the kinase gene by a single base pair. The putative kinase, AaSTPK and phosphatase, AaPPM, were cloned and expressed in E. coli, purified to homogeneity and found to be functional. AaSTPK is a 34-kDa monomer that can use MgATP, MnATP, or MnGTP as co-substrates, although MgATP appears to be the preferred substrate. AaSTPK was autophosphorylated on a threonine residue and was dephosphorylated by AaPPM. AaPPM phosphatase is homologous to the PPM sub-family of Ser/Thr phosphatases and was stimulated by MnCl₂ and CoCl₂ but not MgCl₂. AaSTPK also phosphorylated one threonine residue on the carbamoyl phosphate synthetase, CPS.A subunit. Carbamoyl phosphate synthetase reconstituted with phosphorylated CPS.A had unaltered catalytic activity but allosteric inhibition by UMP and activation by the arginine intermediate, ornithine, were both appreciably attenuated. These changes in allosteric regulation would be expected to activate pyrimidine biosynthesis by releasing the constraints imposed on carbamoyl phosphate synthetase activity by UMP and uncoupling the regulation of pyrimidine and arginine biosynthesis. CPS.A was also dephosphorylated by AaPPM. Aquifex aeolicus occupies the lowest branch on the prokaryotic phylogenetic tree. The Thr/Ser kinase, its cognate phosphatase and a protein substrate may be elements of a simple signaling pathway, perhaps the most primitive example of this mode of regulation described thus far.     

Long-range allosteric transitions in carbamoyl phosphate synthetase

Carbamoyl phosphate synthetase plays a key role in both pyrimidine and arginine biosynthesis by catalyzing the production of carbamoyl phosphate from one molecule of bicarbonate, two molecules of MgATP, and one molecule of glutamine. The enzyme from Escherichia coli consists of two polypeptide chains referred to as the small and large subunits, which contain a total of three separate active sites that are connected by an intramolecular tunnel. The small subunit harbors one of these active sites and is responsible for the hydrolysis of glutamine to glutamate and ammonia. The large subunit binds the two required molecules of MgATP and is involved in assembling the final product. Compounds such as L-ornithine, UMP, and IMP allosterically regulate the enzyme. Here, we report the three-dimensional structure of a site-directed mutant protein of carbamoyl phosphate synthetase from E. coli, where Cys 248 in the small subunit was changed to an aspartate. This residue was targeted for a structural investigation because previous studies demonstrated that the partial glutaminase activity of the C248D mutant protein was increased 40-fold relative to the wild-type enzyme, whereas the formation of carbamoyl phosphate using glutamine as a nitrogen source was completely abolished. Remarkably, although Cys 248 in the small subunit is located at approximately 100 A from the allosteric binding pocket in the large subunit, the electron density map clearly revealed the presence of UMP, although this ligand was never included in the purification or crystallization schemes. The manner in which UMP binds to carbamoyl phosphate synthetase is described.     

Pyrimidine nucleotide biosynthesis in Clonorchis sinensis and Paragonimus ohirai

Kobayashi M., Yokogawa M., Mori M. and Tatibana M. 1978. Pyrimidine nucleotide biosynthesis in Clonorchis sinensis and Paragonimus ohirai. International Journal for Parasitology8: 471–477. A carbamoyl phosphate synthetase was detected in the cytosol fractions of the adult worms of Clonorchis sinensis and Paragonimus ohirai. The enzyme was partially purified and was shown to utilize both l-glutamine and ammonia and does not require $\text{N-}\text{acetyl}\text{-}\text{l}\text{-}\text{glutamate}$. The enzyme was subject to specific feedback inhibition by end products such as UDP, UTP, CDP, dUDP and dCDP and was stimulated by 5-phosphoribosyl-1-pyrophosphate. These properties of the synthetase were similar to those of carbamoyl phosphate synthetase II demonstrated in mammalian tissues Some other enzyme activities of this pathway were also detected in both species. Paragonimus ohirai actively incorporated ¹⁴CO₂ into uridine nucleotides; accumulation of intermediates of the pathway was not seen. These results indicate that the carbamoyl phosphate synthetase plays a key and regulatory step of ^{de novo} pyrimidine nucleotide biosynthesis in these worms.     

