scholarly journals UMP inhibition and sequential firing in aspartate transcarbamoylase open ways to regulate plant growth

2020 ◽  
Author(s):  
Leo Bellin ◽  
Francisco del Caño-Ochoa ◽  
Adrián Velázquez-Campoy ◽  
Torsten Möhlmann ◽  
Santiago Ramón-Maiques
Keyword(s):  
Plant Growth ◽  
Active Sites ◽  
De Novo ◽  
Feedback Inhibition ◽  
Single Point ◽  
Aspartate Transcarbamoylase ◽  
Single Point Mutation ◽  
Carbamoyl Phosphate ◽  
Pyrimidine Nucleotides ◽  
Sequential Firing

ABSTRACTPyrimidine nucleotides are essential to plant development. We proved that Arabidopsis growth can be inhibited or enhanced by down- or upregulating aspartate transcarbamoylase (ATC), the first committed enzyme for de novo biosynthesis of pyrimidines in plants. To understand the unique mechanism of feedback inhibition of this enzyme by uridine 5-monophosphate (UMP), we determined the crystal structure of the Arabidopsis ATC trimer free and bound to UMP, demonstrating that the nucleotide binds and blocks the active site. The regulatory mechanism relies on a loop exclusively conserved in plants, and a single-point mutation (F161A) turns ATC insensitive to UMP. Moreover, the structures in complex with a transition-state analog or with carbamoyl phosphate proved a mechanism in plant ATCs for sequential firing of the active sites. The disclosure of the unique regulatory and catalytic properties suggests new strategies to modulate ATC activity and to control de novo pyrimidine synthesis and plant growth.

scholarly journals Mechanisms of feedback inhibition and sequential firing of active sites in plant aspartate transcarbamoylase

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Leo Bellin ◽  
Francisco Del Caño-Ochoa ◽  
Adrián Velázquez-Campoy ◽  
Torsten Möhlmann ◽  
Santiago Ramón-Maiques
Keyword(s):  
Plant Growth ◽  
Active Sites ◽  
De Novo ◽  
Feedback Inhibition ◽  
Aspartate Transcarbamoylase ◽  
Transition State Analog ◽  
Pyrimidine Synthesis ◽  
De Novo Pyrimidine Synthesis ◽  
Essential Enzyme ◽  
Sequential Firing

AbstractAspartate transcarbamoylase (ATC), an essential enzyme for de novo pyrimidine biosynthesis, is uniquely regulated in plants by feedback inhibition of uridine 5-monophosphate (UMP). Despite its importance in plant growth, the structure of this UMP-controlled ATC and the regulatory mechanism remain unknown. Here, we report the crystal structures of Arabidopsis ATC trimer free and bound to UMP, complexed to a transition-state analog or bearing a mutation that turns the enzyme insensitive to UMP. We found that UMP binds and blocks the ATC active site, directly competing with the binding of the substrates. We also prove that UMP recognition relies on a loop exclusively conserved in plants that is also responsible for the sequential firing of the active sites. In this work, we describe unique regulatory and catalytic properties of plant ATCs that could be exploited to modulate de novo pyrimidine synthesis and plant growth.

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 Repression of the pyr Operon in Lactobacillus plantarum Prevents Its Ability To Grow at Low Carbon Dioxide Levels

2005 ◽  
Vol 187 (6) ◽  
pp. 2093-2104 ◽  
Author(s):  
Hervé Nicoloff ◽  
Aram Elagöz ◽  
Florence Arsène-Ploetze ◽  
Benoît Kammerer ◽  
Jan Martinussen ◽  
...  
Keyword(s):  
Carbon Dioxide ◽  
Lactobacillus Plantarum ◽  
De Novo ◽  
Site Directed Mutagenesis ◽  
Low Carbon ◽  
Wild Type ◽  
Carbamoyl Phosphate ◽  
Phosphoribosyl Pyrophosphate ◽  
Pyrimidine Nucleotides ◽  
Transcription Attenuation

