Escherichia coli K-12 substr. MG1655 Enzyme: phosphoenolpyruvate synthetase

Gene: ppsA Accession Numbers: EG10759 (EcoCyc), b1702, ECK1700

Synonyms: pps

Regulation Summary Diagram: ?

Regulation summary diagram for ppsA

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 [Cooper67a].

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, Cooper67a, 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 [Kao05b]. A ppsA pckA double mutant does not grow on acetate [Oh02] or succinate [Goldie80] 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 [Chen14] 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]

Locations: cytosol

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]

pI: 5.17

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

In Paralogous Gene Group: 336 (3 members) , 337 (3 members)

Gene-Reaction Schematic: ?

Gene-Reaction Schematic

Genetic Regulation Schematic: ?

Genetic regulation schematic for ppsA

GO Terms:

Biological Process: GO:0006094 - gluconeogenesis Inferred from experiment Inferred by computational analysis [UniProtGOA12, Brice67]
GO:0006090 - pyruvate metabolic process Inferred by computational analysis [GOA01a]
GO:0016310 - phosphorylation Inferred by computational analysis [UniProtGOA11a, GOA01a]
Molecular Function: GO:0000287 - magnesium ion binding Inferred from experiment [Cooper67a]
GO:0008986 - pyruvate, water dikinase activity Inferred from experiment Inferred by computational analysis [GOA01, GOA01a, Cooper67a]
GO:0042803 - protein homodimerization activity Inferred from experiment [Narindrasorasak77]
GO:0000166 - nucleotide binding Inferred by computational analysis [UniProtGOA11a]
GO:0003824 - catalytic activity Inferred by computational analysis [GOA01a]
GO:0005524 - ATP binding Inferred by computational analysis [UniProtGOA11a, GOA01a]
GO:0016301 - kinase activity Inferred by computational analysis [UniProtGOA11a, GOA01a]
GO:0016740 - transferase activity Inferred by computational analysis [UniProtGOA11a]
GO:0016772 - transferase activity, transferring phosphorus-containing groups Inferred by computational analysis [GOA01a]
GO:0046872 - metal ion binding Inferred by computational analysis [UniProtGOA11a]
Cellular Component: GO:0005829 - cytosol Inferred from experiment Inferred by computational analysis [DiazMejia09, Zhang07]

MultiFun Terms: metabolism central intermediary metabolism

Essentiality data for ppsA knockouts: ?

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]

Curated 14-Jan-2010 by Keseler I , SRI International
Last-Curated ? 28-May-2015 by Fulcher C , SRI International

Enzymatic reaction of: phosphoenolpyruvate synthetase

Synonyms: pyruvate, water dikinase, phosphoenolpyruvate synthase, PEP synthase, ATP:pyruvate, water phosphotransferase

EC Number:

pyruvate + ATP + H2O <=> phosphoenolpyruvate + AMP + 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 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 [Cook85, Cooper65, Cooper67a, Chulavatnatol73, Chulavatnatol73a, Berman70, Berman70a, Narindrasorasak77, Cooper67, Berman67]. The sequences of the E. coli B and K-12 enzymes are 99% identical.

The pH optimum in vitro is 8.4 in the PEP forming direction. At pH 6.8 pyruvate is formed [Cooper67, Jakeman98]. The enzyme utilized 3-fluoropyruvate, but at a much slower rate [Jakeman98].

Cofactors or Prosthetic Groups: Mg2+ [Berman70, Cooper67a, Cooper65]

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]

Kinetic Parameters:

Km (μM)
[Berman70, BRENDA14]
[Berman70, BRENDA14]

pH(opt) (forward direction): 8.4 [Jakeman98]

pH(opt) (reverse direction): 6.8 [Cooper67]

Sequence Features

Protein sequence of phosphoenolpyruvate synthetase with features indicated

Feature Class Location Citations Comment
Cleavage-of-Initial-Methionine 1
[Niersbach92, UniProt11]
UniProt: Removed.
Chain 2 -> 792
UniProt: Phosphoenolpyruvate synthase;
Sequence-Conflict 194 -> 195
[Holzschu91, UniProt15]
UniProt: (in Ref. 1; AAA24319).
Sequence-Conflict 341 -> 360
[Holzschu91, UniProt15]
UniProt: (in Ref. 1; AAA24319).
Active-Site 421
UniProt: Tele-phosphohistidine intermediate; Non-Experimental Qualifier: by similarity;
Amino-Acid-Sites-That-Bind 511
UniProt: Substrate; Non-Experimental Qualifier: by similarity;
Amino-Acid-Sites-That-Bind 578
UniProt: Substrate; Non-Experimental Qualifier: by similarity;
Metal-Binding-Site 680
UniProt: Magnesium; Non-Experimental Qualifier: by similarity;
Amino-Acid-Sites-That-Bind 701
UniProt: Substrate; via carbonyl oxygen; Non-Experimental Qualifier: by similarity;
Amino-Acid-Sites-That-Bind 702
UniProt: Substrate; via amide nitrogen; Non-Experimental Qualifier: by similarity;
Amino-Acid-Sites-That-Bind 703
UniProt: Substrate; Non-Experimental Qualifier: by similarity;
Metal-Binding-Site 704
UniProt: Magnesium; Non-Experimental Qualifier: by similarity;
Active-Site 751
UniProt: Proton donor; Non-Experimental Qualifier: by similarity;

Gene Local Context (not to scale): ?

