|Gene:||entB||Accession Numbers: EG10260 (MetaCyc), b0595, ECK0588|
Synonyms: entG, holo-EntB
Species: Escherichia coli K-12 substr. MG1655
Component of: enterobactin synthase (extended summary available)
Alternative forms of holo EntB monomer: EntB monomer (extended summary available)
Alternative forms of holo-EntB dimer: 2,3-dihydroxybenzoyl-EntB
The EntB protein is bifunctional, the N-terminal domain contains the isochorismate lyase (isochorismatase) activity and the C-terminus contains the aryl carrier protein (ArCP) domain. The isochorismatase activity of the entB gene product catalyzes the second step in both the overall enterobactin biosynthetic pathway (see pathway enterobactin biosynthesis) and in the first part of the pathway, the conversion of chorismate to 2,3-dihydroxybenzoate (see pathway 2,3-dihydroxybenzoate biosynthesis) [Rusnak90, Staab90].
The ArCP domain of apo-EntB becomes phosphopantetheinylated in a reaction catalyzed by the entD gene product, phosphopantetheinyl transferase. The holo-EntB dimer (holo-EntB) formed can now serve as a substrate for the entE product, a 2,3-dihydroxybenzoate-AMP ligase that activates 2,3-dihydroxybenzoate as the acyl-AMP derivative and transfers the acyl moiety onto holo-EntB. The arylated holo-EntB product (2,3-dihydroxybenzoyl-EntB) then serves as the aryl donor for the amide bond formation in enterobactin assembly [Gehring97, Gehring98].
The crystal structure of this two domain protein has been determined at 2.3 Å resolution. Its two functionally independent domains are joined by a proline-rich linker. Functional analysis of EntB and EntE mutants probed the interaction between these two proteins [Drake06].
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]. Cell lysis and fractionation studies have led to the proposal that a large fraction of the Ent synthase proteins EntB, EntE and EntF is in contact with membranes, or in close proximity to membranes [Hantash00].
A biophysical study of EntE substrate binding and the interaction between EntE and EntB showed that complex formation is most efficient in the presence of 2,3-dihydroxybenzoate [Khalil09]. The thioesterase EntH has been proposed to potentially function in the prevention or reversal of EntB misacylation events [Chen09, Guo09b]. Using combinatorial mutagenesis and selection, the interaction surface between the ArCP domain of holo-EntB and holo-EntF was mapped [Lai06, Lai06a].
Studies of a reduced-genome E. coli suggested that genes entB, marR, dosC, mcbR and yahK were involved in biofilm formation. Overproduction of entB and yahK promoted biofilm development and maturation [May11].
Locations: cytosol, inner membrane
|Map Position: [626,917 -> 627,774]|
Reactions known to consume the compound:
aspyridone A biosynthesis
bassianin and desmethylbassianin biosynthesis
chaetoglobosin A biosynthesis
fusaridione A biosynthesis
fusarin C biosynthesis
Not in pathways:
Reactions known to produce the compound:
(Kdo)2-lipid A biosynthesis I
8-amino-7-oxononanoate biosynthesis I
bassianin and desmethylbassianin biosynthesis
Reactions known to both consume and produce the compound:
fatty acid biosynthesis (plant mitochondria)
fatty acid biosynthesis initiation I
octanoyl-[acyl-carrier protein] biosynthesis (mitochondria, yeast)
a holo-[acyl-carrier protein] + malonyl-CoA ↔ a malonyl-[acp] + coenzyme A
In Reactions of unknown directionality:
Not in pathways:
γ-linolenoyl-CoA + a holo-[acyl-carrier protein] = a γ linolenoyl [acp] + coenzyme A
5-hexenoate + a holo-[acyl-carrier protein] + ATP = a 5-hexenoyl-[acp] + AMP + diphosphate
a medium chain fatty acid + a holo-[acyl-carrier protein] + ATP = a medium-chain acyl-[acp] + AMP + diphosphate
a heptodecanoyl-[acp] + malonyl-CoA + an reduced unknown electron acceptor + H+ = 1-octadecene + 2 CO2 + a holo-[acyl-carrier protein] + coenzyme A + an oxidized unknown electron acceptor
a palmitoyl-[acp] + malonyl-CoA = 1-heptadecene + 2 CO2 + a holo-[acyl-carrier protein] + coenzyme A
