|Gene:||ppsA||Accession Numbers: EG10759 (EcoCyc), b1702, ECK1700|
Subunit composition of
phosphoenolpyruvate synthetase = [PpsA]2
phosphoenolpyruvate synthetase = PpsA
During growth on three-carbon substrates that require the gluconeogenesis pathway (such as lactate or pyruvate), phosphoenolpyruvate (PEP) synthetase provides the ability to generate phosphoenolpyruvate, which is required for the synthesis of precursor metabolites for cellular carbon compounds [Cooper67].
The reaction is thought to proceed in two steps: hydrolysis of ATP, whereby the γ phosphate is released and the β phosphate is transiently attached to a histidine residue within the enzyme, followed by transfer of the phosphate residue to pyruvate, producing PEP [Berman70, Narindrasorasak77].
A regulatory mechanism of the enzymatic activity of PEP synthetase was recently identified. PEP synthetase regulatory protein (PSRP) catalyzes both the Pi-dependent activation and ADP/ATP-dependent inactivation of PEP synthetase. PEP synthetase is protected from inactivation by the presence of pyruvate [Burnell10].
PEP synthetase mutants do not grow on pyruvate, lactate or alanine as the sole source of carbon [Cooper65, Cooper67, Brice67]. The wild-type expression level of ppsA appears to be suboptimal for growth on pyruvate [Chao93]. Expression of ppsA is increased by growth on acetate as the sole source of carbon [Oh00, Oh02, Peng03]. A ppsA mutant shows an increased lag time during the diauxic transition from growth on glucose to growth on acetate [Kao05]. A ppsA pckA double mutant does not grow on acetate [Oh02] or succinate [Goldie80a] as the sole source of carbon.
The effect on carbon flux of overexpressing ppsA in cells grown on glucose has been studied [Patnaik92]. Studies of glucose metabolism in E. coli strains JM109 and BL21 suggested differences in regulation of ppsA and other genes, depending upon glucose supply [Phue05].
Using an antibody that detects protein histidine phosphorylation, it was found that the amount of histidine phosphorylation on PpsA is regulated by nitrogen availability in vivo. In addition, α-ketoglutarate was shown to inhibit phsophotransfer from phosphorylated PpsA to pyruvate [Kee13].
A series of vectors inducibly expressing paired-terminus antisense RNAs was constructed to silence central carbon metabolism in host E. coli K-12 MG1655. A vector that silenced ppsA at 86% efficacy resulted in a defect in carbon catabolite repression [Nakashima14].
A mass spectrometry-based quantitative assay for enzymes involved in central carbon metabolism in E. coli revealed that of 22 enzymes, PpsA was the least abundant protein, present at 491 copies per cell [Trauchessec14].
Ectopic overexpression of ppsA in a glucose-phosphate stressed sgrS mutant rescues this mutant from pyruvate-induced lysis [Richards13]. Due to the precursor role of PEP in the biosynthesis of aromatics and other valuable compounds in E. coli, the amount of available PEP has been manipulated in engineered strains of E. coli by overexpression [Chen14b] or deletion [Sabido14] of several genes including ppsA. Engineering ATP futile cycling by overexpression of ppsA in a high lactate producing strain of E. coli was shown to increase anaerobic lactate production [Hadicke15].