The human Rad9 checkpoint protein stimulates the carbamoyl phosphate synthetase activity of the multifunctional protein CAD

The human Rad9 checkpoint protein is a subunit of the heterotrimeric Rad9-Rad1-Hus1 (9-1-1) complex that plays a role as a damage sensor in the DNA damage checkpoint response. Rad9 has been found to interact with several other proteins outside the context of the 9-1-1 complex with no obvious checkpoint functions. During our studies on the 9-1-1 complex, we found that Rad9 immunoprecipitates contained a 240 kDa protein that was identified as carbamoyl phosphate synthetase/aspartate transcarbamoylase/dihydroorotase (CAD), a multienzymatic protein required for the de novo synthesis of pyrimidine nucleotides and cell growth. Further investigations revealed that only free Rad9, but not Rad9 within the 9-1-1 complex, bound to CAD. The rate-limiting step in de novo pyrimidine nucleotide synthesis is catalyzed by the carbamoyl phosphate synthetase II (CPSase) domain of CAD. We find that Rad9 binds to the CPSase domain, and, moreover, this binding results in a 2-fold stimulation of the CPSase activity of CAD. Similar results were also obtained with an N-terminal Rad9 fragment. These findings suggest that Rad9 may play a role in ribonucleotide biosynthesis.     

氨甲酰磷酸不同合成途径在大肠杆菌中的比较

【背景】氨甲酰磷酸是生物合成代谢中精氨酸与嘧啶的重要前体物质,在工业微生物生产精氨酸与嘧啶及其衍生物中发挥关键作用。【目的】在大肠杆菌Escherichia coli BW25113中比较氨甲酰磷酸不同合成途径的催化效率。【方法】在大肠杆菌Escherichia coli BW25113中过表达鸟氨酸氨甲酰基转移酶(OTC)的基础上,分别过表达大肠杆菌自身的氨基甲酸激酶(CK)和氨甲酰磷酸合酶(CPSⅡ)并表征其反应效果。通过优化底物供应(调整底物浓度与引入L-谷氨酰胺合成酶)对CK与CPSⅡ的催化反应进行优化。【结果】在大肠杆菌中过表达OTC,建立细胞水平氨甲酰磷酸检测体系。在此基础上比较不同来源的CK,发现大肠杆菌来源的CK效果最好,50mmol/LNH4HCO3条件下全细胞催化9h得到2.95±0.15mmol/LL-瓜氨酸;过表达CPSⅡ时,50mmol/LL-谷氨酰胺催化9h得到3.16±0.29 mmol/L L-瓜氨酸。通过改变底物NH4HCO3浓度和引入外源L-谷氨酰胺合成酶(GS)等方式对CK与CPSⅡ的催化反应分别进行优化后,100 mmol/L NH4HCO3条件下,L-瓜氨酸浓度分别提高至4.67±0.55mmol/L和6.12±0.38mmol/L,且过表达GS后CPSⅡ途径可以利用NH3,不需要额外添加L-谷氨酰胺。【结论】引入L-谷氨酰胺合成酶后的CPSⅡ途径合成氨甲酰磷酸的能力优于CK途径,为精氨酸、嘧啶及其衍生物的合成提供了一种更加高效的策略。     

Molecular cloning, identification and characteristics of a novel isoform of carbamyl phosphate synthetase I in human testis

A gene coding a novel isoform of carbamyl phosphate synthetase I (CPS1) was cloned from a human testicular library. As shown by cDNA microarray hybridization, this gene was expressed at a higher level in human adult testes than in fetal testes. The full length of its cDNA was 3831 bp, with a 3149 bp open reading frame, encoding a 1050-amino-acid protein. The cDNA sequence was deposited in the GenBank (AY317138). Sequence analysis showed that it was homologous to the human CPS1 gene. The putative protein contained functional domains composing the intact large subunit of carbamoyl phosphate synthetase, thus indicated it has the capability of arginine biosynthesis. A multiple tissue expression profile showed high expression of this gene in human testis, suggesting the novel alternative splicing form of CPS1 may be correlated with human spermatogenesis.     

