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Escherichia coli K-12 substr. MG1655 Polypeptide: EntB monomer
Inferred from experiment




Gene: entB Accession Numbers: EG10260 (EcoCyc), b0595, ECK0588

Synonyms: entG

Regulation Summary Diagram

Regulation summary diagram for entB

Alternative forms of EntB monomer: holo EntB monomer

Summary:
EntB is a bifunctional enzyme that is involved in the enterobactin biosynthesis pathway. Enterobactin, a siderophore molecule, is synthesized in response to iron deprivation by formation of an amide bond between 2,3-dihydroxybenzoate (2,3-DHB) and L-serine. EntB is an isochorismatase which catalyzes the synthesis of 2,3-DHB and also serves as an aryl carrier protein (ArCP) in the assembly of enterobactin. [Gehring97]

Apo-EntB has an N-terminal isochorismatase domain and a C-terminal apo-aryl carrier protein (apo-ArCP) domain. The apo-ArCP domain undergoes phosphopantetheinylation catalyzed by phosphopantetheinyl transferase EntD, turning EntB into holo-EntB ( holo-EntB dimer), which serves as a substrate for the 2,3-dihydroxybenzoate-AMP ligase, EntE. [Gehring98, Gehring97]

The surface of the ArCP domain has been mapped and several conserved hydrophobic residues as well as protein interaction surfaces were identified [Lai06, Lai06a]. The thioesterase, EntH, interacts specifically with the ArCP domain of EntB during enterobactin production [Leduc07].

Based on gel filtration data, EntB was originally suggested to be a pentamer [Rusnak90] and later suggested to be a trimer [Gehring98]. However subsequent gel filtration and crystallographic data showed it to be a dimer [Drake06]. The crystal structure of an engineered fusion protein containing the EntB aryl-carrier protein and the EntE adenylation domain has also been determined to gain insight into the interaction between these two domains [Sundlov12].

EntB is a cytosolic protein which can be released by osmotic shock. Membrane association of EntB has also been demonstrated. [Hantash00]

Overproduction of EntB in a reduced-genome E. coli, promoted biofilm development and maturation [May11].

Citations: [Zhou07a, Chen09a, Khalil09, Hubrich13]

Gene Citations: [Nahlik89, Ozenberger89, Schultz91]

Locations: inner membrane, cytosol

Map Position: [626,917 -> 627,774] (13.51 centisomes, 49°)
Length: 858 bp / 285 aa

Molecular Weight of Polypeptide: 32.554 kD (from nucleotide sequence)

pI: 5.33

Unification Links: ASAP:ABE-0002052, CGSC:821, DIP:DIP-9512N, EchoBASE:EB0256, EcoGene:EG10260, EcoliWiki:b0595, Mint:MINT-1241343, ModBase:P0ADI4, OU-Microarray:b0595, PortEco:entB, PR:PRO_000022520, Pride:P0ADI4, Protein Model Portal:P0ADI4, RefSeq:NP_415127, RegulonDB:EG10260, SMR:P0ADI4, String:511145.b0595, Swiss-Model:P0ADI4, UniProt:P0ADI4

Relationship Links: InterPro:IN-FAMILY:IPR000868, InterPro:IN-FAMILY:IPR009081, InterPro:IN-FAMILY:IPR016291, PDB:Structure:2FQ1, PDB:Structure:3RG2, Pfam:IN-FAMILY:PF00550, Pfam:IN-FAMILY:PF00857, Prints:IN-FAMILY:PR01398, Prosite:IN-FAMILY:PS50075

In Paralogous Gene Group: 162 (2 members)

Reactions known to consume the compound:

enterobactin biosynthesis :
[EntB aryl-carrier protein] + coenzyme A → a holo-[EntB isochorismatase/aryl-carrier protein] + adenosine 3',5'-bisphosphate + H+

acyl carrier protein metabolism :
an acyl-carrier protein + coenzyme A → adenosine 3',5'-bisphosphate + a holo-[acyl-carrier protein] + H+

Reactions known to produce the compound:

acyl carrier protein metabolism :
a holo-[acyl-carrier protein] + H2O → 4'-phosphopantetheine + an acyl-carrier protein + H+

Gene-Reaction Schematic

Gene-Reaction Schematic

Instance reaction 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)

