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Escherichia coli K-12 substr. MG1655 Enzyme: 2,3-dihydroxybenzoate-AMP ligase

Gene: entE Accession Numbers: EG10263 (EcoCyc), b0594, ECK0587

Synonyms: enterobactin synthetase component E, enterochelin synthase E, enterobactin synthase component E

Regulation Summary Diagram: ?

Component of: enterobactin synthase (extended summary available)

Subunit composition of 2,3-dihydroxybenzoate-AMP ligase = [EntE]2

EntE is an enzyme of the enterobactin biosynthesis pathway that catalyzes the ATP-dependent condensation of 2,3-dihydroxybenzoate (DHB) and EntB isochorismatase / aryl-carrier protein (holo-EntB) to form the covalently arylated form of EntB, aryl-EntB. EntE activity has been characterized as a two-step adenylation-ligation reaction. In the first step it catalyzes the condensation of DHB with ATP to form the adenylate intermediate 2,3-dihydroxybenzoyl-AMP. In the second step DHB is ligated onto the phosphopantetheinyl cofactor of holo-entB to form aryl-entB [Sikora10].

Initial studies showed that EntE is a 2,3-dihydroxybenzoate-AMP ligase, and kinetic data suggested that the (2,3-dihydroxybenzoyl)adenylate intermediate remains bound to the enzyme [Rusnak89]. Later, EntE was found to catalyze a second half-reaction, transfer of the aryl fragment, 2,3-dihydroxybenzoate, via a thioester linkage to the phosphopantetheinyl moiety of holo-EntB [Gehring97]. The kinetic mechanism has been studied in detail, suggesting a bi-uni-uni-b ping pong mechanism [Sikora10]. The adenylation activity of EntE is specific for holo-EntB [Ehmann00]. The interaction between EntE and holo-EntB is remarkably tolerant to point mutations in the predicted interaction surface of EntB [Drake06] and is most efficient in the presence of DHB [Khalil09]. In the absence of holo-EntB, EntE can transfer the adenylate moiety of the (2,3-dihydroxybenzoyl)adenylate intermediate to ATP, generating the stress signaling molecule Ap4A and releasing 2,3-dihydroxybenzoate [Sikora09]. Enhancement of the DHB-AMP ligase activity of EntE by interaction with the DHB-producing enzyme EntA has been demonstrated [Khalil11].

EntE can be released from the cell by osmotic shock, but not by formation of spheroplasts; it was therefore suggested that the enzyme is membrane-associated [Hantash97]. Subsequent cell lysis and fractionation studies have led to the proposal that a large fraction of the Ent synthase proteins EntE, EntB and EntF is in contact with membranes, or in close proximity to membranes [Hantash00]. Gel filtration data disagree on whether EntE is a monomer [Gehring98] or a dimer [Rusnak89] in solution. No stable interaction between the components of the "enterobactin synthase multienzyme complex" can be detected [Gehring98].

Expression of entE is induced under conditions of iron deficiency [Fleming83].

Gene Citations: [Nahlik89, Ozenberger89, Schultz91]

Locations: inner membrane, cytosol, membrane

Map Position: [625,293 -> 626,903] (13.48 centisomes)
Length: 1611 bp / 536 aa

Molecular Weight of Polypeptide: 59.112 kD (from nucleotide sequence), 58.0 kD (experimental) [Nahlik87 ]

Molecular Weight of Multimer: 115.0 kD (experimental) [Rusnak89]

pI: 6.15

Unification Links: ASAP:ABE-0002049 , CGSC:818 , DIP:DIP-9515N , EchoBASE:EB0259 , EcoGene:EG10263 , EcoliWiki:b0594 , Mint:MINT-1228110 , ModBase:P10378 , OU-Microarray:b0594 , PortEco:entE , PR:PRO_000022523 , Pride:P10378 , Protein Model Portal:P10378 , RefSeq:NP_415126 , RegulonDB:EG10263 , SMR:P10378 , String:511145.b0594 , Swiss-Model:P10378 , UniProt:P10378

Relationship Links: InterPro:IN-FAMILY:IPR000873 , InterPro:IN-FAMILY:IPR011963 , InterPro:IN-FAMILY:IPR020845 , InterPro:IN-FAMILY:IPR025110 , PDB:Structure:3RG2 , PDB:Structure:4IZ6 , Pfam:IN-FAMILY:PF00501 , Pfam:IN-FAMILY:PF13193 , Prosite:IN-FAMILY:PS00455

In Paralogous Gene Group: 13 (9 members)

Gene-Reaction Schematic: ?

Genetic Regulation Schematic: ?