Gene local context diagram

Transcription Unit:

Transcription-unit diagram


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

Berman67: Berman K, Itada N, Cohn M (1967). "On the mechanism of ATP cleavage in the phosphoenolpyruvate synthase reaction of Escherichia coli." Biochim Biophys Acta 141(1);214-6. PMID: 4860971

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

BRENDA14: BRENDA team (2014). "Imported from BRENDA version existing on Aug 2014."

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

Chao93: Chao YP, Patnaik R, Roof WD, Young RF, Liao JC (1993). "Control of gluconeogenic growth by pps and pck in Escherichia coli." J Bacteriol 175(21);6939-44. PMID: 8226637

Chen14: 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

Cook85: 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

Cooper65: Cooper RA, Kornberg HL (1965). "Net formation of phosphoenolpyruvate from pyruvate by Escherichia coli." Biochim Biophys Acta 104(2);618-20. PMID: 5322808

Cooper67: Cooper RA, Kornberg HL (1967). "The mechanism of the phosphoenolpyruvate synthase reaction." Biochim Biophys Acta 141(1);211-3. PMID: 4293109

Cooper67a: Cooper RA, Kornberg HL (1967). "The direct synthesis of phosphoenolpyruvate from pyruvate by Escherichia coli." Proc R Soc Lond B Biol Sci 168(12);263-80. PMID: 4383554

Cooper69: Cooper RA, Kornberg HL "Phosphoenolpyruvate Synthetase." Methods Enzymol. 1969;13:309-314.

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

GOA01: GOA, MGI (2001). "Gene Ontology annotation based on Enzyme Commission mapping." Genomics 74;121-128.

GOA01a: GOA, DDB, FB, MGI, ZFIN (2001). "Gene Ontology annotation through association of InterPro records with GO terms."

Goldie80: 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

Holzschu91: Holzschu D.L., McElver J.A., Liao C.C., Berry A. (1991). "The cloning and sequence of the E. coli pps gene." Data submission to EMBL/GenBank/DDBJ databases on 1991-06.

Jakeman98: Jakeman DL, Evans JNS (1998). "Overexpression, Purification, and Use of Phosphoenol Pyruvate Synthetase in the Synthesis of PEP Analogues." Bioorganic Chemistry 26(5);245-253.

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

Kao05b: Kao KC, Tran LM, Liao JC (2005). "A global regulatory role of gluconeogenic genes in Escherichia coli revealed by transcriptome network analysis." J Biol Chem 280(43);36079-87. PMID: 16141204

Kee13: Kee JM, Oslund RC, Perlman DH, Muir TW (2013). "A pan-specific antibody for direct detection of protein histidine phosphorylation." Nat Chem Biol 9(7);416-21. PMID: 23708076

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

Oh00: Oh MK, Liao JC (2000). "Gene expression profiling by DNA microarrays and metabolic fluxes in Escherichia coli." Biotechnol Prog 16(2);278-86. PMID: 10753455

Oh02: Oh MK, Rohlin L, Kao KC, Liao JC (2002). "Global expression profiling of acetate-grown Escherichia coli." J Biol Chem 277(15);13175-83. PMID: 11815613

Patnaik92: Patnaik R, Roof WD, Young RF, Liao JC (1992). "Stimulation of glucose catabolism in Escherichia coli by a potential futile cycle." J Bacteriol 174(23);7527-32. PMID: 1332936

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

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

UniProt10a: UniProt Consortium (2010). "UniProt version 2010-07 released on 2010-06-15 00:00:00." Database.

UniProt11: UniProt Consortium (2011). "UniProt version 2011-06 released on 2011-06-30 00:00:00." Database.

UniProt15: UniProt Consortium (2015). "UniProt version 2015-01 released on 2015-01-16 00:00:00." Database.

UniProtGOA11a: 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."

Zhang07: 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

Other References Related to Gene Regulation

Marzan13: Marzan LW, Hasan CM, Shimizu K (2013). "Effect of acidic condition on the metabolic regulation of Escherichia coli and its phoB mutant." Arch Microbiol 195(3);161-71. PMID: 23274360

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

Shimada11: Shimada T, Yamamoto K, Ishihama A (2011). "Novel Members of the Cra Regulon Involved in Carbon Metabolism in Escherichia coli." J Bacteriol 193(3);649-59. PMID: 21115656

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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Please cite the following article in publications resulting from the use of EcoCyc: Nucleic Acids Research 41:D605-12 2013
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