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Escherichia coli K-12 substr. MG1655 Enzyme: CTP synthase



Gene: pyrG Accession Numbers: EG10810 (EcoCyc), b2780, ECK2774

Regulation Summary Diagram: ?

Subunit composition of CTP synthase = [PyrG]4

Summary:
CTP synthetase catalyzes the glutamine- or ammonia-dependent synthesis of CTP from UTP, the final step in the de novo biosynthesis of CTP. The enzyme is inhibited by its product, CTP [Long67], shows positive cooperativity for its substrates, ATP and UTP [Long67, Levitzki69, Levitzki72a], and is allosterically activated by GTP [Levitzki72].

CTP synthetase contains an N-terminal synthetase domain and a C-terminal glutamine amide transfer (GAT) domain [Weng86]. A G352P mutation specifically inactivates the glutamine amide transfer activity [Weng87]. Site-directed mutagenesis and limited proteolysis of CTP synthetase have allowed identification of residues critical for enzymatic activity and activation [Bearne01, Iyengar03, Simard03, Lunn04, MacLeod06].

The catalytic mechanism and kinetic properties of CTP synthetase have been studied [Anderson83, vonderSaal85, Lewis89]. GTP analogs have been used to study the GTP structural features required for allosteric activation of glutamine hydrolysis, as well as inhibition of glutamine-dependent, or ammonia-dependent CTP formation [Lunn08]. GTP analogs have been used to study the GTP structural features required for allosteric activation of glutamine hydrolysis, as well as inhibition of glutamine-dependent or ammonia-dependent CTP formation [Lunn08]. Inhibition of the enzyme by xanthines, uric acids and their analogs has also been demonstrated [Roy10]. The enzyme is of interest as a drug target.

The native enzyme equilibrates between the monomeric, dimeric and tetrameric forms [Levitzki72a, Long70, Robertson95a]. Site-directed mutagenesis studies have identified Gly142 as critical for nucleotide-dependent formation of the active tetramer [Lunn08a].

Crystal structures of CTP synthetase have been solved at 2.3 and 2.8 Å resolution. The tetrameric structure reveals that essential ATP- and UTP-binding surfaces are contributed by three monomers, providing a structural explanation for the observed cooperativity of the enzyme [Endrizzi04]. A gated channel provides a path for ammonia diffusion between the GAT and synthetase domains, as well as an entry point for exogenous ammonia that overlaps with a predicted GTP-binding site [Endrizzi04]. The structure thus also provides insight into the observed negative cooperativity for glutamine and the effector GTP [Levitzki69] and the observation that high concentrations of GTP inhibit CTP formation, but not glutamine hydrolysis [MacDonnell04]. The location of the CTP binding site suggests a novel strategy for product inhibition [Endrizzi05].

Locations: cytosol

Map Position: [2,906,051 <- 2,907,688] (62.63 centisomes)
Length: 1638 bp / 545 aa

Molecular Weight of Polypeptide: 60.374 kD (from nucleotide sequence), 60 kD (experimental) [Weng86 ]

pI: 6.02

Unification Links: ASAP:ABE-0009112 , CGSC:325 , EchoBASE:EB0803 , EcoGene:EG10810 , EcoliWiki:b2780 , OU-Microarray:b2780 , PortEco:pyrG , PR:PRO_000023662 , Pride:P0A7E5 , Protein Model Portal:P0A7E5 , RefSeq:NP_417260 , RegulonDB:EG10810 , SMR:P0A7E5 , String:511145.b2780 , UniProt:P0A7E5

Relationship Links: InterPro:IN-FAMILY:IPR004468 , InterPro:IN-FAMILY:IPR017456 , InterPro:IN-FAMILY:IPR017926 , InterPro:IN-FAMILY:IPR027417 , Panther:IN-FAMILY:PTHR11550 , PDB:Structure:1S1M , PDB:Structure:2AD5 , Pfam:IN-FAMILY:PF00117 , Pfam:IN-FAMILY:PF06418 , Prosite:IN-FAMILY:PS51273

Gene-Reaction Schematic: ?

Genetic Regulation Schematic: ?