Genetic Regulation Schematic

Genetic regulation schematic for entB


GO Terms:
Biological Process:
Inferred from experimentInferred by computational analysisGO:0009239 - enterobactin biosynthetic process [UniProtGOA12, UniProtGOA11a, Gehring98]
Inferred by computational analysisGO:0008152 - metabolic process [UniProtGOA11a, GOA01a]
Molecular Function:
Inferred from experimentGO:0000287 - magnesium ion binding [Drake06]
Inferred from experimentGO:0005515 - protein binding [Leduc07]
Inferred from experimentInferred by computational analysisGO:0008908 - isochorismatase activity [GOA01, GOA01a, Rusnak90]
Inferred from experimentGO:0016765 - transferase activity, transferring alkyl or aryl (other than methyl) groups [Gehring97]
Inferred from experimentGO:0031177 - phosphopantetheine binding [Sundlov12]
Inferred from experimentGO:0042802 - identical protein binding [Rajagopala14, Leduc07]
Inferred from experimentInferred by computational analysisGO:0047527 - 2,3-dihydroxybenzoate-serine ligase activity [GOA01, Gehring98]
Inferred by computational analysisGO:0003824 - catalytic activity [UniProtGOA11a, GOA01a]
Inferred by computational analysisGO:0016787 - hydrolase activity [UniProtGOA11a]
Inferred by computational analysisGO:0016874 - ligase activity [UniProtGOA11a]
Inferred by computational analysisGO:0046872 - metal ion binding [UniProtGOA11a]
Cellular Component:
Inferred from experimentInferred by computational analysisGO:0005829 - cytosol [DiazMejia09, Ishihama08, Hantash00]
Inferred from experimentGO:0005886 - plasma membrane [Hantash00]
Inferred by computational analysisGO:0005737 - cytoplasm [UniProtGOA11, UniProtGOA11a]

MultiFun Terms: cell structuremembrane
information transferprotein relatedNon-ribosomal peptide synthetase
metabolismbiosynthesis of building blockscofactors, small molecule carriersenterochelin (enterobactin)

Essentiality data for entB knockouts:

Growth Medium Growth? T (°C) O2 pH Osm/L Growth Observations
LB enrichedYes 37 Aerobic 6.95   Yes [Gerdes03, Comment 1]
LB LennoxYes 37 Aerobic 7   Yes [Baba06, Comment 2]
M9 medium with 1% glycerolYes 37 Aerobic 7.2 0.35 Yes [Joyce06, Comment 3]
MOPS medium with 0.4% glucoseYes 37 Aerobic 7.2 0.22 Yes [Baba06, Comment 2]
Yes [Feist07, Comment 4]

Sequence Features

Protein sequence of EntB monomer with features indicated

Feature Class Location Citations Comment
Cleavage-of-Initial-Methionine 1
Inferred from experiment[Rusnak90]
UniProt: Removed.
Protein-Segment 2 -> 213
Author statement[Gehring97]
UniProt: Isochorismatase; Sequence Annotation Type: region of interest.
Chain 2 -> 285
Author statement[UniProt15]
UniProt: Enterobactin synthase component B.
Conserved-Region 214 -> 281
Inferred by computational analysis[UniProt15]
UniProt: Acyl carrier.
Metal-Binding-Site 227
Inferred from experiment[Drake06]
UniProt: Magnesium.
Mutagenesis-Variant 240
Inferred from experiment[Drake06]
UniProt: The catalytic efficiency slightly increases, but a 8-fold decrease of the affinity for the S-dihydroxybenzoyltransferase EntE is observed.
Mutagenesis-Variant 242
Inferred from experiment[Lai06a]
UniProt: Not efficiently phosphopantetheinylated by EntD.
Metal-Binding-Site 242
Inferred from experiment[Drake06]
UniProt: Magnesium; via carbonyl oxygen.
Mutagenesis-Variant 244
Inferred from experiment[Lai06a]
Inferred from experiment[Lai06a]
D → A: Efficiently phosphopantetheinylated by EntD.
D → R: Not efficiently phosphopantetheinylated by EntD.
Metal-Binding-Site 244
Inferred from experiment[Drake06]
UniProt: Magnesium.
Modified-Residue 245
Inferred from experiment[Sundlov12]
UniProt: O-(pantetheine 4'-phosphoryl)serine.
Mutagenesis-Variant 249
Inferred from experiment[Lai06]
UniProt: Unable to recognize EntE and EntF. Exhibits a decrease in the enterobactin production compared to the wild-type. Still able to be phosphopantetheinylated.
Mutagenesis-Variant 263
Inferred from experiment[Drake06]
UniProt: The catalytic efficiency is the same as the wild-type, but a 3-fold decrease of the affinity for the S-dihydroxybenzoyltransferase EntE is observed.
Mutagenesis-Variant 264
Inferred from experiment[Drake06, Lai06]
UniProt: The affinity for EntE and catalytic efficiency of the S-dihydroxybenzoyltransferase EntE are below that of the wild-type.
Mutagenesis-Variant 268
Inferred from experiment[Lai06]
UniProt: Unable to recognize EntE and EntF. Exhibits a decrease in the enterobactin production compared to the wild-type. Still able to be phosphopantetheinylated.
Mutagenesis-Variant 269
Inferred from experiment[Lai06]
UniProt: Behaves similarly to the wild-type.