Gene Citations: [Niersbach92]
|Map Position: [1,782,758 <- 1,785,136] (38.42 centisomes, 138°)||Length: 2379 bp / 792 aa|
Molecular Weight of Polypeptide: 87.435 kD (from nucleotide sequence), 84.0 kD (experimental) [Geerse89 ]
Molecular Weight of Multimer: 150.0 kD (experimental) [Narindrasorasak77]
Unification Links: ASAP:ABE-0005678 , CGSC:367 , DIP:DIP-10552N , EchoBASE:EB0752 , EcoGene:EG10759 , EcoliWiki:b1702 , EcoO157Cyc:PPSA , ModBase:P23538 , OU-Microarray:b1702 , PortEco:ppsA , PR:PRO_000023582 , Pride:P23538 , Protein Model Portal:P23538 , RefSeq:NP_416217 , RegulonDB:EG10759 , SMR:P23538 , String:511145.b1702 , UniProt:P23538
Relationship Links: InterPro:IN-FAMILY:IPR000121 , InterPro:IN-FAMILY:IPR002192 , InterPro:IN-FAMILY:IPR006319 , InterPro:IN-FAMILY:IPR008279 , InterPro:IN-FAMILY:IPR013815 , InterPro:IN-FAMILY:IPR013816 , InterPro:IN-FAMILY:IPR015813 , InterPro:IN-FAMILY:IPR018274 , InterPro:IN-FAMILY:IPR023151 , Pfam:IN-FAMILY:PF00391 , Pfam:IN-FAMILY:PF01326 , Pfam:IN-FAMILY:PF02896 , Prosite:IN-FAMILY:PS00370 , Prosite:IN-FAMILY:PS00742
|Biological Process:||GO:0006094 - gluconeogenesis
GO:0006090 - pyruvate metabolic process [GOA01a]
GO:0016310 - phosphorylation [UniProtGOA11a, GOA01a]
|Molecular Function:||GO:0000287 - magnesium ion binding
GO:0008986 - pyruvate, water dikinase activity [GOA01, GOA01a, Cooper67]
GO:0042803 - protein homodimerization activity [Narindrasorasak77]
GO:0000166 - nucleotide binding [UniProtGOA11a]
GO:0003824 - catalytic activity [GOA01a]
GO:0005524 - ATP binding [UniProtGOA11a, GOA01a]
GO:0016301 - kinase activity [UniProtGOA11a, GOA01a]
GO:0016740 - transferase activity [UniProtGOA11a]
GO:0016772 - transferase activity, transferring phosphorus-containing groups [GOA01a]
GO:0046872 - metal ion binding [UniProtGOA11a]
|Cellular Component:||GO:0005829 - cytosol [DiazMejia09, Zhang07b]|
|MultiFun Terms:||metabolism → central intermediary metabolism|
|Growth Medium||Growth?||T (°C)||O2||pH||Osm/L||Growth Observations|
|LB Lennox||Yes||37||Aerobic||7||Yes [Baba06, Comment 1]|
|M9 medium with 1% glycerol||Yes||37||Aerobic||7.2||0.35||Yes [Joyce06, Comment 2]|
|MOPS medium with 0.4% glucose||Yes||37||Aerobic||7.2||0.22||Yes [Baba06, Comment 1] |
Yes [Feist07, Comment 3]
Enzymatic reaction of: phosphoenolpyruvate synthetase
Synonyms: pyruvate, water dikinase, phosphoenolpyruvate synthase, PEP synthase, ATP:pyruvate, water phosphotransferase
EC Number: 22.214.171.124
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 physiologically favored in the direction shown. [Cooper65]
In Pathways: superpathway of hexitol degradation (bacteria) , superpathway of glycolysis and Entner-Doudoroff , superpathway of glycolysis, pyruvate dehydrogenase, TCA, and glyoxylate bypass , gluconeogenesis I , glycolysis II (from fructose 6-phosphate) , glycolysis I (from glucose 6-phosphate)
Only the enzyme from E. coli B has been biochemically characterized [Cook85a, Cooper65, Cooper67, Chulavatnatol73, Chulavatnatol73a, Berman70, Berman70a, Narindrasorasak77, Cooper67a, Berman67]. The sequences of the E. coli B and K-12 enzymes are 99% identical.
Inhibitors (Unknown Mechanism): oxalate [Narindrasorasak78] , 2-oxoglutarate [Chulavatnatol73] , ADP [Chulavatnatol73] , oxaloacetate [Chulavatnatol73] , fluoride [Cooper65, McCormick15] , p-hydroxymercuribenzoate [Cooper69, Berman70] , AMP [Cooper69, Chulavatnatol73] , phosphoenolpyruvate [Cooper69, Chulavatnatol73]
pH(opt) (forward direction): 8.4 [Jakeman98]
pH(opt) (reverse direction): 6.8 [Cooper67a]
|Chain||2 -> 792|
|Sequence-Conflict||194 -> 195|
|Sequence-Conflict||341 -> 360|
10/20/97 Gene b1702 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG10759.
