|Gene:||pyrG||Accession Numbers: EG10810 (MetaCyc), b2780, ECK2774|
Species: Escherichia coli K-12 substr. MG1655
Subunit composition of CTP synthase = [PyrG]4
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, vonderSaal85a, 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].
|Map Position: [2,906,051 <- 2,907,688]|
Molecular Weight of Polypeptide: 60.374 kD (from nucleotide sequence), 60 kD (experimental) [Weng86 ]
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
|Biological Process:||GO:0006241 - CTP biosynthetic process
GO:0006221 - pyrimidine nucleotide biosynthetic process [UniProtGOA11, GOA06, GOA01]
GO:0006541 - glutamine metabolic process [UniProtGOA11, GOA06]
GO:0044210 - 'de novo' CTP biosynthetic process [UniProtGOA12]
|Molecular Function:||GO:0003883 - CTP synthase activity
[GOA06, GOA01a, GOA01, Chakraborty61, Iyengar03]
GO:0042802 - identical protein binding [Robertson95a]
GO:0000166 - nucleotide binding [UniProtGOA11]
GO:0005524 - ATP binding [UniProtGOA11]
GO:0016874 - ligase activity [UniProtGOA11]
|Cellular Component:||GO:0005829 - cytosol [DiazMejia09, Ishihama08, LopezCampistrou05, Lasserre06]|
|MultiFun Terms:||metabolism → biosynthesis of building blocks → nucleotides → pyrimidine ribonucleotide biosynthesis|
|metabolism → central intermediary metabolism → nucleotide and nucleoside conversions|
Enzymatic reaction of: CTP synthase
Synonyms: CTP synthetase, UTP-ammonia ligase, UTP:ammonia ligase (ADP-forming)
EC Number: 184.108.40.206
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
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].
Primary Physiological Regulators of Enzyme Activity: CTP
|Protein-Segment||2 -> 253|
|Chain||2 -> 545|
|Conserved-Region||291 -> 542|
10/20/97 Gene b2780 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG10810; confirmed by SwissProt match.
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
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
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
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
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
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
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
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
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
vonderSaal85a: 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
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