GO Terms:

Biological Process: GO:0006241 - CTP biosynthetic process Inferred from experiment [Iyengar03]
GO:0006221 - pyrimidine nucleotide biosynthetic process Inferred by computational analysis [UniProtGOA11, GOA06, GOA01]
GO:0006541 - glutamine metabolic process Inferred by computational analysis [UniProtGOA11, GOA06]
GO:0044210 - 'de novo' CTP biosynthetic process Inferred by computational analysis [UniProtGOA12]
Molecular Function: GO:0003883 - CTP synthase activity Inferred from experiment Inferred by computational analysis [GOA06, GOA01a, GOA01, Chakraborty61, Iyengar03]
GO:0042802 - identical protein binding Inferred from experiment [Robertson95a]
GO:0000166 - nucleotide binding Inferred by computational analysis [UniProtGOA11]
GO:0005524 - ATP binding Inferred by computational analysis [UniProtGOA11]
GO:0016874 - ligase activity Inferred by computational analysis [UniProtGOA11]
Cellular Component: GO:0005829 - cytosol Inferred from experiment Inferred by computational analysis [DiazMejia09, Ishihama08, LopezCampistrou05, Lasserre06]

MultiFun Terms: metabolism biosynthesis of building blocks nucleotides pyrimidine ribonucleotide biosynthesis
metabolism central intermediary metabolism nucleotide and nucleoside conversions

Essentiality data for pyrG knockouts: ?

Growth Medium Growth? T (°C) O2 pH Osm/L Growth Observations
LB Lennox No 37 Aerobic 7   No [Baba06, Comment 1]

Credits:
Curated 04-Dec-2006 by Keseler I , SRI International
Last-Curated ? 13-Jan-2012 by Fulcher C , SRI International


Enzymatic reaction of: CTP synthase

Synonyms: CTP synthetase, UTP-ammonia ligase, UTP:ammonia ligase (ADP-forming)

EC Number: 6.3.4.2

ATP + UTP + L-glutamine + H2O <=> ADP + CTP + L-glutamate + phosphate + 2 H+

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction of enzyme catalysis.

The reaction is irreversible in the direction shown.

Alternative Substrates for L-glutamine: ammonia

In Pathways: glutamine degradation I , superpathway of histidine, purine, and pyrimidine biosynthesis , superpathway of pyrimidine deoxyribonucleotides de novo biosynthesis , superpathway of pyrimidine ribonucleotides de novo biosynthesis , superpathway of pyrimidine ribonucleosides salvage , superpathway of pyrimidine nucleobases salvage , UTP and CTP de novo biosynthesis

Summary:
Early assays of the enzymatic activity (e.g. [Lieberman56, Chakraborty61, Long67, Levitzki69, Long70, Levitzki72, Levitzki72a]) were performed with enzyme purified from E. coli B.

Kinetic parameters of the enzyme differ depending on the presence or absence of GTP. Parameters measured in the presence of 0.2 mM GTP [Lewis89] are shown here. The presence of GTP has no effect on the ammonia-dependent reaction [Lewis89].

Cofactors or Prosthetic Groups: Mg2+ [Long70]

Activators (Allosteric): GTP [Levitzki72, Comment 2]

Inhibitors (Competitive): 2-thiouridine 5'-triphosphate [Scheit82] , CTP [Long67]

Inhibitors (Irreversible): thiourea dioxide [Robertson92]

Inhibitors (Other): L-glutamate-5-semialdehyde [Bearne01]

Primary Physiological Regulators of Enzyme Activity: CTP

Kinetic Parameters:

Substrate
Km (μM)
kcat (sec-1)
kcat/Km (sec-1 μM-1)
Citations
L-glutamine
170.0
[Lewis89]
L-glutamine
354.0
6.1
[Lunn04, BRENDA14]
ATP
54.0, 116.0
[Lewis89, BRENDA14]
ATP
6.26, 10.8, 12.8
[MacLeod06, BRENDA14]
UTP
27.0, 71.0
[Lewis89, BRENDA14]
UTP
5.0
[Lunn08a, BRENDA14]
UTP
6.9, 13.7, 14.0
[MacLeod06, BRENDA14]

pH(opt): 7.5 [BRENDA14, Zalkin85], 8 [BRENDA14, Robertson93], 10.3 [BRENDA14, Zalkin85]


