EntE (2,3-dihydroxybenzoate-AMP ligase; enterobactin synthase component E) is the stand-alone adenylation enzyme of the Escherichia coli enterobactin nonribosomal peptide synthetase. A soluble cytoplasmic member of the ANL (ATP-dependent AMP-binding) superfamily, it uses ATP to activate 2,3-dihydroxybenzoate (DHB) as an enzyme-bound DHB-adenylate and then transfers the DHB moiety onto the phosphopantetheine arm of the aryl carrier protein domain of holo-EntB (EC 6.2.1.71). DHB-loaded EntB is the aryl donor used by EntF, which forms the amide and ester bonds of the cyclic tris(N-2,3-dihydroxybenzoyl-L-serine) siderophore. EntE is produced under iron limitation (Fur-regulated); its activity is stimulated by interaction with the upstream DHB-producing dehydrogenase EntA, and DHB binding primes complex formation with EntB. In the absence of holo-EntB the bound DHB-adenylate can react with ATP to form Ap4A in vitro. A minor fraction of the protein associates with membranes.
Summary: EntE-EntB interaction (IPI) reflecting EntE engaging its carrier-protein substrate, holo-EntB ArCP, to transfer DHB-AMP.
Reason: Generic protein binding is uninformative. The paper shows that DHB-loaded EntE forms a complex with EntB to channel DHB-AMP onto EntB; the informative molecular function underlying this interaction is EntE's DHB--[aryl-carrier protein] ligase activity (GO:0008668), already annotated to EntE. The interaction itself is not in doubt.
binding of 2,3-DHB to EntE and EntB primes these proteins for efficient complexation, thus facilitating direct channeling of the siderophore precursor 2,3-DHB-AMP.
Belonging to the family of aryl acid adenylating enzymes (AAAE), EntE is characterized by a two-step adenylation/ligation reaction: first, the enzyme catalyzes the condensation of DHB and ATP to form an adenylate intermediate, followed by the ligation of DHB onto the phosphopantetheinylated cofactor that is bound to the ArCP of EntB
IPI PMID:21166461 Enzymatic adenylation of 2,3-dihydroxybenzoate is enhanced b...
REMOVE
Summary: EntE-EntB interaction (IPI) attributed to the EntA-EntE paper; presumably from pull-down experiments in the full text, which is not cached.
Reason: Bare protein binding is uninformative, and this row's own evidence does not support a more informative molecular function. PMID:21166461 is an EntA-EntE interaction study: its abstract reports EntA-EntE complex formation and stimulation of EntE, and mentions holo-EntB only as background. Nothing visible in the cached abstract assays EntE catalysis on EntB, so this row cannot be refined to GO:0008668. That activity is annotated separately from the dedicated EntE/EntB enzymology papers. Removal does not dispute the EntE-EntB interaction, which may come from pull-down data in the full text, not cached here.
IPI PMID:21166461 Enzymatic adenylation of 2,3-dihydroxybenzoate is enhanced b...
REMOVE
Summary: EntE-EntA interaction (IPI); tetrameric EntA binds EntE and stimulates DHB adenylation about 6-fold.
Reason: Removal reflects only that bare GO:0005515 carries no functional information; the EntA-EntE interaction is well supported (pull-downs, cross-linking, ITC). No GO MF term captures 'binds and is stimulated by the upstream pathway enzyme' more informatively for EntE; the stimulation is a regulatory property of EntE activity rather than a separate function of EntE.
IPI PMID:24561554 The binary protein-protein interaction landscape of Escheric...
REMOVE
Summary: EntE-EntB interaction detected in the proteome-wide E. coli binary interaction (yeast two-hybrid) screen.
Reason: A high-throughput Y2H hit gives no information about molecular function, so bare GO:0005515 is uninformative. The EntE-EntB interaction is biologically real (EntB is EntE's carrier-protein substrate), and that function is represented by GO:0008668. Removal does not mean the interaction is false.
binding of 2,3-DHB to EntE and EntB primes these proteins for efficient complexation, thus facilitating direct channeling of the siderophore precursor 2,3-DHB-AMP.
IDA PMID:18304323 Protein abundance profiling of the Escherichia coli cytosol.
ACCEPT
Summary: Cytosol localisation from a whole-cytosol proteomics survey; EntE is a soluble cytoplasmic enzyme.
Reason: EntE is a soluble cytoplasmic enzyme (described as cytoplasmic in multiple papers) and was detected in the cytosolic fraction proteome. This is the principal location of its activity.
Biosynthesis of enterobactin, the Escherichia coli catecholate siderophore, requires adenylation of 2,3-dihydroxybenzoic acid (2,3-DHB) by the cytoplasmic enzyme EntE.