ABSTRACT Carbamoyl phosphate is a precursor for both arginine and pyrimidine biosynthesis. In Lactobacillus plantarum, carbamoyl phosphate is synthesized from glutamine, ATP, and carbon dioxide by two sets of identified genes encoding carbamoyl phosphate synthase (CPS). The expression of the carAB operon (encoding CPS-A) responds to arginine availability, whereas pyrAaAb (encoding CPS-P) is part of the pyrR1BCAaAbDFE operon coding for the de novo pyrimidine pathway repressed by exogenous uracil. The pyr operon is regulated by transcription attenuation mediated by a trans-acting repressor that binds to the pyr mRNA attenuation site in response to intracellular UMP/phosphoribosyl pyrophosphate pools. Intracellular pyrimidine triphosphate nucleoside pools were lower in mutant FB335 (carAB deletion) harboring only CPS-P than in the wild-type strain harboring both CPS-A and CPS-P. Thus, CPS-P activity is the limiting step in pyrimidine synthesis. FB335 is unable to grow in the presence of uracil due to a lack of sufficient carbamoyl phosphate required for arginine biosynthesis. Forty independent spontaneous FB335-derived mutants that have lost regulation of the pyr operon were readily obtained by their ability to grow in the presence of uracil and absence of arginine; 26 harbored mutations in the pyrR1-pyrB loci. One was a prototroph with a deletion of both pyrR1 and the transcription attenuation site that resulted in large amounts of excreted pyrimidine nucleotides and increased intracellular UTP and CTP pools compared to wild-type levels. Low pyrimidine-independent expression of the pyr operon was obtained by antiterminator site-directed mutagenesis. The resulting AE1023 strain had reduced UTP and CTP pools and had the phenotype of a high-CO2-requiring auxotroph, since it was able to synthesize sufficient arginine and pyrimidines only in CO2-enriched air. Therefore, growth inhibition without CO2 enrichment may be due to low carbamoyl phosphate pools from lack of CPS activity.

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.

Physiological consequences of mutation for ALS-inhibitor resistance

Weed Science ◽  
1999 ◽  
Vol 47 (4) ◽  
pp. 383-392 ◽  
Author(s):  
Charlotte V. Eberlein ◽  
Mary J. Guttieri ◽  
Philip H. Berger ◽  
John K. Fellman ◽  
Carol A. Mallory-Smith ◽  
...  
Keyword(s):  
Specific Activity ◽  
Feedback Inhibition ◽  
Sequence Data ◽  
Single Point ◽  
Acetolactate Synthase ◽  
Acid Synthesis ◽  
Single Point Mutation ◽  
Resistance Allele ◽  
Amino Acid Synthesis ◽  
Lactuca Serriola

Biochemical and physiological effects of target site resistance to herbicides inhibiting acetolactate synthase (ALS) were evaluated using sulfonylurea-resistant (R) and -susceptible (S) near isonuclearLactuca sativa‘Bibb’ lines derived by backcrossing the resistance allele fromLactuca serriolaL. intoL. sativa.Sequence data suggest that resistance inL. sativais conferred by a single-point mutation that encodes a proline197to histidine substitution in Domain A of the ALS protein; this is the same substitution observed in RL. serriola. Kmapp(pyruvate) values for ALS isolated from R and SL. sativawere 7.3 and 11.1 mM, respectively, suggesting that the resistance allele did not alter the pyruvate binding domain on the ALS enzyme. Both R and S ALS had greater affinity for 2-oxobutyrate than for pyruvate at the second substrate site. Ratios of acetohydroxybutyrate: acetolactate produced by R ALS across a range of 2-oxobutyrate concentrations were similar to acetohydroxybutyrate: acetolactate ratios produced by S ALS. Specific activity of ALS from RL. sativawas 46% of the specific activity from SL. sativa, suggesting that the resistance allele has detrimental effects on enzyme function, expression, or stability. ALS activity from R plants was less sensitive to feedback inhibition by valine, leucine, and isoleucine than ALS from S plants. Valine, leucine, and isoleucine concentrations were about 1.5 times higher in R seed than in S seed on a per gram of seed basis, and concentrations of valine and leucine were 1.3 and 1.6 times higher, respectively, in R leaves than in S leaves. Findings suggest that the mutation for resistance results in altered regulation of branched-chain amino acid synthesis.

Structure and function of aspartate transcarbamoylase studied using chymotrypsin as a probe

1978 ◽  
Vol 56 (6) ◽  
pp. 654-658 ◽  
Author(s):  
William W.-C. Chan ◽  
Caroline A. Enns
Keyword(s):  
Active Sites ◽  
Sodium Dodecyl ◽  
Peptide Bond ◽  
Structure And Function ◽  
Catalytic Subunit ◽  
Native Enzyme ◽  
Aspartate Transcarbamoylase ◽  
Carbamoyl Phosphate ◽  
And Function ◽  
Extended Exposure

Aspartate transcarbamoylase from Escherichia coli is composed of six catalytic (c) and six regulatory (r) polypeptides. We have studied the structure and function of this enzyme using chymotrypsin as a probe. The protease inactivates the isolated catalytic subunit (c3) but has no effects on the native enzyme (c6r6). Under identical conditions, the c3r6 complex is inactivated at a much slower rate than c3. The presence of the substrate analogue succinate together with carbamoyl phosphate reduces substantially the rate of inactivation. Extended exposure to chymotrypsin converts the catalytic subunit into a partially active derivative with a fourfold higher Michaelis constant. This derivative is indistinguishable from the unmodified catalytic subunit in gel electrophoresis under nondenaturing conditions. However, in the presence of sodium dodecyl sulfate, the major fragment in the electropherogram is smaller than that of the intact catalytic polypeptide. The results could be explained by postulating the presence of a chymotrypsin-sensitive peptide bond at or near the active site. Since X-ray crystallographic studies have indicated that the active sites are located in a central cavity, the resistance of the native enzyme towards inactivation may be due to the inability of chymotrypsin to enter this cavity.