GO Terms:

Biological Process: GO:0009239 - enterobactin biosynthetic process Inferred from experiment Inferred by computational analysis [UniProtGOA12, UniProtGOA11a, Luke71]
GO:0008152 - metabolic process Inferred by computational analysis [UniProtGOA11a, GOA01a]
GO:0019290 - siderophore biosynthetic process Inferred by computational analysis [GOA01a]
Molecular Function: GO:0008668 - (2,3-dihydroxybenzoyl)adenylate synthase activity Inferred from experiment Inferred by computational analysis [GOA01, GOA01a, Rusnak89]
GO:0000166 - nucleotide binding Inferred by computational analysis [UniProtGOA11a]
GO:0003824 - catalytic activity Inferred by computational analysis [UniProtGOA11a, GOA01a]
GO:0005524 - ATP binding Inferred by computational analysis [UniProtGOA11a]
GO:0016740 - transferase activity Inferred by computational analysis [UniProtGOA11a]
GO:0016746 - transferase activity, transferring acyl groups Inferred by computational analysis [UniProtGOA11a]
GO:0016874 - ligase activity Inferred by computational analysis [UniProtGOA11a]
Cellular Component: GO:0005829 - cytosol Inferred from experiment Inferred by computational analysis [DiazMejia09, Ishihama08, Hantash00]
GO:0016020 - membrane Inferred from experiment [Hantash00]
GO:0005737 - cytoplasm
GO:0005886 - plasma membrane

MultiFun Terms: cell structure membrane
metabolism biosynthesis of building blocks cofactors, small molecule carriers enterochelin (enterobactin)

Essentiality data for entE 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 1% glycerol Yes 37 Aerobic 7.2 0.35 Yes [Joyce06, Comment 3]
MOPS medium with 0.4% glucose Yes 37 Aerobic 7.2 0.22 Yes [Baba06, Comment 2]
Yes [Feist07, Comment 4]

Curated 15-Apr-2010 by Keseler I , SRI International
Last-Curated ? 12-Jun-2012 by Fulcher C , SRI International

Enzymatic reaction of: S-dihydroxybenzoyltransferase (2,3-dihydroxybenzoate-AMP ligase)

EC Number: 2.5.1.-

EntB isochorismatase / aryl-carrier protein + 2,3-dihydroxybenzoate + ATP <=> aryl-EntB + AMP + diphosphate

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction in which it was curated.

Reversibility of this reaction is unspecified.

Enzymatic reaction of: 2,3-dihydroxybenzoate-AMP ligase

Synonyms: 2,3-DHB-AMP ligase, dihydroxybenzoic acid activating enzyme

EC Number:

ATP + 2,3-dihydroxybenzoate + H+ <=> 2,3-dihydroxybenzoyl-AMP + diphosphate

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 physiologically favored in the direction shown.

Alternative Substrates for 2,3-dihydroxybenzoate [Comment 5 ]: salicylate [Rusnak89 ] , 2,4-dihydroxybenzoate [Rusnak89 ] , gentisate [Rusnak89 ] , 2,3,4-trihydroxybenzoate [Rusnak89 ]

The equilibrium binding constant Kd of 2,3-dihydroxybenzoate to EntE is 7.3 µM [Khalil09].

Cofactors or Prosthetic Groups: Mg2+ [Rusnak89]

Inhibitors (Competitive): 2,3-dihydroxybenzohydroxamoyl adenylate [Callahan06]

Inhibitors (Unknown Mechanism): N-ethylmaleimide [Bryce72]

Kinetic Parameters:

Km (μM)

Enzymatic reaction of: Ap4A synthetase (2,3-dihydroxybenzoate-AMP ligase)

EC Number: 2.7.7.-

ATP + ATP + H+ <=> 5',5'''-diadenosine tetraphosphate + diphosphate

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction in which it was curated.

Reversibility of this reaction is unspecified.

Kinetic Parameters:

Km (μM)
5',5'''-diadenosine tetraphosphate

Subunit of: enterobactin synthase

Synonyms: enterobactin synthetase multienzyme complex

Subunit composition of enterobactin synthase = [EntB isochorismatase / aryl-carrier protein][EntD][EntF][(EntE)2]
         phosphopantetheinyl transferase = EntD (extended summary available)
         aryl carrier protein / L-seryl-AMP synthase = EntF (extended summary available)
         2,3-dihydroxybenzoate-AMP ligase = (EntE)2 (extended summary available)

Enterobactin contains three units of 2,3-dihydroxybenzoylserine joined in a cyclic structure by lactone linkages. Studies have suggested that the later steps of enterobactin synthesis are carried out by a multienzyme complex consisting of the entD, entE, entF and entB gene products [Hantash97].