EXP PMID:10688898 The EntF and EntE adenylation domains of Escherichia coli en...
ACCEPT
Summary: Experimental support for EntE's core activity: ATP-dependent adenylation of 2,3-DHB and transfer of the DHB moiety onto the phosphopantetheine of holo-EntB ArCP.
Reason: GO:0008668 precisely describes EntE's catalytic function (EC 6.2.1.71). EntE adenylates DHB and ligates it to the phosphopantetheinylated ArCP of EntB, as shown by purified-enzyme studies from several laboratories. Abstract-only papers are deferred to the curator; the activity is independently established by full-text papers (PMID:20359185, PMID:19852513).
Enterobactin, the tris-(N-(2,3-dihydroxybenzoyl)serine) trilactone siderophore of Escherichia coli, is synthesized by a three-protein (EntE, B, F) six-module nonribosomal peptide synthetase (NRPS).
Belonging to the family of aryl acid adenylating enzymes (AAAE), EntE is characterized by a two-step adenylation/ligation reaction: first, the enzyme catalyzes the condensation of DHB and ATP to form an adenylate intermediate, followed by the ligation of DHB onto the phosphopantetheinylated cofactor that is bound to the ArCP of EntB
EXP PMID:16567620 A protein interaction surface in nonribosomal peptide synthe...
ACCEPT
Summary: Experimental support for EntE's core activity: ATP-dependent adenylation of 2,3-DHB and transfer of the DHB moiety onto the phosphopantetheine of holo-EntB ArCP.
Reason: GO:0008668 precisely describes EntE's catalytic function (EC 6.2.1.71). EntE adenylates DHB and ligates it to the phosphopantetheinylated ArCP of EntB, as shown by purified-enzyme studies from several laboratories. Abstract-only papers are deferred to the curator; the activity is independently established by full-text papers (PMID:20359185, PMID:19852513).
We report the use of combinatorial mutagenesis coupled with in vivo selection for the production of the Escherichia coli NRPS product enterobactin to map the surface of the aryl carrier protein (ArCP) domain of EntB that interacts with the downstream elongation module EntF.
Belonging to the family of aryl acid adenylating enzymes (AAAE), EntE is characterized by a two-step adenylation/ligation reaction: first, the enzyme catalyzes the condensation of DHB and ATP to form an adenylate intermediate, followed by the ligation of DHB onto the phosphopantetheinylated cofactor that is bound to the ArCP of EntB
EXP PMID:19852513 Enterobactin synthetase-catalyzed formation of P(1),P(3)-dia...
ACCEPT
Summary: Experimental support for EntE's core activity: ATP-dependent adenylation of 2,3-DHB and transfer of the DHB moiety onto the phosphopantetheine of holo-EntB ArCP.
Reason: GO:0008668 precisely describes EntE's catalytic function (EC 6.2.1.71). EntE adenylates DHB and ligates it to the phosphopantetheinylated ArCP of EntB, as shown by purified-enzyme studies from several laboratories. Abstract-only papers are deferred to the curator; the activity is independently established by full-text papers (PMID:20359185, PMID:19852513).
EntE ligase catalyzes the ATP-dependent transfer of DHB onto the phosphopantothenylated cofactor that is bound to the aryl carrier protein domain (ArCP) of EntB to yield the covalently arylated EntB.
EXP PMID:20359185 Kinetic and inhibition studies of dihydroxybenzoate-AMP liga...
ACCEPT
Summary: Experimental support for EntE's core activity: ATP-dependent adenylation of 2,3-DHB and transfer of the DHB moiety onto the phosphopantetheine of holo-EntB ArCP.
Reason: GO:0008668 precisely describes EntE's catalytic function (EC 6.2.1.71). EntE adenylates DHB and ligates it to the phosphopantetheinylated ArCP of EntB, as shown by purified-enzyme studies from several laboratories. Abstract-only papers are deferred to the curator; the activity is independently established by full-text papers (PMID:20359185, PMID:19852513).
Belonging to the family of aryl acid adenylating enzymes (AAAE), EntE is characterized by a two-step adenylation/ligation reaction: first, the enzyme catalyzes the condensation of DHB and ATP to form an adenylate intermediate, followed by the ligation of DHB onto the phosphopantetheinylated cofactor that is bound to the ArCP of EntB
EXP PMID:20845982 MbtH-like proteins as integral components of bacterial nonri...
ACCEPT
Summary: Experimental support for EntE's core activity: ATP-dependent adenylation of 2,3-DHB and transfer of the DHB moiety onto the phosphopantetheine of holo-EntB ArCP.
