Characterization of pyrimidine metabolism in the cellular slime mold, Dictyostelium discoideum

1989 ◽  
Vol 35 (4) ◽  
pp. 432-438 ◽  
Author(s):  
Melinda E. Wales ◽  
Mary G. Mann-Dean ◽  
James R. Wild
Keyword(s):  
Dictyostelium Discoideum ◽  
De Novo ◽  
Nucleotide Metabolism ◽  
Pyrimidine Biosynthesis ◽  
Metabolic Energy ◽  
Aspartate Transcarbamoylase ◽  
Adenylate Energy Charge ◽  
Carbamoyl Phosphate Synthetase ◽  
Pyrimidine Metabolism ◽  
Carbamoyl Phosphate

The arginine-independent, de novo biosynthetic pathway of pyrrolidines in Dictyostelium discoideum is initiated by a class II carbamoyl-phosphate synthetase (EC 6.3.5.5) specific for pyrimidine biosynthesis which utilized L-glutamine as its N donor and was partially inhibited by both UTP and CTP. The second step in the de novo pathway was provided by an unregulated aspartate transcarbamoylase (EC 2.1.3.2) which primarily appeared as a multimeric enzyme of 105 kilodaltons. The next enzyme, dihydroorotase (EC 3.5.2.3), was approximately 90–100 kilodaltons. Although the early enzymatic activities of the pyrimidine pathway appeared to reside in independent protein complexes, various unstable molecular species were observed. These structural variants may represent proteolytic fragments of a multienzyme complex. In addition to de novo synthesis, the amoeba demonstrated the capacity for salvage utilization of uracil, uridine, and cytidine. Upon starvation on a solid substratum, axenically grown amoebas began a concerted developmental program accompanied by a restructuring of nucleotide metabolism. The absolute levels of the ribonucleotide pools droppedby 98% within 30 h; however, both the adenylate energy charge and the GTP/ATP ratios were maintained for 50 h after the initiation of development. The maintenance of these metabolic energy parameters required the tight cell–cell contact necessary for development, and the capacity for pyrimidine metabolism was maintained throughout developmental morphogenesis.Key words: aspartate transcarbamoylase, carbamoyl phosphate synthetase, development, pyrimidine biosynthesis.

scholarly journals Crystal structure of truncated aspartate transcarbamoylase fromPlasmodium falciparum

2016 ◽  
Vol 72 (7) ◽  
pp. 523-533 ◽  
Author(s):  
Sergey Lunev ◽  
Soraya S. Bosch ◽  
Fernando de Assis Batista ◽  
Carsten Wrenger ◽  
Matthew R. Groves
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Active Sites ◽  
Catalytic Domain ◽  
De Novo ◽  
Pyrimidine Biosynthesis ◽  
Aspartate Transcarbamoylase ◽  
Carbamoyl Phosphate ◽  
Cell Parameters ◽  
High Degree

Thede novopyrimidine-biosynthesis pathway ofPlasmodium falciparumis a promising target for antimalarial drug discovery. The parasite requires a supply of purines and pyrimidines for growth and proliferation and is unable to take up pyrimidines from the host. Direct (or indirect) inhibition ofde novopyrimidine biosynthesisviadihydroorotate dehydrogenase (PfDHODH), the fourth enzyme of the pathway, has already been shown to be lethal to the parasite. In the second step of the plasmodial pyrimidine-synthesis pathway, aspartate and carbamoyl phosphate are condensed toN-carbamoyl-L-aspartate and inorganic phosphate by aspartate transcarbamoylase (PfATC). In this paper, the 2.5 Å resolution crystal structure ofPfATC is reported. The space group of thePfATC crystals was determined to be monoclinicP21, with unit-cell parametersa= 87.0,b= 103.8,c= 87.1 Å, α = 90.0, β = 117.7, γ = 90.0°. The presentedPfATC model shares a high degree of homology with the catalytic domain ofEscherichia coliATC. There is as yet no evidence of the existence of a regulatory domain inPfATC. Similarly toE. coliATC,PfATC was modelled as a homotrimer in which each of the three active sites is formed at the oligomeric interface. Each active site comprises residues from two adjacent subunits in the trimer with a high degree of evolutional conservation. Here, the activity loss owing to mutagenesis of the key active-site residues is also described.

scholarly journals A Carbamoyl Phosphate Synthetase II (CPSII) Deletion Mutant of Toxoplasma gondii Induces Partial Protective Immunity in Mice