Sequence Features

Feature Class Location Citations Comment
Cleavage-of-Initial-Methionine 1
[Link97, UniProt11a]
UniProt: Removed.
Protein-Segment 2 -> 253
[UniProt10]
UniProt: Aminator domain; Sequence Annotation Type: region of interest;
Chain 2 -> 545
[UniProt09]
UniProt: CTP synthase;
Acetylation-Modification 239
[Yu08]
 
Conserved-Region 291 -> 542
[UniProt09]
UniProt: Glutamine amidotransferase type-1;
Sequence-Conflict 338
[Weng86, UniProt10a]
Alternate sequence: V → L; UniProt: (in Ref. 1; AAA24485);
Mutagenesis-Variant 349
[Weng87, UniProt11a]
Alternate sequence: V → S; UniProt: 30% increase in both glutamine- dependent and ammonia-dependent activities.
Mutagenesis-Variant 352
[Weng87, UniProt11a]
Alternate sequence: G → P; UniProt: Loss of glutamine-dependent activity, but no change in ammonia- dependent activity.
Mutagenesis-Variant 379
[Bearne01, UniProt11a]
Alternate sequence: C → S; UniProt: Loss of glutamine-dependent activity, but no change in ammonia- dependent activity.
Alternate sequence: C → A; UniProt: Loss of glutamine-dependent activity, but no change in ammonia- dependent activity.
Active-Site 379
[UniProt10]
UniProt: Nucleophile; Non-Experimental Qualifier: probable;
Sequence-Conflict 476
[Weng86, UniProt10a]
Alternate sequence: M → S; UniProt: (in Ref. 1; AAA24485);
Active-Site 515
[UniProt10]
UniProt: Non-Experimental Qualifier: by similarity;
Active-Site 517
[UniProt10]
UniProt: Non-Experimental Qualifier: by similarity;


Gene Local Context (not to scale): ?

Transcription Units:

Notes:

History:
10/20/97 Gene b2780 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG10810; confirmed by SwissProt match.


References

Anderson83: Anderson PM (1983). "CTP synthetase from Escherichia coli: an improved purification procedure and characterization of hysteretic and enzyme concentration effects on kinetic properties." Biochemistry 22(13);3285-92. PMID: 6349684

Baba06: Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H (2006). "Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection." Mol Syst Biol 2;2006.0008. PMID: 16738554

Bearne01: Bearne SL, Hekmat O, Macdonnell JE (2001). "Inhibition of Escherichia coli CTP synthase by glutamate gamma-semialdehyde and the role of the allosteric effector GTP in glutamine hydrolysis." Biochem J 356(Pt 1);223-32. PMID: 11336655

BRENDA14: BRENDA team (2014). "Imported from BRENDA version existing on Aug 2014." http://www.brenda-enzymes.org.

Chakraborty61: Chakraborty KP, Hurlbert RB (1961). "Role of glutamine in the biosynthesis of cytidine nucleotides in Escherichia coli." Biochim Biophys Acta 47;607-9. PMID: 13692169

DiazMejia09: Diaz-Mejia JJ, Babu M, Emili A (2009). "Computational and experimental approaches to chart the Escherichia coli cell-envelope-associated proteome and interactome." FEMS Microbiol Rev 33(1);66-97. PMID: 19054114

Endrizzi04: Endrizzi JA, Kim H, Anderson PM, Baldwin EP (2004). "Crystal structure of Escherichia coli cytidine triphosphate synthetase, a nucleotide-regulated glutamine amidotransferase/ATP-dependent amidoligase fusion protein and homologue of anticancer and antiparasitic drug targets." Biochemistry 43(21);6447-63. PMID: 15157079

Endrizzi05: Endrizzi JA, Kim H, Anderson PM, Baldwin EP (2005). "Mechanisms of product feedback regulation and drug resistance in cytidine triphosphate synthetases from the structure of a CTP-inhibited complex." Biochemistry 44(41);13491-9. PMID: 16216072

GOA01: GOA, DDB, FB, MGI, ZFIN (2001). "Gene Ontology annotation through association of InterPro records with GO terms."

GOA01a: GOA, MGI (2001). "Gene Ontology annotation based on Enzyme Commission mapping." Genomics 74;121-128.

GOA06: GOA, SIB (2006). "Electronic Gene Ontology annotations created by transferring manual GO annotations between orthologous microbial proteins."