Reason: GO:0008668 precisely describes EntE's catalytic function (EC 6.2.1.71). EntE adenylates DHB and ligates it to the phosphopantetheinylated ArCP of EntB, as shown by purified-enzyme studies from several laboratories. Abstract-only papers are deferred to the curator; the activity is independently established by full-text papers (PMID:20359185, PMID:19852513).
We overexpressed and purified EntE, an A domain specific for 2,3-dihydroxybenzoate (2,3-Dhb), and EntF, a multidomain NRPS with an A domain specific for L-Ser, in the presence or absence of YbdZ.
IDA PMID:2531000 Subcloning, expression, and purification of the enterobactin...
ACCEPT
Summary: Experimental support for EntE's core activity: ATP-dependent adenylation of 2,3-DHB and transfer of the DHB moiety onto the phosphopantetheine of holo-EntB ArCP.
Reason: GO:0008668 precisely describes EntE's catalytic function (EC 6.2.1.71). EntE adenylates DHB and ligates it to the phosphopantetheinylated ArCP of EntB, as shown by purified-enzyme studies from several laboratories. Abstract-only papers are deferred to the curator; the activity is independently established by full-text papers (PMID:20359185, PMID:19852513).
Summary: Electronic annotation from InterPro IPR011963 / RHEA:61652 / EC 6.2.1.71; matches EntE's experimentally established activity.
Reason: The overall reaction DHB + ATP + holo-[ACP] = DHB-[ACP] + AMP + PPi is exactly EntE's two-step adenylation/ligation reaction onto holo-EntB ArCP. Consistent with the experimental rows.
Belonging to the family of aryl acid adenylating enzymes (AAAE), EntE is characterized by a two-step adenylation/ligation reaction: first, the enzyme catalyzes the condensation of DHB and ATP to form an adenylate intermediate, followed by the ligation of DHB onto the phosphopantetheinylated cofactor that is bound to the ArCP of EntB
file:ECOLI/entE/entE-uniprot.txt
Belongs to the ATP-dependent AMP-binding enzyme family.
Summary: Phylogenetic inference that EntE-clade adenylation enzymes act in enterobactin biosynthesis; EntE itself is among the experimentally grounded members.
Reason: EntE catalyses the DHB activation/loading step of enterobactin assembly. The presence of P10378 in WITH/FROM reflects its own experimental grounding.
Belonging to the family of aryl acid adenylating enzymes (AAAE), EntE is characterized by a two-step adenylation/ligation reaction: first, the enzyme catalyzes the condensation of DHB and ATP to form an adenylate intermediate, followed by the ligation of DHB onto the phosphopantetheinylated cofactor that is bound to the ArCP of EntB
The holo-EntF monomer acts as the catalyst for the formation of the three amide and three ester bonds in enterobactin using ATP, L-serine, and acyl-holo-EntB, acylated with 2,3-dihydroxybenzoate by EntE, as substrates
IDA PMID:19699210 Ligand-induced conformational rearrangements promote interac...
ACCEPT
Summary: EntE directly participates in enterobactin biosynthesis by activating DHB and loading it onto holo-EntB.
Reason: EntE performs a defined catalytic step of enterobactin biosynthesis (DHB adenylation and transfer to EntB ArCP), which is participation in the process, not mere necessity.
Biosynthesis of enterobactin, the Escherichia coli catecholate siderophore, requires adenylation of 2,3-dihydroxybenzoic acid (2,3-DHB) by the cytoplasmic enzyme EntE.
binding of 2,3-DHB to EntE and EntB primes these proteins for efficient complexation, thus facilitating direct channeling of the siderophore precursor 2,3-DHB-AMP.
IDA PMID:21166461 Enzymatic adenylation of 2,3-dihydroxybenzoate is enhanced b...
ACCEPT
Summary: EntE directly participates in enterobactin biosynthesis by activating DHB and loading it onto holo-EntB.
Reason: EntE performs a defined catalytic step of enterobactin biosynthesis (DHB adenylation and transfer to EntB ArCP), which is participation in the process, not mere necessity.
The holo-EntF monomer acts as the catalyst for the formation of the three amide and three ester bonds in enterobactin using ATP, L-serine, and acyl-holo-EntB, acylated with 2,3-dihydroxybenzoate by EntE, as substrates
IMP PMID:4939766 Location of three genes concerned with the conversion of 2,3...
ACCEPT
Summary: entE mutants cannot convert DHB into enterochelin (classic genetic complementation study).
Reason: Loss-of-function mutants lacking EntE are blocked in enterobactin formation from DHB. The broad acts_upstream_of_or_within qualifier is appropriate for this genetic evidence; EntE directly catalyses a step of the pathway.