Proteins EntB, EntD, EntE and EntF of the enterobactin synthase multienzyme complex have been purified and characterized, but no evidence has been obtained for the existence of a stable multienzyme complex. These proteins are required for the ATP-dependent conversion of three molecules each of 2,3-dihydroxybenzoate and L-serine to enterobactin [Gehring97, Gehring98, Drake06].

Proteins EntB, EntE and EntF together contain domains that comprise a nonribosomal peptide synthase (NRPS). EntE provides an adenylation domain, EntB provides an aryl carrier protein domain (located at its C-terminus), and EntF provides condensation, adenylation, peptidyl carrier protein, and chain-releasing thioesterase domains. Thus, six domains of three proteins comprise a two-module NRPS [Ehmann00]. EntD is a phosphopantetheinyl transferase that adds this cofactor to the peptidyl carrier protein domains of EntB and EntF [Gehring97]. The activities of EntE, the EntB C-terminal domain, and EntF assemble enterobactin in an iterative manner [Drake06, Ehmann00].

Last-Curated ? 13-Jun-2012 by Fulcher C , SRI International

Enzymatic reaction of: enterobactin synthase

EC Number:

6 ATP + 3 L-serine + 3 2,3-dihydroxybenzoate <=> 6 AMP + enterobactin + 6 diphosphate + 3 H+

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 physiologically favored in the direction shown.

In Pathways: superpathway of chorismate metabolism , enterobactin biosynthesis

Sequence Features

Feature Class Location Citations Comment
Cleavage-of-Initial-Methionine 1
Sequence-Conflict 369 -> 378
[Staab89, UniProt10a]
Alternate sequence: DAEGNPLPQG → ECRRKSTAAR; UniProt: (in Ref. 1; CAA33158);

Gene Local Context (not to scale): ?

Transcription Units:


10/20/97 Gene b0594 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG10263; confirmed by SwissProt match.


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

Bryce72: Bryce GF, Brot N (1972). "Studies on the enzymatic synthesis of the cyclic trimer of 2,3-dihydroxy-N-benzoyl-L-serine in Escherichia coli." Biochemistry 1972;11(9);1708-15. PMID: 4337557

Callahan06: Callahan BP, Lomino JV, Wolfenden R (2006). "Nanomolar inhibition of the enterobactin biosynthesis enzyme, EntE: synthesis, substituent effects, and additivity." Bioorg Med Chem Lett 16(14);3802-5. PMID: 16678412

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

Drake06: Drake EJ, Nicolai DA, Gulick AM (2006). "Structure of the EntB multidomain nonribosomal peptide synthetase and functional analysis of its interaction with the EntE adenylation domain." Chem Biol 13(4);409-19. PMID: 16632253

Ehmann00: Ehmann DE, Shaw-Reid CA, Losey HC, Walsh CT (2000). "The EntF and EntE adenylation domains of Escherichia coli enterobactin synthetase: sequestration and selectivity in acyl-AMP transfers to thiolation domain cosubstrates." Proc Natl Acad Sci U S A 97(6);2509-14. PMID: 10688898

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

Fleming83: Fleming TP, Nahlik MS, McIntosh MA (1983). "Regulation of enterobactin iron transport in Escherichia coli: characterization of ent::Mu d(Apr lac) operon fusions." J Bacteriol 156(3);1171-7. PMID: 6227609

Gehring97: Gehring AM, Bradley KA, Walsh CT (1997). "Enterobactin biosynthesis in Escherichia coli: isochorismate lyase (EntB) is a bifunctional enzyme that is phosphopantetheinylated by EntD and then acylated by EntE using ATP and 2,3-dihydroxybenzoate." Biochemistry 1997;36(28);8495-503. PMID: 9214294

Gehring98: Gehring AM, Mori I, Walsh CT (1998). "Reconstitution and characterization of the Escherichia coli enterobactin synthetase from EntB, EntE, and EntF." Biochemistry 1998;37(8);2648-59. PMID: 9485415

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

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."

Greenwood76: Greenwood KT, Luke RJ (1976). "Studies on the enzymatic synthesis of enterochelin in Escherichia coli K-12. Four polypeptides involved in the conversion of 2,3-dihydroxybenzoate to enterochelin." Biochim Biophys Acta 1976;454(2);285-97. PMID: 136989

Hantash00: Hantash FM, Earhart CF (2000). "Membrane association of the Escherichia coli enterobactin synthase proteins EntB/G, EntE, and EntF." J Bacteriol 182(6);1768-73. PMID: 10692387

Hantash97: Hantash FM, Ammerlaan M, Earhart CF (1997). "Enterobactin synthase polypeptides of Escherichia coli are present in an osmotic-shock-sensitive cytoplasmic locality." Microbiology 1997;143 ( Pt 1);147-56. PMID: 9025288