2021 ◽  
Vol 11 ◽  
Author(s):  
Xunhui Zhuo ◽  
Kaige Du ◽  
Haojie Ding ◽  
Di Lou ◽  
Bin Zheng ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
De Novo ◽  
Parasite Growth ◽  
Pyrimidine Biosynthesis ◽  
Mrna Levels ◽  
Biosynthesis Pathway ◽  
Carbamoyl Phosphate Synthetase ◽  
Carbamoyl Phosphate

Toxoplasma gondii is an obligate intracellular protozoan parasite. T. gondii primarily infection in pregnant women may result in fetal abortion, and infection in immunosuppressed population may result in toxoplasmosis. Carbamoyl phosphate synthetase II (CPSII) is a key enzyme in the de novo pyrimidine-biosynthesis pathway, and has a crucial role in parasite replication. We generated a mutant with complete deletion of CPSII via clustered regularly interspaced short palindromic repeats (CRISPR)/cas9 in type-1 RH strain of T. gondii. We tested the intracellular proliferation of this mutant and found that it showed significantly reduced replication in vitro, though CPSII deletion did not completely stop the parasite growth. The immune responses induced by the infection of RHΔCPSII tachyzoites in mice were evaluated. During infection in mice, the RHΔCPSII mutant displayed notable defects in replication and virulence, and significantly enhanced the survival of mice compared with survival of RH-infected mice. We tracked parasite propagation from ascitic fluid in mice infected with the RHΔCPSII mutant, and few tachyzoites were observed at early infection. We also observed that the RHΔCPSII mutant induced greater accumulation of neutrophils. The mutant induced a higher level of T-helper type-1 cytokines [interferon (IFN)-γ, interleukin (IL)-12]. The mRNA levels of signal transducer and activator of transcription cellular transcription factor 1 and IFN regulatory factor 8 were significantly higher in the RHΔCPSII mutant-infected group. Together, these data suggest that CPSII is crucial for parasite growth, and that strains lack the de novo pyrimidine biosynthesis pathway and salvage pathway may become a promising live attenuated vaccine to prevent infection with T. gondii.

Site-directed mutagenesis studies on the uridine monophosphate binding sites of feedback inhibition in carbamoyl phosphate synthetase and effects on cytidine production by Bacillus amyloliquefaciens

2013 ◽  
Vol 59 (6) ◽  
pp. 374-379 ◽  
Author(s):  
Haitian Fang ◽  
Huiyan Liu ◽  
Ning Chen ◽  
Chenglin Zhang ◽  
Xixian Xie ◽  
...  
Keyword(s):  
Binding Sites ◽  
Bacillus Amyloliquefaciens ◽  
De Novo ◽  
Feedback Inhibition ◽  
Large Subunit ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Carbamoyl Phosphate Synthetase ◽  
Carbamoyl Phosphate ◽  
Uridine Monophosphate

A major problem when pyrimidine de novo biosynthesis is used for cytidine production is the existence of many negative regulatory factors. Cytidine biosynthesis in Bacillus amyloliquefaciens proceeds via a pathway that is controlled by uridine monophosphate (UMP) through feedback inhibition of carbamoyl phosphate synthetase (CPS), the enzyme that converts CO2, NH3, and glutamine to carbamoyl phosphate. In this study, the gene carB encoding the large subunit of CPS from B. amyloliquefaciens CYT1 was site directed, and the UMP binding sites of feedback inhibition in Bam-CPS are described. The residues Thr-941, Thr-970, and Lys-986 in CPS from B. amyloliquefaciens were subjected to site-directed mutagenesis to alter UMP’s feedback inhibition of CPS. To find feedback-resistant B. amyloliquefaciens, the influence of the T941F, T970A, K986I, T941F/K986I, and T941F/T970A/K986I mutations on CPS enzymatic properties was studied. The recombinant B. amyloliquefaciens with mutated T941F/K986I and T941F/T970A/K986I CPS showed a 3.7- and 5.7-fold increase, respectively, in cytidine production in comparison with the control expressing wild-type CPS, which was more suitable for further application of the cytidine synthesis. To a certain extent, the 5 mutations were found to release the enzyme from UMP inhibition and to improve B. amyloliquefaciens cytidine-producing strains.

scholarly journals Pyrimidine nucleotide biosynthesis in Phaseolus aureus. Enzymic aspects of the control of carbamoyl phosphate synthesis and utilization