No detectable diffusible intermediate in the formation of enterochelin from 2,3-dihydroxybenzoate was formed by cell extracts of mutants carrying mutations in the entD, entE, or entF genes.
EXP PMID:10692387 Membrane association of the Escherichia coli enterobactin sy...
KEEP AS NON CORE
Summary: A small fraction of EntE fractionates with a minor membrane fraction intermediate in density between cytoplasmic and outer membranes.
Reason: The experiment supports peripheral association of a minor fraction of EntE with a membrane fraction; EntE is predominantly cytoplasmic and has no transmembrane segments. 'membrane' is as specific as this evidence allows (the fraction is not assigned to a defined membrane), so it is kept, but as non-core.
Here, consistent with the idea that cytoplasmic proteins found in shockates have an affinity for membranes, a small fraction of each was found in membrane preparations.
Summary: UniProt SubCell mapping of 'Membrane' (itself based on PMID:10692387).
Reason: Same claim as the EXP row: a minor membrane-associated fraction of a mainly cytosolic enzyme. Kept as non-core for consistency with the EXP row.
Here, consistent with the idea that cytoplasmic proteins found in shockates have an affinity for membranes, a small fraction of each was found in membrane preparations.
Summary: IBA transfer of CoA-ligase activity from acyl-CoA synthetase members of the ANL (AMP-binding enzyme) superfamily.
Reason: EntE is an ANL-superfamily adenylate-forming enzyme, so the essence (acyl-adenylate formation followed by thioester formation) is sound, but its thiol acceptor is the phosphopantetheine arm of the holo-EntB aryl carrier protein, not free coenzyme A. No CoA-ligase activity has been reported for EntE. The accurate term is GO:0008668, which the gene already carries with experimental evidence.
Propagation Review
Root cause:TERM SCOPING PROBLEM
Failure modes:FUNCTIONAL DIVERGENCE
Sources checked:
PANTHER:PTN000756557SUPPORTS SOURCE BUT NOT TARGET
The deep ANL-superfamily node carries CoA ligase activity from acyl-CoA synthetase members, but EntE belongs to the aryl acid adenylating enzyme lineage whose thiol acceptor is a carrier-protein phosphopantetheine rather than free CoA. The adenylate-forming thioester-ligase chemistry transfers; the CoA acceptor does not.
Belonging to the family of aryl acid adenylating enzymes (AAAE), EntE is characterized by a two-step adenylation/ligation reaction: first, the enzyme catalyzes the condensation of DHB and ATP to form an adenylate intermediate, followed by the ligation of DHB onto the phosphopantetheinylated cofactor that is bound to the ArCP of EntB
file:ECOLI/entE/entE-uniprot.txt
Belongs to the ATP-dependent AMP-binding enzyme family.
Summary: ARBA electronic annotation to the broad acid-thiol ligase class.
Reason: True but general: GO:0008668 is an is_a descendant of GO:0016878 and is the specific activity supported for EntE (already annotated with experimental evidence).
Belonging to the family of aryl acid adenylating enzymes (AAAE), EntE is characterized by a two-step adenylation/ligation reaction: first, the enzyme catalyzes the condensation of DHB and ATP to form an adenylate intermediate, followed by the ligation of DHB onto the phosphopantetheinylated cofactor that is bound to the ArCP of EntB
Summary: InterPro IPR011963-based annotation to siderophore biosynthesis.
Reason: Correct but general: the siderophore made with EntE is enterobactin, and GO:0009239 enterobactin biosynthetic process (an is_a descendant of GO:0019290) is already annotated with experimental evidence.
Biosynthesis of enterobactin, the Escherichia coli catecholate siderophore, requires adenylation of 2,3-dihydroxybenzoic acid (2,3-DHB) by the cytoplasmic enzyme EntE.
EXP PMID:10688898 The EntF and EntE adenylation domains of Escherichia coli en...
MODIFY
Summary: Experimental annotation to the enterobactin synthetase DHB-serine ligase reaction; EntE catalyses only the DHB activation/loading half of this multi-protein reaction.
Reason: GO:0047527 is defined by the product N-(2,3-dihydroxybenzoyl)-L-serine, i.e. the DHB-serine amide bond. That bond (and the ester bonds) is made by EntF using DHB-loaded holo-EntB, not by EntE. EntE's own experimentally demonstrated reaction is DHB adenylation and transfer onto holo-EntB ArCP (GO:0008668). The curators' evidence is sound for EntE's contribution to the enterobactin synthetase reaction (EC 6.3.2.14, which UniProt assigns to all synthetase components), so the essence is retained and refined to EntE's actual step rather than removed. The same reconstitution study found no stable complex of the synthetase components, so EntE cannot be treated as a subunit of a single enzyme that carries out the DHB-serine ligation as a whole. Its contribution is the separate upstream DHB-loading reaction.