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

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

Khalil09: Khalil S, Pawelek PD (2009). "Ligand-induced conformational rearrangements promote interaction between the Escherichia coli enterobactin biosynthetic proteins EntE and EntB." J Mol Biol 393(3);658-71. PMID: 19699210

Khalil11: Khalil S, Pawelek PD (2011). "Enzymatic adenylation of 2,3-dihydroxybenzoate is enhanced by a protein-protein interaction between Escherichia coli 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase (EntA) and 2,3-dihydroxybenzoate-AMP ligase (EntE)." Biochemistry 50(4);533-45. PMID: 21166461

Luke71: Luke RK, Gibson F (1971). "Location of three genes concerned with the conversion of 2,3-dihydroxybenzoate into enterochelin in Escherichia coli K-12." J Bacteriol 107(2);557-62. PMID: 4939766

Nahlik87: Nahlik MS, Fleming TP, McIntosh MA (1987). "Cluster of genes controlling synthesis and activation of 2,3-dihydroxybenzoic acid in production of enterobactin in Escherichia coli." J Bacteriol 1987;169(9);4163-70. PMID: 3040680

Nahlik89: Nahlik MS, Brickman TJ, Ozenberger BA, McIntosh MA (1989). "Nucleotide sequence and transcriptional organization of the Escherichia coli enterobactin biosynthesis cistrons entB and entA." J Bacteriol 171(2);784-90. PMID: 2521621

Neres08: Neres J, Wilson DJ, Celia L, Beck BJ, Aldrich CC (2008). "Aryl acid adenylating enzymes involved in siderophore biosynthesis: fluorescence polarization assay, ligand specificity, and discovery of non-nucleoside inhibitors via high-throughput screening." Biochemistry 47(45);11735-49. PMID: 18928302

Ozenberger89: Ozenberger BA, Brickman TJ, McIntosh MA (1989). "Nucleotide sequence of Escherichia coli isochorismate synthetase gene entC and evolutionary relationship of isochorismate synthetase and other chorismate-utilizing enzymes." J Bacteriol 1989;171(2);775-83. PMID: 2536681

Rusnak89: Rusnak F, Faraci WS, Walsh CT (1989). "Subcloning, expression, and purification of the enterobactin biosynthetic enzyme 2,3-dihydroxybenzoate-AMP ligase: demonstration of enzyme-bound (2,3-dihydroxybenzoyl)adenylate product." Biochemistry 1989;28(17);6827-35. PMID: 2531000

Schultz91: Schultz JE, Matin A (1991). "Molecular and functional characterization of a carbon starvation gene of Escherichia coli." J Mol Biol 1991;218(1);129-40. PMID: 1848300

Sikora09: Sikora AL, Cahill SM, Blanchard JS (2009). "Enterobactin synthetase-catalyzed formation of P(1),P(3)-diadenosine-5'-tetraphosphate." Biochemistry 48(46);10827-9. PMID: 19852513

Sikora10: Sikora AL, Wilson DJ, Aldrich CC, Blanchard JS (2010). "Kinetic and Inhibition Studies of Dihydroxybenzoate-AMP Ligase from Escherichia coli." Biochemistry. PMID: 20359185

Staab89: Staab JF, Elkins MF, Earhart CF (1989). "Nucleotide sequence of the Escherichia coli entE gene." FEMS Microbiol Lett 50(1-2);15-9. PMID: 2525505

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

Woodrow79: Woodrow GC, Young IG, Gibson F (1979). "Biosynthesis of enterochelin in Escherichia coli K-12: separation of the polypeptides coded for by the entD, E, F and G genes." Biochim Biophys Acta 582(1);145-53. PMID: 216414

Other References Related to Gene Regulation

Brickman90: Brickman TJ, Ozenberger BA, McIntosh MA (1990). "Regulation of divergent transcription from the iron-responsive fepB-entC promoter-operator regions in Escherichia coli." J Mol Biol 1990;212(4);669-82. PMID: 2139473

Chen07: Chen Z, Lewis KA, Shultzaberger RK, Lyakhov IG, Zheng M, Doan B, Storz G, Schneider TD (2007). "Discovery of Fur binding site clusters in Escherichia coli by information theory models." Nucleic Acids Res 35(20);6762-77. PMID: 17921503

Newman99: Newman DL, Shapiro JA (1999). "Differential fiu-lacZ fusion regulation linked to Escherichia coli colony development." Mol Microbiol 33(1);18-32. PMID: 10411720

Zhang05a: Zhang Z, Gosset G, Barabote R, Gonzalez CS, Cuevas WA, Saier MH (2005). "Functional interactions between the carbon and iron utilization regulators, Crp and Fur, in Escherichia coli." J Bacteriol 187(3);980-90. PMID: 15659676

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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 Sat Dec 20, 2014, biocyc12.