Ishihama08: Ishihama Y, Schmidt T, Rappsilber J, Mann M, Hartl FU, Kerner MJ, Frishman D (2008). "Protein abundance profiling of the Escherichia coli cytosol." BMC Genomics 9;102. PMID: 18304323

Iyengar03: Iyengar A, Bearne SL (2003). "Aspartate-107 and leucine-109 facilitate efficient coupling of glutamine hydrolysis to CTP synthesis by Escherichia coli CTP synthase." Biochem J 369(Pt 3);497-507. PMID: 12383057

Koshland74: Koshland DE, Levitzki A "CTP Synthetase and Related Enzymes." Academic Press, New York 1974;3:539-559.

Lasserre06: Lasserre JP, Beyne E, Pyndiah S, Lapaillerie D, Claverol S, Bonneu M (2006). "A complexomic study of Escherichia coli using two-dimensional blue native/SDS polyacrylamide gel electrophoresis." Electrophoresis 27(16);3306-21. PMID: 16858726

Levitzki69: Levitzki A, Koshland DE (1969). "Negative cooperativity in regulatory enzymes." Proc Natl Acad Sci U S A 62(4);1121-8. PMID: 5256410

Levitzki72: Levitzki A, Koshland DE (1972). "Role of an allosteric effector. Guanosine triphosphate activation in cytosine triphosphate synthetase." Biochemistry 11(2);241-6. PMID: 4550559

Levitzki72a: Levitzki A, Koshland DE (1972). "Ligand-induced dimer-to-tetramer transformation in cytosine triphosphate synthetase." Biochemistry 11(2);247-53. PMID: 4550560

Lewis89: Lewis DA, Villafranca JJ (1989). "Investigation of the mechanism of CTP synthetase using rapid quench and isotope partitioning methods." Biochemistry 28(21);8454-9. PMID: 2532543

Lieberman56: Lieberman I (1956). "Enzymatic amination of uridine triphosphate to cytidine triphosphate." J Biol Chem 222(2);765-75. PMID: 13367044

Link97: Link AJ, Robison K, Church GM (1997). "Comparing the predicted and observed properties of proteins encoded in the genome of Escherichia coli K-12." Electrophoresis 18(8);1259-313. PMID: 9298646

Long67: Long CW, Pardee AB (1967). "Cytidine triphosphate synthetase of Escherichia coli B. I. Purification and kinetics." J Biol Chem 242(20);4715-21. PMID: 4862983

Long70: Long CW, Levitzki A, Koshland DE (1970). "The subunit structure and subunit interactions of cytidine triphosphate synthetase." J Biol Chem 245(1);80-7. PMID: 5411547

LopezCampistrou05: Lopez-Campistrous A, Semchuk P, Burke L, Palmer-Stone T, Brokx SJ, Broderick G, Bottorff D, Bolch S, Weiner JH, Ellison MJ (2005). "Localization, annotation, and comparison of the Escherichia coli K-12 proteome under two states of growth." Mol Cell Proteomics 4(8);1205-9. PMID: 15911532

Lunn04: Lunn FA, Bearne SL (2004). "Alternative substrates for wild-type and L109A E. coli CTP synthases: kinetic evidence for a constricted ammonia tunnel." Eur J Biochem 271(21);4204-12. PMID: 15511226

Lunn08: Lunn FA, MacDonnell JE, Bearne SL (2008). "Structural requirements for the activation of Escherichia coli CTP synthase by the allosteric effector GTP are stringent, but requirements for inhibition are lax." J Biol Chem 283(4);2010-20. PMID: 18003612

Lunn08a: Lunn FA, Macleod TJ, Bearne SL (2008). "Mutational analysis of conserved glycine residues 142, 143 and 146 reveals Gly(142) is critical for tetramerization of CTP synthase from Escherichia coli." Biochem J 412(1);113-21. PMID: 18260824

MacDonnell04: MacDonnell JE, Lunn FA, Bearne SL (2004). "Inhibition of E. coli CTP synthase by the "positive" allosteric effector GTP." Biochim Biophys Acta 1699(1-2);213-20. PMID: 15158730

