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Escherichia coli K-12 substr. MG1655 Enzyme: chorismate mutase / prephenate dehydratase



Gene: pheA Accession Numbers: EG10707 (EcoCyc), b2599, ECK2596

Synonyms: chorismate mutase-prephenate hydrolyase(decarboxylating), P-protein

Regulation Summary Diagram: ?

Subunit composition of chorismate mutase / prephenate dehydratase = [PheA]2

Summary:
Bifunctional chorismate mutase / prephenate dehydratase (PheA) carries out the shared first step in the parallel biosynthetic pathways for the aromatic amino acids tyrosine and phenylalanine, as well as the second step in phenylalanine biosynthesis. The native enzyme is a dimer of identical subunits each containing a dehydratase active site, a mutase active site and a phenylalanine binding site [Gething77]. L-phenylalanine was shown to feedback inhibit both the chorismate mutase and prephenate dehydratase activities of the enzyme by an allosteric mechanism [Dopheide72].

Early chemical modification studies suggested distinct, or slightly overlapping active sites with a separate phenylalanine binding site [Gething77a, Gething77]. Kinetic studies using radioactive chorismate also suggested two active sites and that prephenate, which is formed from chorismate, dissociates from the mutase site and equilibrates with the bulk medium before combining at the dehydratase site [Duggleby78]. Differential inactivation of the dehydratase and mutase activities by inhibitors also provided evidence that the two activities are catalyzed at separate sites on the enzyme, or at sites that only slightly overlap [Baldwin83]. Evidence was also presented for two cysteine residues at or close to the prephenate dehydratase active site, both of which may be essential for prephenate dehydratase activity [Ma85]. Kinetic and structural analysis of mutants lacking either, or both activities also suggested separability of the two active sites [Baldwin81].

The functional regions of PheA were more definitively mapped by recombinantly expressing, purifying and characterizing polypeptide segments derived from PheA. Chorismate mutase activity was previously localized to residues 1-109 and the crystal structure of this polypeptide was determined [Lee95]. In another study, prephenate dehydratase activity was localized to residues 101-285, and residues 286-386 were found to be crucial for the phenylalanine feedback resistance regulatory activity. Three distinct domains were defined as the mutase domain, the dehydratase domain and the R-domain (regulatory domain). Dimerization of PheA was localized to the mutase domain [Zhang98b]. An engineered chorismate mutase domain linked directly to the regulatory domain was constructed and found to bind phenylalanine. Surprisingly, it exhibited allosteric activation, rather than inhibition [Zhang03h].

Determination of the thermodynamic parameters for transition state analog binding to an engineered, monofunctional chorismate mutase derivative of PheA showed that amino acid residue Glu88 plays an important role in catalysis [Lee98]. To examine the catalytic mechanism of the prephenate dehydratase domain, site directed mutagenesis of selected residues was carried out, followed by biochemical and biophysical characterization of the mutants. This identified residues important for catalysis and identified mutants with enhanced prephenate dehydratase activity [Zhang00a].

Mutations in the E. coli pheA gene that result in enzymes highly resistant to feedback inhibition by high concentrations of phenylalanine have been isolated and characterized [Nelms92]. Such mutants are of interest in the industrial production of phenylalanine, an important amino acid in the food and pharmaceutical industries [BaezViveros04]. Large-scale bioreactor production of phenylalanine using an E. coli K-12-derived strain has been demonstrated [Wahl04].

Bifunctional PheA is considered to be an important model enzyme and has been the subject of computational enzyme design studies. Site-directed mutagenesis of secondary active site residues in the engineered chorismate mutase domain has demonstrated their importance in determining enzymatic activity [Lassila07]. Computational protein design followed by experimental testing allowed identification of active site mutations that enhance catalytic activity and efficiency [Lassila05].