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
Berman70: Berman KM, Cohn M (1970). "Phosphoenolpyruvate synthetase of Escherichia coli. Purification, some properties, and the role of divalent metal ions." J Biol Chem 245(20);5309-18. PMID: 4319237
Berman70a: Berman KM, Cohn M (1970). "Phosphoenolpyruvate synthetase. Partial reactions studied with adenosine triphosphate analogues and the inorganic phosphate-H2 18O exchange reaction." J Biol Chem 245(20);5319-25. PMID: 4319238
Brice67: Brice CB, Kornberg HL (1967). "Location of a gene specifying phosphopyruvate synthase activity on the genome of Escherichia coli, K12." Proc R Soc Lond B Biol Sci 168(12);281-92. PMID: 4383555
Burnell10: Burnell JN (2010). "Cloning and characterization of Escherichia coli DUF299: a bifunctional ADP-dependent kinase - phosphate-dependent pyrophosphorylase from bacteria." BMC Biochem 11(1);1. PMID: 20044937
Chen14b: Chen X, Li M, Zhou L, Shen W, Algasan G, Fan Y, Wang Z (2014). "Metabolic engineering of Escherichia coli for improving shikimate synthesis from glucose." Bioresour Technol 166;64-71. PMID: 24905044
Chulavatnatol73: Chulavatnatol M, Atkinson DE (1973). "Phosphoenolpyruvate synthetase from Escherichia coli. Effects of adenylate energy charge and modifier concentrations." J Biol Chem 248(8);2712-5. PMID: 4572511
Chulavatnatol73a: Chulavatnatol M, Atkinson DE (1973). "Kinetic competition in vitro between phosphoenolpyruvate synthetase and the pyruvate dehydrogenase complex from Escherichia coli." J Biol Chem 248(8);2716-21. PMID: 4572512
Cook85a: Cook AG, Knowles JR (1985). "Phosphoenolpyruvate synthetase and pyruvate, orthophosphate dikinase: stereochemical consequences at both the beta-phospho and gamma-phospho groups of ATP." Biochemistry 24(1);51-8. PMID: 2986676
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
Feist07: Feist AM, Henry CS, Reed JL, Krummenacker M, Joyce AR, Karp PD, Broadbelt LJ, Hatzimanikatis V, Palsson BO (2007). "A genome-scale metabolic reconstruction for Escherichia coli K-12 MG1655 that accounts for 1260 ORFs and thermodynamic information." Mol Syst Biol 3;121. PMID: 17593909
Geerse89: Geerse RH, van der Pluijm J, Postma PW (1989). "The repressor of the PEP:fructose phosphotransferase system is required for the transcription of the pps gene of Escherichia coli." Mol Gen Genet 218(2);348-52. PMID: 2674659
Goldie80a: Goldie AH, Sanwal BD (1980). "Genetic and physiological characterization of Escherichia coli mutants deficient in phosphoenolpyruvate carboxykinase activity." J Bacteriol 141(3);1115-21. PMID: 6988403
Hadicke15: Hadicke O, Bettenbrock K, Klamt S (2015). "Enforced ATP futile cycling increases specific productivity and yield of anaerobic lactate production in Escherichia coli." Biotechnol Bioeng. PMID: 25899755
Joyce06: Joyce AR, Reed JL, White A, Edwards R, Osterman A, Baba T, Mori H, Lesely SA, Palsson BO, Agarwalla S (2006). "Experimental and computational assessment of conditionally essential genes in Escherichia coli." J Bacteriol 188(23);8259-71. PMID: 17012394
McCormick15: McCormick NE, Jakeman DL (2015). "On the mechanism of phosphoenolpyruvate synthetase (PEPs) and its inhibition by sodium fluoride: potential magnesium and aluminum fluoride complexes of phosphoryl transfer." Biochem Cell Biol 93(3);236-40. PMID: 25707819
Nakashima14: Nakashima N, Ohno S, Yoshikawa K, Shimizu H, Tamura T (2014). "A vector library for silencing central carbon metabolism genes with antisense RNAs in Escherichia coli." Appl Environ Microbiol 80(2);564-73. PMID: 24212579
