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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 holo-EntB dimer (holo-EntB) to form the covalently arylated form of EntB, 2,3-dihydroxybenzoyl-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: ?

Gene-Reaction Schematic

Instance reactions of [an acyl-carrier protein + coenzyme A → adenosine 3',5'-bisphosphate + a holo-[acyl-carrier protein] + H+] (2.7.8.7):
i1: [EntB aryl-carrier protein] + coenzyme A → a holo EntB isochorismatase/aryl-carrier protein + adenosine 3',5'-bisphosphate + H+ (2.7.8.7)

i2: AsbD acyl-carrier protein + coenzyme A → adenosine 3',5'-bisphosphate + a holo-AsbD acyl-carrier protein + H+ (no EC#)

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:0047527 - 2,3-dihydroxybenzoate-serine ligase activity Inferred by computational analysis Inferred from experiment [Gehring98, GOA01]
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)

Credits:
Imported from EcoCyc 27-Jan-2015 by Paley S , SRI International


Enzymatic reaction of: 2,3-dihydroxybenzoate-adenylate:[EntB] 2,3-dihydroxybenzoate transferase (2,3-dihydroxybenzoate-AMP ligase)

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

EC Number: 2.5.1.-

2,3-dihydroxybenzoyl adenylate + a holo EntB isochorismatase/aryl-carrier protein <=> a 2,3-dihydroxybenzoyl-[EntB isochorismatase/aryl-carrier protein] + AMP + H+

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.

The reaction is favored in the direction shown.

In Pathways: superpathway of chorismate metabolism , enterobactin biosynthesis

Credits:
Imported from MetaCyc 25-Sep-2014 by Caspi R , SRI International
Imported from EcoCyc 27-Jan-2015 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]


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 adenylate + 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.

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 ]

In Pathways: superpathway of chorismate metabolism , enterobactin biosynthesis

Credits:
Imported from EcoCyc 27-Jan-2015 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 27-Jan-2015 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)2][EntD][EntF][(EntE)2]
         holo-EntB dimer = (EntB)2 (extended summary available)
                 holo EntB monomer = EntB
         phosphopantetheinyl transferase = EntD (extended summary available)
         holo [EntF peptidyl-carrier protein] = 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 27-Jan-2015 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 direction in which it was curated.

The reaction is physiologically favored in the direction shown.

In Pathways: superpathway of chorismate metabolism , enterobactin biosynthesis

Credits:
Imported from EcoCyc 27-Jan-2015 by Paley S , SRI International


Sequence Features

Feature Class Location Citations Comment
Cleavage-of-Initial-Methionine 1
[Rusnak89]
 
Sequence-Conflict 369 -> 378
[Staab89, UniProt10]
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, 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

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

UniProt10: 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


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Please cite the following article in publications resulting from the use of MetaCyc: Caspi et al, Nucleic Acids Research 42:D459-D471 2014
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