Gene Citations: [Hudson84a]

Locations: cytosol

Map Position: [2,735,767 -> 2,736,927] (58.96 centisomes)
Length: 1161 bp / 386 aa

Molecular Weight of Polypeptide: 43.111 kD (from nucleotide sequence), 40.0 kD (experimental) [Davidson72 ]

Molecular Weight of Multimer: 85.0 kD (experimental) [Davidson72]

Unification Links: ASAP:ABE-0008545 , CGSC:403 , DIP:DIP-36017N , EchoBASE:EB0701 , EcoGene:EG10707 , EcoliWiki:b2599 , Mint:MINT-1248118 , ModBase:P0A9J8 , OU-Microarray:b2599 , PortEco:pheA , PR:PRO_000023528 , Pride:P0A9J8 , Protein Model Portal:P0A9J8 , RefSeq:NP_417090 , RegulonDB:EG10707 , SMR:P0A9J8 , String:511145.b2599 , Swiss-Model:P0A9J8 , UniProt:P0A9J8

Relationship Links: InterPro:IN-FAMILY:IPR001086 , InterPro:IN-FAMILY:IPR002701 , InterPro:IN-FAMILY:IPR008242 , InterPro:IN-FAMILY:IPR010952 , InterPro:IN-FAMILY:IPR018528 , InterPro:IN-FAMILY:IPR020822 , PDB:Structure:1ECM , Pfam:IN-FAMILY:PF00800 , Pfam:IN-FAMILY:PF01817 , Prosite:IN-FAMILY:PS00857 , Prosite:IN-FAMILY:PS00858 , Prosite:IN-FAMILY:PS51168 , Prosite:IN-FAMILY:PS51171 , Prosite:IN-FAMILY:PS51671 , Smart:IN-FAMILY:SM00830

In Paralogous Gene Group: 438 (2 members)

Gene-Reaction Schematic: ?

GO Terms:

Biological Process: GO:0006571 - tyrosine biosynthetic process Inferred from experiment [Baldwin81]
GO:0009094 - L-phenylalanine biosynthetic process Inferred from experiment Inferred by computational analysis [UniProtGOA12, UniProtGOA11a, GOA01a, Baldwin81]
GO:0008152 - metabolic process Inferred by computational analysis [UniProtGOA11a, GOA01a]
GO:0008652 - cellular amino acid biosynthetic process Inferred by computational analysis [UniProtGOA11a]
GO:0009073 - aromatic amino acid family biosynthetic process Inferred by computational analysis [UniProtGOA11a]
GO:0046417 - chorismate metabolic process Inferred by computational analysis [GOA01a]
Molecular Function: GO:0004106 - chorismate mutase activity Inferred from experiment Inferred by computational analysis [GOA01, GOA01a, Zhang98b, Baldwin81]
GO:0004664 - prephenate dehydratase activity Inferred from experiment Inferred by computational analysis [GOA01, GOA01a, Zhang98b, Baldwin81]
GO:0042803 - protein homodimerization activity Inferred from experiment [Davidson72]
GO:0003824 - catalytic activity Inferred by computational analysis [UniProtGOA11a]
GO:0016597 - amino acid binding Inferred by computational analysis [GOA01a]
GO:0016829 - lyase activity Inferred by computational analysis [UniProtGOA11a]
GO:0016853 - isomerase activity Inferred by computational analysis [UniProtGOA11a]
Cellular Component: GO:0005737 - cytoplasm Inferred by computational analysis [UniProtGOA11, UniProtGOA11a, GOA01a]
GO:0005829 - cytosol Inferred by computational analysis [DiazMejia09]

MultiFun Terms: metabolism biosynthesis of building blocks amino acids phenylalanine
metabolism biosynthesis of building blocks amino acids tyrosine

Essentiality data for pheA knockouts: ?

Growth Medium Growth? T (°C) O2 pH Osm/L Growth Observations
LB enriched Yes 37 Aerobic 6.95   Yes [Gerdes03, Comment 1]
LB Lennox Yes 37 Aerobic 7   Yes [Baba06, Comment 2]
M9 medium with 0.4% glucose No 37 Aerobic 7.2 0.27 No [Patrick07, Comment 3]
M9 medium with 1% glycerol No 37 Aerobic 7.2 0.35 No [Joyce06]
MOPS medium with 0.4% glucose Indeterminate 37 Aerobic 7.2 0.22 Yes [Baba06, Comment 2]
No [Feist07, Comment 4]

Credits:
Last-Curated ? 21-Jan-2010 by Fulcher C , SRI International


Enzymatic reaction of: chorismate mutase

EC Number: 5.4.99.5

chorismate <=> prephenate

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the Enzyme Commission system.