a stearoyl-[acp] + malonyl-CoA + 3 H+ = 1-nonadecene + 2 CO2 + a holo-[acyl-carrier protein] + coenzyme A
a 2,3,4-saturated fatty acyl-[acp] + a 2,3,4-saturated 2-lysophosphatidate = a 2,3,4-saturated L-phosphatidate + a holo-[acyl-carrier protein]
an acetyl-[acp] + 9 malonyl-CoA + H2O + 8 H+ = 3,5,7,9,11,13,15,17,19-nonaoxoicosanoate + a holo-[acyl-carrier protein] + 9 CO2 + 9 coenzyme A
[glycine cleavage system lipoyl-carrier protein]-L-lysine + a lipoyl-[acp] = a [glycine-cleavage complex H protein] N6-lipoyl-L-lysine + a holo-[acyl-carrier protein] + H+
an acyl-[acyl-carrier protein] + a malonyl-[acp] + H+ = a 3-oxoacyl-[acp] + CO2 + a holo-[acyl-carrier protein]
a myristoyl-[acp] + coenzyme A = a holo-[acyl-carrier protein] + myristoyl-CoA
a (3R)-3-hydroxybutanoyl-[acp] + S-adenosyl-L-methionine = a holo-[acyl-carrier protein] + S-methyl-5'-thioadenosine + HAI-1 + H+
an octanoyl-[acp] + S-adenosyl-L-methionine = a holo-[acyl-carrier protein] + S-methyl-5'-thioadenosine + VAI-1-2 + H+
a butyryl-[acp] + S-adenosyl-L-methionine = a holo-[acyl-carrier protein] + S-methyl-5'-thioadenosine + PAI-1-2 + H+
a 3-oxo-dodecanoyl-[acp] + S-adenosyl-L-methionine = a holo-[acyl-carrier protein] + S-methyl-5'-thioadenosine + PAI-1 + H+
a 3-oxo-hexanoyl-[acp] + S-adenosyl-L-methionine = a holo-[acyl-carrier protein] + S-methyl-5'-thioadenosine + VAI-1 + H+
a 3-oxo-octanoyl-[acp] + S-adenosyl-L-methionine = a holo-[acyl-carrier protein] + S-methyl-5'-thioadenosine + AAI-1 + H+
a (3R)-3-hydroxyacyl-[acyl-carrier protein] + coenzyme A = a (3R)-3-hydroxyacyl-CoA + a holo-[acyl-carrier protein]
an arachidoyl-[acp] + coenzyme A = icosanoyl-CoA + a holo-[acyl-carrier protein]
an sn-3-D-galactosyl-sn-2-acylglycerol + an acyl-[acyl-carrier protein] = a holo-[acyl-carrier protein] + a β-monogalactosyldiacylglycerol
Instance reactions of [an acyl-carrier protein + coenzyme A → adenosine 3',5'-bisphosphate + a holo-[acyl-carrier protein] + H+] (126.96.36.199):
|Biological Process:||GO:0009239 - enterobactin biosynthetic process [Staab90, Gehring98]|
|Molecular Function:||GO:0008908 - isochorismatase activity
GO:0031177 - phosphopantetheine binding [Gehring97]
|Cellular Component:||GO:0005829 - cytosol
GO:0005886 - plasma membrane [Hantash00]
GO:0009366 - enterobactin synthetase complex [Gehring98]
|MultiFun Terms:||cell structure → membrane|
|information transfer → protein related → Non-ribosomal peptide synthetase|
|metabolism → biosynthesis of building blocks → cofactors, small molecule carriers → enterochelin (enterobactin)|
Enzymatic reaction of: isochorismatase (holo-EntB dimer)
Synonyms: isochorismate pyruvate-hydrolyase, 2,3-dihydro-2,3-dihydroxybenzoate synthase
EC Number: 188.8.131.52
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.
Alternative Substrates [Comment 1]:
Isochorismatase activity was assayed with either a coupled assay using lactate dehydrogenase, or a coupled assay using 2,3-dihydro-2,3-dihycroxybenzoate dehydrogenase [Rusnak90].
pH(opt): 6.5-7.5 [Rusnak90]
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)
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].
Enzymatic reaction of: enterobactin synthase
EC Number: 184.108.40.206
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.
10/20/97 Gene b0595 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG10260; confirmed by SwissProt match.
Chen09: 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
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
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
Guo09b: Guo ZF, Sun Y, Zheng S, Guo Z (2009). "Preferential hydrolysis of aberrant intermediates by the type II thioesterase in Escherichia coli nonribosomal enterobactin synthesis: substrate specificities and mutagenic studies on the active-site residues." Biochemistry 48(8);1712-22. PMID: 19193103
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
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
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
©2015 SRI International, 333 Ravenswood Avenue, Menlo Park, CA 94025-3493