Narindrasorasak77: Narindrasorasak S, Bridger WA (1977). "Phosphoenolpyruvate Synthetase of Escherichia coli. Molecular weight, subunit composition, and identification of phosphohistidine in phosphoenzyme intermediate." J Biol Chem 252(10):3121-3127. PMID: 16880
Narindrasorasak78: Narindrasorasak S, Bridger WA (1978). "Probes of the structure of phosphoenolpyruvate synthetase: effects of a transition state analogue on enzyme conformation." Can J Biochem 56(8);816-9. PMID: 210911
Niersbach92: Niersbach M, Kreuzaler F, Geerse RH, Postma PW, Hirsch HJ (1992). "Cloning and nucleotide sequence of the Escherichia coli K-12 ppsA gene, encoding PEP synthase." Mol Gen Genet 1992;231(2);332-6. PMID: 1310524
Peng03: Peng L, Shimizu K (2003). "Global metabolic regulation analysis for Escherichia coli K12 based on protein expression by 2-dimensional electrophoresis and enzyme activity measurement." Appl Microbiol Biotechnol 61(2);163-78. PMID: 12655459
Phue05: Phue JN, Noronha SB, Hattacharyya R, Wolfe AJ, Shiloach J (2005). "Glucose metabolism at high density growth of E. coli B and E. coli K: differences in metabolic pathways are responsible for efficient glucose utilization in E. coli B as determined by microarrays and Northern blot analyses." Biotechnol Bioeng 90(7);805-20. PMID: 15806547
Richards13: Richards GR, Patel MV, Lloyd CR, Vanderpool CK (2013). "Depletion of glycolytic intermediates plays a key role in glucose-phosphate stress in Escherichia coli." J Bacteriol 195(21);4816-25. PMID: 23995640
Sabido14: Sabido A, Sigala JC, Hernandez-Chavez G, Flores N, Gosset G, Bolivar F (2014). "Physiological and transcriptional characterization of Escherichia coli strains lacking interconversion of phosphoenolpyruvate and pyruvate when glucose and acetate are coutilized." Biotechnol Bioeng 111(6);1150-60. PMID: 24375081
Trauchessec14: Trauchessec M, Jaquinod M, Bonvalot A, Brun V, Bruley C, Ropers D, de Jong H, Garin J, Bestel-Corre G, Ferro M (2014). "Mass spectrometry-based workflow for accurate quantification of Escherichia coli enzymes: how proteomics can play a key role in metabolic engineering." Mol Cell Proteomics 13(4);954-68. PMID: 24482123
Zhang07b: Zhang N, Chen R, Young N, Wishart D, Winter P, Weiner JH, Li L (2007). "Comparison of SDS- and methanol-assisted protein solubilization and digestion methods for Escherichia coli membrane proteome analysis by 2-D LC-MS/MS." Proteomics 7(4);484-93. PMID: 17309111
Negre98: Negre D, Oudot C, Prost JF, Murakami K, Ishihama A, Cozzone AJ, Cortay JC (1998). "FruR-mediated transcriptional activation at the ppsA promoter of Escherichia coli." J Mol Biol 1998;276(2);355-65. PMID: 9512708
Ramseier93: Ramseier TM, Negre D, Cortay JC, Scarabel M, Cozzone AJ, Saier MH (1993). "In vitro binding of the pleiotropic transcriptional regulatory protein, FruR, to the fru, pps, ace, pts and icd operons of Escherichia coli and Salmonella typhimurium." J Mol Biol 234(1);28-44. PMID: 8230205
Sarkar08: Sarkar D, Siddiquee KA, Arauzo-Bravo MJ, Oba T, Shimizu K (2008). "Effect of cra gene knockout together with edd and iclR genes knockout on the metabolism in Escherichia coli." Arch Microbiol 190(5);559-71. PMID: 18648770
Son11: Son YJ, Phue JN, Trinh LB, Lee SJ, Shiloach J (2011). "The role of Cra in regulating acetate excretion and osmotic tolerance in E. coli K-12 and E. coli B at high density growth." Microb Cell Fact 10;52. PMID: 21718532
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