Sequence Pfam Features

Protein sequence of EntB monomer with features indicated

Feature Class Location Citations Comment
Pfam PF00857 32 -> 204
Inferred by computational analysis[Finn14]
Isochorismatase : Isochorismatase family
Pfam PF00550 217 -> 276
Inferred by computational analysis[Finn14]
PP-binding : Phosphopantetheine attachment site


Gene Local Context (not to scale -- see Genome Browser for correct scale)

Gene local context diagram

Transcription Units

Transcription-unit diagram

Transcription-unit diagram

Notes:

History:
10/20/97 Gene b0595 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG10260; 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

Chen09a: Chen D, Wu R, Bryan TL, Dunaway-Mariano D (2009). "In vitro kinetic analysis of substrate specificity in enterobactin biosynthetic lower pathway enzymes provides insight into the biochemical function of the hot dog-fold thioesterase EntH." Biochemistry 48(3);511-3. PMID: 19119850

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

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

Finn14: Finn RD, Bateman A, Clements J, Coggill P, Eberhardt RY, Eddy SR, Heger A, Hetherington K, Holm L, Mistry J, Sonnhammer EL, Tate J, Punta M (2014). "Pfam: the protein families database." Nucleic Acids Res 42(Database issue);D222-30. PMID: 24288371

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."

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

Hubrich13: Hubrich F, Mordhorst S, Andexer JN (2013). "Cinnamic acid derivatives as inhibitors for chorismatases and isochorismatases." Bioorg Med Chem Lett 23(5);1477-81. PMID: 23380376

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

Lai06: Lai JR, Fischbach MA, Liu DR, Walsh CT (2006). "A protein interaction surface in nonribosomal peptide synthesis mapped by combinatorial mutagenesis and selection." Proc Natl Acad Sci U S A 103(14);5314-9. PMID: 16567620

Lai06a: Lai JR, Fischbach MA, Liu DR, Walsh CT (2006). "Localized protein interaction surfaces on the EntB carrier protein revealed by combinatorial mutagenesis and selection." J Am Chem Soc 128(34);11002-3. PMID: 16925399

Leduc07: Leduc D, Battesti A, Bouveret E (2007). "The Hotdog thioesterase EntH (YbdB) plays a role in vivo in optimal enterobactin biosynthesis by interacting with the ArCP domain of EntB." J Bacteriol 189(19):7112-26. PMID: 17675380

May11: May T, Okabe S (2011). "Enterobactin is required for biofilm development in reduced-genome Escherichia coli." Environ Microbiol 13(12);3149-62. PMID: 21980953

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

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

Rajagopala14: Rajagopala SV, Sikorski P, Kumar A, Mosca R, Vlasblom J, Arnold R, Franca-Koh J, Pakala SB, Phanse S, Ceol A, Hauser R, Siszler G, Wuchty S, Emili A, Babu M, Aloy P, Pieper R, Uetz P (2014). "The binary protein-protein interaction landscape of Escherichia coli." Nat Biotechnol 32(3);285-90. PMID: 24561554

Rusnak90: Rusnak F, Liu J, Quinn N, Berchtold GA, Walsh CT (1990). "Subcloning of the enterobactin biosynthetic gene entB: expression, purification, characterization, and substrate specificity of isochorismatase." Biochemistry 1990;29(6);1425-35. PMID: 2139796

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

Sundlov12: Sundlov JA, Shi C, Wilson DJ, Aldrich CC, Gulick AM (2012). "Structural and functional investigation of the intermolecular interaction between NRPS adenylation and carrier protein domains." Chem Biol 19(2);188-98. PMID: 22365602

UniProt15: UniProt Consortium (2015). "UniProt version 2015-08 released on 2015-07-22." 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."

Zhou07a: Zhou Z, Lai JR, Walsh CT (2007). "Directed evolution of aryl carrier proteins in the enterobactin synthetase." Proc Natl Acad Sci U S A 104(28);11621-6. PMID: 17606920

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
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