scholarly journals Specificity of molecular recognition in oligomerization of bacterial L-asparaginases

2012 ◽  
Vol 58 (1) ◽  
pp. 50-64 ◽  
Author(s):  
Yu.V. Mezentsev ◽  
A.A. Molnar ◽  
N.N. Sokolov ◽  
V.B. Lisitsina ◽  
A.S. Ivanov ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Molecular Recognition ◽  
Point Mutation ◽  
Active Sites ◽  
Single Point ◽  
Single Point Mutation ◽  
High Avidity ◽  
Salt Bridges ◽  
Antitumor Therapy ◽  
Tetrameric Complex

Bacterial L-asparaginases, which are widely used in the antitumor therapy, act only as homotetramers, because their active sites are located at the interface between the subunits of the enzyme. Since salt bridges substantially stabilize L-asparaginase tetramers, we have supposed that oligomerization of bacterial L-asparaginase is a high-avidity process. This assumption was proved by bioinformatic and biosensoric methods. It was shown, that a stable tetrameric complex can be formed only by the subunits of the same L-asparaginase. Using two mutants of L-asparaginase Helicobacter pylori it was shown that specificity of molecular recognition is significantly reduced even by single point mutation at the interface of high-homologous closely-related subunits.

scholarly journals Recurrent de novo single point mutation on the gene encoding Na+/K+ pump results in epilepsy

2021 ◽  
Author(s):  
Hong-Ming Li ◽  
Wen-Bao Hu ◽  
Chun-Gu Hong ◽  
Ran Duan ◽  
Meng-Lu Chen ◽  
...  
Keyword(s):  
Mouse Model ◽  
Point Mutation ◽  
De Novo ◽  
Memory Function ◽  
Single Point ◽  
Idiopathic Epilepsy ◽  
Single Point Mutation ◽  
Gene Encoding ◽  
Binding Function ◽  
Potassium Binding

AbstractThe etiology of epilepsy remains undefined in two-thirds of patients. Here, we identified a de novo mutation of ATP1A2 (c.2426 T>G, p.Leu809Arg), which encodes the α2 subunit of Na+/K+-ATPase, from a family with idiopathic epilepsy. This mutation caused seizures in the study patients. We generated the point mutation mouse model Atp1a2L809R, which recapitulated the epilepsy observed in the study patients. In Atp1a2L809R/WT mice, convulsions were observed and cognitive and memory function was impaired. This mutation affected the potassium binding function of the protein, disabling its ion transport ability, thereby increasing the frequency of nerve impulses. Our work revealed that ATP1A2L809R mutations cause a predisposition to epilepsy. Moreover, we first provide a point mutation mouse model for epilepsy research and drug screening.

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 The effect of glutamine concentration on the activity of carbamoyl-phosphate synthase II and on the incorporation of [3H]thymidine into DNA in rat mesenteric lymphocytes stimulated by phytohaemagglutinin

1989 ◽  
Vol 261 (3) ◽  
pp. 979-983 ◽  
Author(s):  
Z Szondy ◽  
E A Newsholme
Keyword(s):  
De Novo ◽  
Glutamine Concentration ◽  
Carbamoyl Phosphate ◽  
Purine Nucleotides ◽  
Pyrimidine Nucleotides ◽  
Nucleotide Synthesis ◽  
Pyrimidine Synthesis ◽  
Pyrimidine Nucleotide ◽  
Formation De Novo ◽  
Phosphate Synthase

The maximum catalytic activities of carbamoyl-phosphate synthase II, a limiting enzyme for pyrimidine nucleotide synthesis, are very much less than those of glutaminase, a limiting enzyme for glutamine utilization, in lymphocytes and macrophages; and the flux through the pathway for pyrimidine formation de novo is only about 0.4% of the rate of glutamine utilization by lymphocytes. The Km of synthase II for glutamine is about 16 microM and the concentration of glutamine necessary to stimulate lymphocyte proliferation half-maximally is about 21 microM. This agreement suggests that the importance of glutamine for these cells is provision of nitrogen for biosynthesis of pyrimidine nucleotides (and probably purine nucleotides). However, the glutamine concentration necessary for half-maximal stimulation of glutamine utilization (glutaminolysis) by the lymphocytes is 2.5 mM. The fact that the rate of glutamine utilization by lymphocytes is markedly in excess of the rate of the pathway for pyrimidine nucleotide synthesis de novo and that the Km and ‘half-maximal concentration’ values are so different, suggests that the glutaminolytic pathway is independent of the use of glutamine nitrogen for pyrimidine synthesis.

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