Enterobactin, the tris-(N-(2,3-dihydroxybenzoyl)serine) trilactone siderophore of Escherichia coli, is synthesized by a three-protein (EntE, B, F) six-module nonribosomal peptide synthetase (NRPS).
The holo-EntF monomer acts as the catalyst for the formation of the three amide and three ester bonds in enterobactin using ATP, L-serine, and acyl-holo-EntB, acylated with 2,3-dihydroxybenzoate by EntE, as substrates
EXP PMID:16567620 A protein interaction surface in nonribosomal peptide synthe...
MODIFY
Summary: Experimental annotation to the enterobactin synthetase DHB-serine ligase reaction; EntE catalyses only the DHB activation/loading half of this multi-protein reaction.
Reason: GO:0047527 is defined by the product N-(2,3-dihydroxybenzoyl)-L-serine, i.e. the DHB-serine amide bond. That bond (and the ester bonds) is made by EntF using DHB-loaded holo-EntB, not by EntE. EntE's own experimentally demonstrated reaction is DHB adenylation and transfer onto holo-EntB ArCP (GO:0008668). The curators' evidence is sound for EntE's contribution to the enterobactin synthetase reaction (EC 6.3.2.14, which UniProt assigns to all synthetase components), so the essence is retained and refined to EntE's actual step rather than removed. The same reconstitution study found no stable complex of the synthetase components, so EntE cannot be treated as a subunit of a single enzyme that carries out the DHB-serine ligation as a whole. Its contribution is the separate upstream DHB-loading reaction.
We report the use of combinatorial mutagenesis coupled with in vivo selection for the production of the Escherichia coli NRPS product enterobactin to map the surface of the aryl carrier protein (ArCP) domain of EntB that interacts with the downstream elongation module EntF.
The holo-EntF monomer acts as the catalyst for the formation of the three amide and three ester bonds in enterobactin using ATP, L-serine, and acyl-holo-EntB, acylated with 2,3-dihydroxybenzoate by EntE, as substrates
EXP PMID:19852513 Enterobactin synthetase-catalyzed formation of P(1),P(3)-dia...
MODIFY
Summary: Experimental annotation to the enterobactin synthetase DHB-serine ligase reaction; EntE catalyses only the DHB activation/loading half of this multi-protein reaction.
Reason: GO:0047527 is defined by the product N-(2,3-dihydroxybenzoyl)-L-serine, i.e. the DHB-serine amide bond. That bond (and the ester bonds) is made by EntF using DHB-loaded holo-EntB, not by EntE. EntE's own experimentally demonstrated reaction is DHB adenylation and transfer onto holo-EntB ArCP (GO:0008668). The curators' evidence is sound for EntE's contribution to the enterobactin synthetase reaction (EC 6.3.2.14, which UniProt assigns to all synthetase components), so the essence is retained and refined to EntE's actual step rather than removed. The same reconstitution study found no stable complex of the synthetase components, so EntE cannot be treated as a subunit of a single enzyme that carries out the DHB-serine ligation as a whole. Its contribution is the separate upstream DHB-loading reaction.
EntE ligase catalyzes the ATP-dependent transfer of DHB onto the phosphopantothenylated cofactor that is bound to the aryl carrier protein domain (ArCP) of EntB to yield the covalently arylated EntB.
The holo-EntF monomer acts as the catalyst for the formation of the three amide and three ester bonds in enterobactin using ATP, L-serine, and acyl-holo-EntB, acylated with 2,3-dihydroxybenzoate by EntE, as substrates
EXP PMID:20359185 Kinetic and inhibition studies of dihydroxybenzoate-AMP liga...
MODIFY
Summary: Experimental annotation to the enterobactin synthetase DHB-serine ligase reaction; EntE catalyses only the DHB activation/loading half of this multi-protein reaction.
Reason: GO:0047527 is defined by the product N-(2,3-dihydroxybenzoyl)-L-serine, i.e. the DHB-serine amide bond. That bond (and the ester bonds) is made by EntF using DHB-loaded holo-EntB, not by EntE. EntE's own experimentally demonstrated reaction is DHB adenylation and transfer onto holo-EntB ArCP (GO:0008668). The curators' evidence is sound for EntE's contribution to the enterobactin synthetase reaction (EC 6.3.2.14, which UniProt assigns to all synthetase components), so the essence is retained and refined to EntE's actual step rather than removed. The same reconstitution study found no stable complex of the synthetase components, so EntE cannot be treated as a subunit of a single enzyme that carries out the DHB-serine ligation as a whole. Its contribution is the separate upstream DHB-loading reaction.