1972 ◽  
Vol 129 (3) ◽  
pp. 583-593 ◽  
Author(s):  
B. L. Ong ◽  
J. F. Jackson
Keyword(s):  
Inhibitory Effect ◽  
Aspartate Transcarbamoylase ◽  
Synthetase Activity ◽  
Carbamoyl Phosphate Synthetase ◽  
Phaseolus Aureus ◽  
Carbamoyl Phosphate ◽  
Purine Nucleotides ◽  
Nucleotide Biosynthesis ◽  
Pyrimidine Nucleotide ◽  
Ornithine Transcarbamoylase

1. Carbamoyl phosphate synthetase activity of Phaseolus aureus extracts was assayed by coupling it to the catalytic subunit of Escherichia coli aspartate transcarbamoylase and determining the [14C]carbamoylaspartate so formed. The stability of the activity was improved by the addition of ornithine and dimethyl sulphoxide to the extraction medium. 2. The synthetase activity was found to utilize either glutamine or ammonia as amino donor, the Michaelis constants being 0.17±0.03mm and 6.1±1.0mm respectively. N-Acetylglutamate did not significantly alter the rate with either substrate, and azaserine inhibited the reaction with both amino donors to the same extent. 3. Ornithine was shown to stimulate the activity, and to counteract inhibition by UMP. The purine nucleotides IMP and GMP enhanced carbamoyl phosphate formation, whereas AMP had an inhibitory effect. 4. The Michaelis constant for carbamoyl phosphate was determined in concentrated extracts for both aspartate transcarbamoylase and ornithine transcarbamoylase activities, and was 0.13±0.03mm and 1.58±0.16mm respectively. The ratio of the activities of these two enzymes, determined at near-saturating substrate concentrations, was 1:3 (aspartate transcarbamoylase/ornithine transcarbamoylase). 5. It is concluded that in this plant tissue there is one enzyme, carbamoyl phosphate synthetase, supplying carbamoyl phosphate to both the pyrimidine and arginine pathways, that the pyrimidine pathway claims most of the available carbamoyl phosphate (depending on the concentration of the nucleotide effectors) when this intermediate is present at low concentrations; and that when the carbamoyl phosphate concentration is increased, possibly by ornithine stimulation, a larger proportion can be taken up by the arginine pathway.

scholarly journals Substitutions in hamster CAD carbamoyl-phosphate synthetase alter allosteric response to 5-phosphoribosyl-alpha-pyrophosphate (PRPP) and UTP

2004 ◽  
Vol 378 (3) ◽  
pp. 991-998 ◽  
Author(s):  
Christine Q. SIMMONS ◽  
Alan J. SIMMONS ◽  
Aaron HAUBNER ◽  
Amber REAM ◽  
Jeffrey N. DAVIDSON
Keyword(s):  
De Novo ◽  
Binding Pocket ◽  
Tryptophan Residue ◽  
Carbamoyl Phosphate Synthetase ◽  
Carbamoyl Phosphate ◽  
Aspartate Transcarbamylase ◽  
Inosine Monophosphate ◽  
E Coli ◽  
Mammalian Enzyme ◽  
Cad Protein

CPSase (carbamoyl-phosphate synthetase II), a component of CAD protein (multienzymic protein with CPSase, aspartate transcarbamylase and dihydro-orotase activities), catalyses the regulated steps in the de novo synthesis of pyrimidines. Unlike the orthologous Escherichia coli enzyme that is regulated by UMP, inosine monophosphate and ornithine, the mammalian CPSase is allosterically inhibited by UTP, and activated by PRPP (5-phosphoribosyl-α-pyrophosphate) and phosphorylation. Four residues (Thr974, Lys993, Lys954 and Thr977) are critical to the E. coli inosine monophosphate/UMP-binding pocket. In the present study, three of the corresponding residues in the hamster CPSase were altered to determine if they affect either PRPP activation or UTP inhibition. Substitution of the hamster residue, positionally equivalent to Thr974 in the E. coli enzyme, with alanine residue led to an enzyme with 5-fold lower activity and a near loss of PRPP activation. Whereas replacement of the tryptophan residue at position 993 had no effect, an Asp992→Asn substitution yielded a much-activated enzyme that behaved as if PRPP was present. The substitution Lys954→Glu had no effect on PRPP stimulation. Only modest decreases in UTP inhibitions were observed with each of the altered CPSases. The results also show that while PRPP and UTP can act simultaneously, PRPP activation is dominant. Apparently, UTP and PRPP have distinctly different associations within the mammalian enzyme. The findings of the present study may prove relevant to the neuropathology of Lesch–Nyhan syndrome.

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