The reaction is favored in the direction shown.

In Pathways: superpathway of chorismate metabolism , superpathway of phenylalanine, tyrosine, and tryptophan biosynthesis , phenylalanine biosynthesis I , tyrosine biosynthesis I

Summary:
In this enzyme reaction, chorismate undergoes a Claisen rearrangement to form prephenate (in [Lassila05]).

Inhibitors (Allosteric): L-phenylalanine [Baldwin81, Dopheide72]

Inhibitors (Competitive): citrate [Baldwin83] , prephenate [Gething77a, Duggleby78]

Inhibitors (Unknown Mechanism): 2,4,6-trinitrobenzene sulfonate [Gething77] , (S)-2-hydroxyglutarate [Baldwin83]

Primary Physiological Regulators of Enzyme Activity: L-phenylalanine

Kinetic Parameters:

Substrate
Km (μM)
Citations
chorismate
24.0
[Duggleby78]

pH(opt): 7.3 [Dopheide72]


Enzymatic reaction of: prephenate dehydratase

EC Number: 4.2.1.51

prephenate + H+ <=> 2-oxo-3-phenylpropanoate + CO2 + H2O

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the Enzyme Commission system.

The reaction is favored in the direction shown.

In Pathways: superpathway of chorismate metabolism , superpathway of phenylalanine, tyrosine, and tryptophan biosynthesis , phenylalanine biosynthesis I

Inhibitors (Allosteric): L-phenylalanine [Baldwin81, Dopheide72]

Inhibitors (Competitive): α-oxo-1-carboxy-4-tetrahydrothiopyranpropanoate S-oxide [Bushweller89, Helmward89] , α-oxo-1-carboxy-5,6-dihydrothiopyranpropanoate S-oxide [Helmward89, Bushweller89] , trans-aconitate [Baldwin83] , cis-aconitate [Baldwin83]

Inhibitors (Unknown Mechanism): N-ethylmaleimide [Ma85] , 5,5'-dithio-bis-2-nitrobenzoate [Gething77a] , chorismate [Duggleby78] , tetranitromethane [Gething77]

Primary Physiological Regulators of Enzyme Activity: L-phenylalanine

Kinetic Parameters:

Substrate
Km (μM)
Citations
prephenate
470.0
[Duggleby78]


Sequence Features

Feature Class Location Citations Comment
Conserved-Region 1 -> 92
[UniProt09]
UniProt: Chorismate mutase;
Mutagenesis-Variant 11
[Liu96, UniProt12b]
Alternate sequence: R → K; UniProt: Important decrease in catalytic efficiency and affinity.
Alternate sequence: R → A; UniProt: Important decrease in catalytic efficiency and affinity.
Amino-Acid-Sites-That-Bind 11
[UniProt12b]
UniProt: Substrate.
Mutagenesis-Variant 28
[Liu96, UniProt12b]
Alternate sequence: R → K; UniProt: Important decrease in catalytic efficiency and affinity.
Alternate sequence: R → A; UniProt: Important decrease in catalytic efficiency and affinity.
Amino-Acid-Sites-That-Bind 28
[UniProt12b]
UniProt: Substrate.
Mutagenesis-Variant 39
[Liu96, UniProt12b]
Alternate sequence: K → R; UniProt: Important decrease in catalytic efficiency and affinity.
Alternate sequence: K → Q; UniProt: Important decrease in catalytic efficiency and affinity.
Alternate sequence: K → A; UniProt: Important decrease in catalytic efficiency and affinity.
Amino-Acid-Sites-That-Bind 39
[UniProt12b]
UniProt: Substrate.
Amino-Acid-Sites-That-Bind 48
[UniProt12b]
UniProt: Substrate.
Mutagenesis-Variant 52
[Liu96, UniProt12b]
Alternate sequence: E → Q; UniProt: Important decrease in catalytic efficiency and affinity.
Alternate sequence: E → D; UniProt: Important decrease in catalytic efficiency and affinity.
Alternate sequence: E → A; UniProt: Important decrease in catalytic efficiency and affinity.
Amino-Acid-Sites-That-Bind 52
[UniProt12b]
UniProt: Substrate.
Amino-Acid-Sites-That-Bind 84
[UniProt12b]
UniProt: Substrate.
Mutagenesis-Variant 88
[Liu96, UniProt12b]
Alternate sequence: Q → K; UniProt: Important decrease in catalytic efficiency and affinity.
Alternate sequence: Q → E; UniProt: Important decrease in catalytic efficiency and affinity.
Alternate sequence: Q → A; UniProt: Important decrease in catalytic efficiency and affinity.
Amino-Acid-Sites-That-Bind 88
[UniProt12b]
UniProt: Substrate.
Conserved-Region 105 -> 285
[UniProt09]
UniProt: Prephenate dehydratase;
Amino-Acid-Site 278
[UniProt10a]
UniProt: Essential for prephenate dehydratase activity; Sequence Annotation Type: site; Non-Experimental Qualifier: potential;
Protein-Segment 286 -> 386
[UniProt10a]
UniProt: Regulatory (Phe-binding); Sequence Annotation Type: region of interest;
Conserved-Region 299 -> 376
[UniProt13]
UniProt: ACT.