Belonging to the family of aryl acid adenylating enzymes (AAAE), EntE is characterized by a two-step adenylation/ligation reaction: first, the enzyme catalyzes the condensation of DHB and ATP to form an adenylate intermediate, followed by the ligation of DHB onto the phosphopantetheinylated cofactor that is bound to the ArCP of EntB
The holo-EntF monomer acts as the catalyst for the formation of the three amide and three ester bonds in enterobactin using ATP, L-serine, and acyl-holo-EntB, acylated with 2,3-dihydroxybenzoate by EntE, as substrates
EXP PMID:20845982 MbtH-like proteins as integral components of bacterial nonri...
MODIFY
Summary: Experimental annotation to the enterobactin synthetase DHB-serine ligase reaction; EntE catalyses only the DHB activation/loading half of this multi-protein reaction.
Reason: GO:0047527 is defined by the product N-(2,3-dihydroxybenzoyl)-L-serine, i.e. the DHB-serine amide bond. That bond (and the ester bonds) is made by EntF using DHB-loaded holo-EntB, not by EntE. EntE's own experimentally demonstrated reaction is DHB adenylation and transfer onto holo-EntB ArCP (GO:0008668). The curators' evidence is sound for EntE's contribution to the enterobactin synthetase reaction (EC 6.3.2.14, which UniProt assigns to all synthetase components), so the essence is retained and refined to EntE's actual step rather than removed. The same reconstitution study found no stable complex of the synthetase components, so EntE cannot be treated as a subunit of a single enzyme that carries out the DHB-serine ligation as a whole. Its contribution is the separate upstream DHB-loading reaction.
We overexpressed and purified EntE, an A domain specific for 2,3-dihydroxybenzoate (2,3-Dhb), and EntF, a multidomain NRPS with an A domain specific for L-Ser, in the presence or absence of YbdZ.
The holo-EntF monomer acts as the catalyst for the formation of the three amide and three ester bonds in enterobactin using ATP, L-serine, and acyl-holo-EntB, acylated with 2,3-dihydroxybenzoate by EntE, as substrates
EXP PMID:2531000 Subcloning, expression, and purification of the enterobactin...
MODIFY
Summary: Experimental annotation to the enterobactin synthetase DHB-serine ligase reaction; EntE catalyses only the DHB activation/loading half of this multi-protein reaction.
Reason: GO:0047527 is defined by the product N-(2,3-dihydroxybenzoyl)-L-serine, i.e. the DHB-serine amide bond. That bond (and the ester bonds) is made by EntF using DHB-loaded holo-EntB, not by EntE. EntE's own experimentally demonstrated reaction is DHB adenylation and transfer onto holo-EntB ArCP (GO:0008668). The curators' evidence is sound for EntE's contribution to the enterobactin synthetase reaction (EC 6.3.2.14, which UniProt assigns to all synthetase components), so the essence is retained and refined to EntE's actual step rather than removed. The same reconstitution study found no stable complex of the synthetase components, so EntE cannot be treated as a subunit of a single enzyme that carries out the DHB-serine ligation as a whole. Its contribution is the separate upstream DHB-loading reaction.
The holo-EntF monomer acts as the catalyst for the formation of the three amide and three ester bonds in enterobactin using ATP, L-serine, and acyl-holo-EntB, acylated with 2,3-dihydroxybenzoate by EntE, as substrates
EXP PMID:9214294 Enterobactin biosynthesis in Escherichia coli: isochorismate...
MODIFY
Summary: Experimental annotation to the enterobactin synthetase DHB-serine ligase reaction; EntE catalyses only the DHB activation/loading half of this multi-protein reaction.
Reason: GO:0047527 is defined by the product N-(2,3-dihydroxybenzoyl)-L-serine, i.e. the DHB-serine amide bond. That bond (and the ester bonds) is made by EntF using DHB-loaded holo-EntB, not by EntE. EntE's own experimentally demonstrated reaction is DHB adenylation and transfer onto holo-EntB ArCP (GO:0008668). The curators' evidence is sound for EntE's contribution to the enterobactin synthetase reaction (EC 6.3.2.14, which UniProt assigns to all synthetase components), so the essence is retained and refined to EntE's actual step rather than removed. The same reconstitution study found no stable complex of the synthetase components, so EntE cannot be treated as a subunit of a single enzyme that carries out the DHB-serine ligation as a whole. Its contribution is the separate upstream DHB-loading reaction.
