Metabolic Modeling Tutorial
discounted EARLY registration ends Dec 31, 2014
Metabolic Modeling Tutorial
discounted EARLY registration ends Dec 31, 2014
Metabolic Modeling Tutorial
discounted EARLY registration ends Dec 31, 2014
Metabolic Modeling Tutorial
discounted EARLY registration ends Dec 31, 2014
Metabolic Modeling Tutorial
discounted EARLY registration ends Dec 31, 2014
twitter
MetaCyc Enzyme: 2,3-dihydroxybenzoate-AMP ligase

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

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

Species: Escherichia coli K-12 substr. MG1655

Component of: enterobactin synthase (extended summary available)

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

Summary:
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].

Locations: inner membrane, cytosol, membrane

Map Position: [625,293 -> 626,903]

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

Gene-Reaction Schematic: ?

GO Terms:

Biological Process: GO:0009239 - enterobactin biosynthetic process Inferred from experiment Inferred by computational analysis [UniProtGOA12, UniProtGOA11, Luke71]
GO:0008152 - metabolic process Inferred by computational analysis [UniProtGOA11, GOA01]
GO:0019290 - siderophore biosynthetic process Inferred by computational analysis [GOA01]
Molecular Function: GO:0008668 - (2,3-dihydroxybenzoyl)adenylate synthase activity Inferred from experiment Inferred by computational analysis [GOA01a, GOA01, Rusnak89]
GO:0000166 - nucleotide binding Inferred by computational analysis [UniProtGOA11]
GO:0003824 - catalytic activity Inferred by computational analysis [UniProtGOA11, GOA01]
GO:0005524 - ATP binding Inferred by computational analysis [UniProtGOA11]
GO:0016740 - transferase activity Inferred by computational analysis [UniProtGOA11]
GO:0016746 - transferase activity, transferring acyl groups Inferred by computational analysis [UniProtGOA11]
GO:0016874 - ligase activity Inferred by computational analysis [UniProtGOA11]
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)

Credits:
Imported from EcoCyc 16-Sep-2014 by Paley S , SRI International


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

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

EC Number: 2.7.7.58

2,3-dihydroxybenzoate + ATP + 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 1 ]: salicylate [Rusnak89 ] , 2,4-dihydroxybenzoate [Rusnak89 ] , gentisate [Rusnak89 ] , 2,3,4-trihydroxybenzoate [Rusnak89 ]

Credits:
Imported from EcoCyc 16-Sep-2014 by Paley S , SRI International

Summary:
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:

Substrate
Km (μM)
Citations
ATP
1120.0
[Rusnak89]
2,3-dihydroxybenzoate
2.7
[Rusnak89]


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.

Credits:
Imported from EcoCyc 16-Sep-2014 by Paley S , SRI International

Kinetic Parameters:

Substrate
Km (μM)
Citations
5',5'''-diadenosine tetraphosphate
400.0
[Sikora09]


Subunit of: enterobactin synthase

Synonyms: enterobactin synthetase multienzyme complex

Species: Escherichia coli K-12 substr. MG1655

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)

Summary:
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].

Credits:
Imported from EcoCyc 16-Sep-2014 by Paley S , SRI International


Enzymatic reaction of: enterobactin synthase

EC Number: 6.3.2.14

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

Credits:
Imported from EcoCyc 16-Sep-2014 by Paley S , SRI International


Sequence Features

Feature Class Location Citations Comment
Cleavage-of-Initial-Methionine 1
[Rusnak89]
 
Sequence-Conflict 369 -> 378
[Staab89, UniProt10a]
Alternate sequence: ECRRKSTAAR; UniProt: (in Ref. 1; CAA33158);

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


References

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

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

GOA01: GOA, DDB, FB, MGI, ZFIN (2001). "Gene Ontology annotation through association of InterPro records with GO terms."

GOA01a: GOA, MGI (2001). "Gene Ontology annotation based on Enzyme Commission mapping." Genomics 74;121-128.

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

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

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

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

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 49(17);3648-57. 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.

UniProtGOA11: 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


Report Errors or Provide Feedback
Please cite the following article in publications resulting from the use of MetaCyc: Caspi et al, Nucleic Acids Research 42:D459-D471 2014
Page generated by SRI International Pathway Tools version 18.5 on Mon Dec 29, 2014, BIOCYC15B.