Gene Local Context (not to scale): ?

Transcription Unit:

Notes:

History:
10/20/97 Gene b2599 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG10707; confirmed by SwissProt match.


References

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

BaezViveros04: Baez-Viveros JL, Osuna J, Hernandez-Chavez G, Soberon X, Bolivar F, Gosset G (2004). "Metabolic engineering and protein directed evolution increase the yield of L-phenylalanine synthesized from glucose in Escherichia coli." Biotechnol Bioeng 87(4);516-24. PMID: 15286989

Baldwin81: Baldwin GS, Davidson BE (1981). "A kinetic and structural comparison of chorismate mutase/prephenate dehydratase from mutant strains of Escherichia coli K 12 defective in the PheA gene." Arch Biochem Biophys 1981;211(1);66-75. PMID: 7030214

Baldwin83: Baldwin GS, Davidson BE (1983). "Kinetic studies on the mechanism of chorismate mutase/prephenate dehydratase from Escherichia coli K12." Biochim Biophys Acta 1983;742(2);374-83. PMID: 6337635

Bushweller89: Bushweller JH, Bartlett PA "Sulfoxide analogues of dihydro- and tetrahydroprephenate as inhibitors of prephenate dehydratase." J Org Chem 1989; 54:2404-2409.

Davidson72: Davidson BE, Blackburn EH, Dopheide TA (1972). "Chorismate mutase-prephenate dehydratase from Escherichia coli K-12. I. Purification, molecular weight, and amino acid composition." J Biol Chem 247(14);4441-6. PMID: 4557843

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

Dopheide72: Dopheide TA, Crewther P, Davidson BE (1972). "Chorismate mutase-prephenate dehydratase from Escherichia coli K-12. II. Kinetic properties." J Biol Chem 247(14);4447-52. PMID: 4261395

Duggleby78: Duggleby RG, Sneddon MK, Morrison JF (1978). "Chorismate mutase-prephenate dehydratase from Escherichia coli: active sites of a bifunctional enzyme." Biochemistry 1978;17(8);1548-54. PMID: 348236

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

Gerdes03: Gerdes SY, Scholle MD, Campbell JW, Balazsi G, Ravasz E, Daugherty MD, Somera AL, Kyrpides NC, Anderson I, Gelfand MS, Bhattacharya A, Kapatral V, D'Souza M, Baev MV, Grechkin Y, Mseeh F, Fonstein MY, Overbeek R, Barabasi AL, Oltvai ZN, Osterman AL (2003). "Experimental determination and system level analysis of essential genes in Escherichia coli MG1655." J Bacteriol 185(19);5673-84. PMID: 13129938

Gething77: Gething MJ, Davidson BE (1977). "Chorismate mutase/prephenate dehydratase from Escherichia coli K12. Effects of chemical modification on the enzymic activities and allosteric inhibition." Eur J Biochem 1977;78(1);111-7. PMID: 334530

Gething77a: Gething MJ, Davidson BE (1977). "Chorismate mutase/prephenate dehydratase from Escherichia coli K12. Modification with 5,5'-dithio-bis(2-nitrobenzoic acid)." Eur J Biochem 1977;78(1);103-10. PMID: 334529

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."