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.     

Pyrimidine biosynthesis in Aspergillus nidulans. Isolation and characterisation of mutants resistant to fluoropyrimidines.

Summary Mutants resistant to 5-fluorouracil, 5-fluorouridine and 5-fluorodeoxyuridine have been selected in Aspergillus nidulans. Growth tests combined with genetic analysis showed that mutations conferring resistance to fluoropyrimidines could occur in at least seven genes. Three of these, fulE, fulF and furA were concerned with either the uptake of pyrimidines or their conversion to uridine monophosphate. The other four genes did not affect these functions. Mutations in fulA probably confer resistance by lowering ornithine transcarbamoylase, thereby making the normally arginine-specific carbamoyl phosphate pool available for increased uracil synthesis. Mutations in fulD may make the arginine-specific carbamoyl phosphate synthetase insensitive to inhibition or repression by arginine, and so lead to increased carbamoyl phosphate pool sizes, and increased uracil synthesis. Both fulA and fulD mutants suppress pyrA mutants which lack the uracil-specific carbamoyl phosphate synthetase. Mutations in fulB and fulC do not suppress pyrA, and so may act more directly to increase uracil synthesis. The synthesis of aspartate carbamoyl transferase in fulB7 strains is not repressed by uracil. fulC mutants are closely linked to the pyrA, B, C, N region which codes for the first two enzymes of pyrimidine biosynthesis, and may result in these enzymes being less sensitive to inhibition by uracil.Abbreviations used 5FU 5-fluorouracil - 5FUR 5-fluorouridine - 5FdUR 5-fluorodeoxyuridine     

Factors influencing the development of urea-synthesizing enzymes in rat liver

1. The activities of enzymes of the urea cycle, carbamoyl phosphate synthetase, ornithine transcarbamoylase, argininosuccinate synthetase, argininosuccinase (the last two comprising the arginine synthetase system) and arginase, were measured in the liver during development of the rat. All five enzymes exhibited relatively low activities in foetal liver and a rapid postnatal increase was found. The rate-limiting enzyme of urea synthesis in the rat, the condensing enzyme of the arginine synthetase system, showed the lowest activity at birth and the most rapid postnatal increase, a fivefold increase within 24hr. after birth. A second increase of activity was noted after the tenth day. These results suggest that the postnatal increase of arginine synthetase activity initiates the ability for urea synthesis in the rat. 2. Some factors influencing the development of the rate-limiting arginine synthetase system were studied in more detail. (a) Intraperitoneal administration of puromycin inhibited the postnatal increaseof the enzyme activity. (b) Starvation of newborn animals for 24hr. after birth had no effect on the postnatal development of the enzyme. (c) Bilateral adrenalectomy at birth caused a marked diminution in the postnatal increase of the enzyme activity and injections of triamcinolone were effective in preventing the effect of adrenalectomy. (d) Administration of triamcinolone alone had a marked stimulatory effect on the postnatal development of this enzyme. (e) Premature and postmature birth had virtually no effect on the developmental pattern of the arginine synthetase activity, suggesting that the increase of this enzyme activity after birth is not initiated by the birth process.     