MacLeod06: MacLeod TJ, Lunn FA, Bearne SL (2006). "The role of lysine residues 297 and 306 in nucleoside triphosphate regulation of E. coli CTP synthase: inactivation by 2',3'-dialdehyde ATP and mutational analyses." Biochim Biophys Acta 1764(2);199-210. PMID: 16427816

Robertson92: Robertson JG, Sparvero LJ, Villafranca JJ (1992). "Inactivation and covalent modification of CTP synthetase by thiourea dioxide." Protein Sci 1(10);1298-307. PMID: 1303749

Robertson93: Robertson JG, Villafranca JJ (1993). "Characterization of metal ion activation and inhibition of CTP synthetase." Biochemistry 32(14);3769-77. PMID: 8385490

Robertson95a: Robertson JG (1995). "Determination of subunit dissociation constants in native and inactivated CTP synthetase by sedimentation equilibrium." Biochemistry 34(22);7533-41. PMID: 7779798

Roy10: Roy AC, Lunn FA, Bearne SL (2010). "Inhibition of CTP synthase from Escherichia coli by xanthines and uric acids." Bioorg Med Chem Lett 20(1);141-4. PMID: 20004571

Scheit82: Scheit KH, Linke HJ (1982). "Substrate specificity of CTP synthetase from Escherichia coli." Eur J Biochem 126(1);57-60. PMID: 6751817

Simard03: Simard D, Hewitt KA, Lunn F, Iyengar A, Bearne SL (2003). "Limited proteolysis of Escherichia coli cytidine 5'-triphosphate synthase. Identification of residues required for CTP formation and GTP-dependent activation of glutamine hydrolysis." Eur J Biochem 270(10);2195-206. PMID: 12752439

UniProt09: UniProt Consortium (2009). "UniProt version 15.8 released on 2009-10-01 00:00:00." Database.

UniProt10: UniProt Consortium (2010). "UniProt version 2010-07 released on 2010-06-15 00:00:00." Database.

UniProt10a: UniProt Consortium (2010). "UniProt version 2010-11 released on 2010-11-02 00:00:00." Database.

UniProt11a: UniProt Consortium (2011). "UniProt version 2011-06 released on 2011-06-30 00:00:00." Database.

UniProtGOA11: UniProt-GOA (2011). "Gene Ontology annotation based on manual assignment of UniProtKB keywords in UniProtKB/Swiss-Prot entries."

UniProtGOA12: UniProt-GOA (2012). "Gene Ontology annotation based on UniPathway vocabulary mapping."

vonderSaal85: von der Saal W, Anderson PM, Villafranca JJ (1985). "Mechanistic investigations of Escherichia coli cytidine-5'-triphosphate synthetase. Detection of an intermediate by positional isotope exchange experiments." J Biol Chem 260(28);14993-7. PMID: 2933396

Weng86: Weng M, Makaroff CA, Zalkin H (1986). "Nucleotide sequence of Escherichia coli pyrG encoding CTP synthetase." J Biol Chem 1986;261(12);5568-74. PMID: 3514618

Weng87: Weng ML, Zalkin H (1987). "Structural role for a conserved region in the CTP synthetase glutamine amide transfer domain." J Bacteriol 169(7);3023-8. PMID: 3298209

Yu08: Yu BJ, Kim JA, Moon JH, Ryu SE, Pan JG (2008). "The diversity of lysine-acetylated proteins in Escherichia coli." J Microbiol Biotechnol 18(9);1529-36. PMID: 18852508

Zalkin85: Zalkin H (1985). "CTP synthetase." Methods Enzymol 113;282-7. PMID: 3911002

Other References Related to Gene Regulation

MendozaVargas09: Mendoza-Vargas A, Olvera L, Olvera M, Grande R, Vega-Alvarado L, Taboada B, Jimenez-Jacinto V, Salgado H, Juarez K, Contreras-Moreira B, Huerta AM, Collado-Vides J, Morett E (2009). "Genome-wide identification of transcription start sites, promoters and transcription factor binding sites in E. coli." PLoS One 4(10);e7526. PMID: 19838305


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Please cite the following article in publications resulting from the use of EcoCyc: Nucleic Acids Research 41:D605-12 2013
Page generated by SRI International Pathway Tools version 18.5 on Mon Nov 24, 2014, BIOCYC13B.