The holo-EntF monomer acts as the catalyst for the formation of the three amide and three ester bonds in enterobactin using ATP, L-serine, and acyl-holo-EntB, acylated with 2,3-dihydroxybenzoate by EntE, as substrates
IDA PMID:9485415 Reconstitution and characterization of the Escherichia coli ...
MODIFY
Summary: Experimental annotation to the enterobactin synthetase DHB-serine ligase reaction; EntE catalyses only the DHB activation/loading half of this multi-protein reaction.
Reason: GO:0047527 is defined by the product N-(2,3-dihydroxybenzoyl)-L-serine, i.e. the DHB-serine amide bond. That bond (and the ester bonds) is made by EntF using DHB-loaded holo-EntB, not by EntE. EntE's own experimentally demonstrated reaction is DHB adenylation and transfer onto holo-EntB ArCP (GO:0008668). The curators' evidence is sound for EntE's contribution to the enterobactin synthetase reaction (EC 6.3.2.14, which UniProt assigns to all synthetase components), so the essence is retained and refined to EntE's actual step rather than removed. The same reconstitution study found no stable complex of the synthetase components, so EntE cannot be treated as a subunit of a single enzyme that carries out the DHB-serine ligation as a whole. Its contribution is the separate upstream DHB-loading reaction.
The holo-EntF monomer acts as the catalyst for the formation of the three amide and three ester bonds in enterobactin using ATP, L-serine, and acyl-holo-EntB, acylated with 2,3-dihydroxybenzoate by EntE, as substrates
Reason: GO:0047527 is defined by the product N-(2,3-dihydroxybenzoyl)-L-serine, i.e. the DHB-serine amide bond. That bond (and the ester bonds) is made by EntF using DHB-loaded holo-EntB, not by EntE. EntE's own experimentally demonstrated reaction is DHB adenylation and transfer onto holo-EntB ArCP (GO:0008668). The curators' evidence is sound for EntE's contribution to the enterobactin synthetase reaction (EC 6.3.2.14, which UniProt assigns to all synthetase components), so the essence is retained and refined to EntE's actual step rather than removed. The same reconstitution study found no stable complex of the synthetase components, so EntE cannot be treated as a subunit of a single enzyme that carries out the DHB-serine ligation as a whole. Its contribution is the separate upstream DHB-loading reaction.
The holo-EntF monomer acts as the catalyst for the formation of the three amide and three ester bonds in enterobactin using ATP, L-serine, and acyl-holo-EntB, acylated with 2,3-dihydroxybenzoate by EntE, as substrates
Belonging to the family of aryl acid adenylating enzymes (AAAE), EntE is characterized by a two-step adenylation/ligation reaction: first, the enzyme catalyzes the condensation of DHB and ATP to form an adenylate intermediate, followed by the ligation of DHB onto the phosphopantetheinylated cofactor that is bound to the ArCP of EntB
There is no evidence for a stable complex of the enterobactin synthetase components.
Core Functions
ATP-dependent adenylation of 2,3-dihydroxybenzoate and transfer of the DHB moiety onto the phosphopantetheinylated aryl carrier protein domain of holo-EntB, loading the aryl donor for EntF-catalysed enterobactin assembly in the cytosol.
Belonging to the family of aryl acid adenylating enzymes (AAAE), EntE is characterized by a two-step adenylation/ligation reaction: first, the enzyme catalyzes the condensation of DHB and ATP to form an adenylate intermediate, followed by the ligation of DHB onto the phosphopantetheinylated cofactor that is bound to the ArCP of EntB
The holo-EntF monomer acts as the catalyst for the formation of the three amide and three ester bonds in enterobactin using ATP, L-serine, and acyl-holo-EntB, acylated with 2,3-dihydroxybenzoate by EntE, as substrates
Q: Should EntE's experimental GO:0047527 (2,3-dihydroxybenzoate-serine ligase activity) annotations be converted to GO:0008668, or to contributes_to GO:0047527, given that EntF, not EntE, forms the DHB-serine amide bond?
Q: Does EntE-catalysed Ap4A formation occur at physiologically meaningful levels in iron-starved cells when holo-EntB is limiting, and does it feed into Ap4A stress signalling?
Q: Is the minor membrane-associated pool of EntE functionally linked to enterobactin export, as proposed from the fractionation data?
Suggested Experiments
Experiment: Measure Ap4A in iron-limited wild type, entE deletion, and entB deletion strains (the last expected to raise EntE-dependent Ap4A by removing the acceptor), and complement with catalytically inactive EntE.
Hypothesis: Ap4A produced by EntE contributes to the cellular Ap4A pool under iron starvation.
Type: metabolite profiling and genetics
External Prediction Reviews
These computational predictions are reviewed separately from the GOA annotation set used for this review. The assessments below are from this project and do not constitute official GO annotations or endorsement by GO/UniProt. They are not included in the existing annotation review above.