ATP6v0d2 deficiency increases bone mass, but does not influence ovariectomy-induced bone loss

SIR2 protein, an NAD-dependent deacetylase, is localized to nucleus and is involved in life span extension by calorie restriction in yeast. In mammals, among the seven SIR2 homologues (SIRT1-7), SIRT3, 4, and 5 are localized to mitochondria. As SIRT5 mRNA levels in liver are increased by fasting, the physiological role of SIRT5 was investigated in liver of SIRT5-overexpressing transgenic (SIRT5 Tg) mice. We identified carbamoyl phosphate synthetase 1 (CPS1), a key enzyme of the urea cycle that catalyzes condensation of ammonia with bicarbonate to form carbamoyl phosphate, as a target of SIRT5 by two-dimensional electrophoresis comparing mitochondrial proteins in livers of SIRT5 Tg and wild-type mice. CPS1 protein was more deacetylated and activated in liver of SIRT5 Tg mice than in wild-type. In addition, urea production was upregulated in hepatocytes of SIRT5 Tg mice. These results agree with those of a previous study using SIRT5 knockout (KO) mice. Because ammonia generated during fasting is toxic, SIRT5 protein might play a protective role by converting ammonia to non-toxic urea through deacetylation and activation of CPS1.     

Modulation of intestinal urea cycle by dietary spermine in suckling rat

Argininosuccinate synthetase, an ubiquitous enzyme in mammals, catalyses the formation of argininosuccinate, the precursor of arginine. Arginine is recognised as an essential amino acid in foetuses and neonates, but also as a conditionally essential amino acid in adults. Argininosuccinate synthetase is initially expressed in enterocytes during the developmental period, it disappeared from this organ then appeared in the kidneys. Although the importance of both intestinal and renal argininosuccinate synthetases has been recognised for a long time, nutrients have not yet been identified as inducers of the gene expression. In the context of a proteomic screening of intestinal modifications induced by dietary spermine in suckling rats, we showed that argininosuccinate synthetase and carbamoyl phosphate synthase disappeared from enterocytes after this treatment. The disappearance of argininosuccinate synthetase in small intestine was confirmed by immunodetection. Expression of carbamoyl phosphate synthase and argininosuccinate synthetase coding genes decreased also after spermine administration. Expression of other urea cycle enzyme coding genes was modulated by spermine administration: argininosuccinate lyase decreased and arginase increased. Our results fit with the developmental variation of argininosuccinate synthetase and carbamoyl phosphate synthase. Modulation of the gene expression for several urea cycle enzymes suggests a coordination between all the pathway steps and switch toward polyamine (or proline and glutamate) biosynthesis from ornithine.     

Dissection of the functional domains of Escherichia coli carbamoyl phosphate synthetase by site-directed mutagenesis

The catalytic functions of the amino-terminal and carboxyl-terminal halves of the large subunit of carbamoyl phosphate synthetase from Escherichia coli have been identified using site-directed mutagenesis. Glycine residues at positions 176, 180, and 722 within the putative mononucleotide-binding site were replaced with isoleucine residues. Each of these mutations resulted in at least a 1 order of magnitude reduction in the Vmax for carbamoyl phosphate synthesis. The mutations on the amino-terminal half, G176I and G180I, caused slight reduction in the rate of synthesis of ATP from ADP and carbamoyl phosphate (the partial ATP synthesis reaction) but the bicarbonate-dependent ATPase reaction velocity was reduced to less than 10% of the wild-type rate. The mutant G722I, which is on the carboxy-terminal half, caused the partial ATP synthesis reaction to be reduced by 1 order of magnitude but the bicarbonate-dependent ATPase reaction was reduced only slightly. All three mutations are within regions which show homology to the putative glycine-rich loops of many ATP-binding proteins. These results have been interpreted to suggest that the two homologous halves of the large subunit of carbamoyl phosphate synthetase each contain a binding site for ATP. The NH2-terminal domain contains the portion of the large subunit that is primarily involved with the phosphorylation of bicarbonate to carboxy phosphate while the COOH-terminal domain contains the region of the enzyme that catalyzes the phosphorylation of carbamate to carbamoyl phosphate.