Helmward89: Helmward Z "Handbook of Enzyme Inhibitors. 2nd, revised and enlarged edition." Weinheim, Federal Republic of Germany ; New York, NY, USA , 1989.

Hudson84a: Hudson GS, Davidson BE (1984). "Nucleotide sequence and transcription of the phenylalanine and tyrosine operons of Escherichia coli K12." J Mol Biol 1984;180(4);1023-51. PMID: 6396419

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

Lassila05: Lassila JK, Keeffe JR, Oelschlaeger P, Mayo SL (2005). "Computationally designed variants of Escherichia coli chorismate mutase show altered catalytic activity." Protein Eng Des Sel 18(4);161-3. PMID: 15820980

Lassila07: Lassila JK, Keeffe JR, Kast P, Mayo SL (2007). "Exhaustive mutagenesis of six secondary active-site residues in Escherichia coli chorismate mutase shows the importance of hydrophobic side chains and a helix N-capping position for stability and catalysis." Biochemistry 46(23);6883-91. PMID: 17506527

Lee95: Lee AY, Karplus PA, Ganem B, Clardy J (1995). "Atomic structure of the buried catalytic pocket of Escherichia coli chorismate mutase." J. Am. Chem. Soc., 117 3627-3628.

Lee98: Lee AY, Zhang S, Kongsaeree P, Clardy J, Ganem B, Erickson JW, Xie D (1998). "Thermodynamics of a transition state analogue inhibitor binding to Escherichia coli chorismate mutase: probing the charge state of an active site residue and its role in inhibitor binding and catalysis." Biochemistry 37(25);9052-7. PMID: 9636050

Liu96: Liu D.R., Cload S.T., Pastor R.M., Schultz P.G. (1996). "Analysis of active site residues in Escherichia coli chorismate mutase by site-directed mutagenesis." J. Am. Chem. Soc., Volume 118, page(s) 1789-1790.

Ma85: Ma KH, Davidson BE (1985). "The reactivity of the sulphydryl groups of chorismate mutase/prephenate dehydratase--a bifunctional enzyme of phenylalanine biosynthesis in Escherichia coli K12." Biochim Biophys Acta 1985;827(1);1-7. PMID: 3881132

Nelms92: Nelms J, Edwards RM, Warwick J, Fotheringham I (1992). "Novel mutations in the pheA gene of Escherichia coli K-12 which result in highly feedback inhibition-resistant variants of chorismate mutase/prephenate dehydratase." Appl Environ Microbiol 58(8);2592-8. PMID: 1514806

Patrick07: Patrick WM, Quandt EM, Swartzlander DB, Matsumura I (2007). "Multicopy suppression underpins metabolic evolvability." Mol Biol Evol 24(12);2716-22. PMID: 17884825

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.

UniProt12b: UniProt Consortium (2012). "UniProt version 2012-02 released on 2012-02-29 00:00:00." Database.

UniProt13: UniProt Consortium (2013). "UniProt version 2013-08 released on 2013-08-01 00:00:00." Database.

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

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."

Wahl04: Wahl A, El Massaoudi M, Schipper D, Wiechert W, Takors R (2004). "Serial 13C-based flux analysis of an L-phenylalanine-producing E. coli strain using the sensor reactor." Biotechnol Prog 20(3);706-14. PMID: 15176872

Zhang00a: Zhang S, Wilson DB, Ganem B (2000). "Probing the catalytic mechanism of prephenate dehydratase by site-directed mutagenesis of the Escherichia coli P-protein dehydratase domain." Biochemistry 39(16);4722-8. PMID: 10769128

Zhang03h: Zhang S, Wilson DB, Ganem B (2003). "An engineered chorismate mutase with allosteric regulation." Bioorg Med Chem 11(14);3109-14. PMID: 12818673

Zhang98b: Zhang S, Pohnert G, Kongsaeree P, Wilson DB, Clardy J, Ganem B (1998). "Chorismate mutase-prephenate dehydratase from Escherichia coli. Study of catalytic and regulatory domains using genetically engineered proteins." J Biol Chem 273(11);6248-53. PMID: 9497350


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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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