NCBI PGAP names WP_000026812.1 "(2,3-dihydroxybenzoyl)adenylate synthase EntE" from BlastRule NBR012093 and assigns three EC numbers (2.5.1.-, 6.2.1.71, 6.3.2.14) but no GO terms. The GO gap comes from how PGAP propagates GO: it passes on GO only from the model that names the protein, and NBR012093 carries none. EntE also matches two equivalog HMMs with the same product name, NF008192 and TIGR02275, and both carry GO:0008668 (2,3-dihydroxybenzoate--[aryl-carrier protein] ligase activity) and GO:0019290 (siderophore biosynthetic process). Because a BlastRule outranks them for naming, their GO is discarded, so PGAP omits EntE's core molecular function. GOA supports that function with five EXP annotations and one IDA annotation. Judged against the literature, EC 6.2.1.71 is exactly EntE's activity: ATP-dependent adenylation of 2,3-dihydroxybenzoate (DHB) and transfer onto holo-EntB ArCP. EC 6.3.2.14, the overall enterobactin synthase reaction, is defensible only as a component-level assignment, since EntF forms the amide and ester bonds; UniProt makes the same assignment. EC 2.5.1.- (transferase of alkyl or aryl groups other than methyl) is not supported. DHB transfer onto the phosphopantetheine thiol is an aroyl (acyl) transfer, and it is the second half-reaction of EC 6.2.1.71, not a separate alkyl/aryl-transferase activity.
Prediction method: NCBI PGAP · Version: current WP_ records (wp); GO from WP_ record · PMID:33270901
CURATION MISTAKE
Review rationale: EC 2.5.1 covers transfer of alkyl or aryl groups other than methyl. EntE has no such activity. Its second half-reaction moves the 2,3-dihydroxybenzoyl group from DHB-AMP onto the phosphopantetheine thiol of holo-EntB. That is an aroyl (acyl) transfer, which UniProt records as "S-dihydroxybenzoyltransferase" kinetics with no EC number, and it is already part of the EC 6.2.1.71 overall reaction. The EC 2.5.1.- assignment looks like an outdated or misclassified label for that half-reaction, carried by the naming BlastRule. Its exact provenance is not recorded in the evidence files. It should be dropped, not replaced.
PMID:9214294: "This holo-EntB is then acylated with 2,3-DHB in a reaction catalyzed by EntE, previously described as the 2,3-DHB-AMP ligase, with a kcat of 100 min-1"
Prediction method: NCBI PGAP · Version: current WP_ records (wp); GO from WP_ record · PMID:33270901
Review rationale: Correct. EC 6.2.1.71 (2,3-dihydroxybenzoate--[aryl-carrier protein] ligase) is EntE's experimentally established activity: ATP-dependent adenylation of DHB and transfer of the DHB moiety onto the phosphopantetheine of holo-EntB ArCP. The equivalog HMMs NF008192 and TIGR02275 carry the same EC. UniProt already holds it with experimental evidence, and GOA holds the corresponding GO:0008668, so the prediction is correct but not novel. PGAP misses the matching GO term only because the naming BlastRule has no GO.
PMID:20359185: "Belonging to the family of aryl acid adenylating enzymes (AAAE), EntE is characterized by a two-step adenylation/ligation reaction: first, the enzyme catalyzes the condensation of DHB and ATP to form an adenylate intermediate, followed by the ligation of DHB onto the phosphopantetheinylated cofactor that is bound to the ArCP of EntB"
Prediction method: NCBI PGAP · Version: current WP_ records (wp); GO from WP_ record · PMID:33270901
Review rationale: Defensible as a component-level assignment, not as EntE's own catalytic activity. EC 6.3.2.14 (enterobactin synthase) is the overall reaction 3 DHB + 3 L-serine + 6 ATP -> enterobactin, carried out jointly by EntB, EntD, EntE and EntF. EntF makes the amide and ester bonds, using the DHB-loaded holo-EntB that EntE produces. UniProt follows a component-level convention here: it assigns the synthase EC 6.3.2.14 to each component of the system, EntE included, citing experimental evidence. The PGAP prediction therefore matches the existing record under that convention, but it should not be read as EntE catalysing the full reaction by itself. For GO, this reaction is better represented on EntE by GO:0008668 than by GO:0047527.
PMID:9485415: "The holo-EntF monomer acts as the catalyst for the formation of the three amide and three ester bonds in enterobactin using ATP, L-serine, and acyl-holo-EntB, acylated with 2,3-dihydroxybenzoate by EntE, as substrates"