Escherichia coli K-12 substr. MG1655 Enzyme: proofreading thioesterase in enterobactin biosynthesis

Gene: entH Accession Numbers: EG11105 (EcoCyc), b0597, ECK0590

Synonyms: ybdB

Regulation Summary Diagram: ?

Regulation summary diagram for entH

Subunit composition of proofreading thioesterase in enterobactin biosynthesis = [EntH]4
         proofreading thioesterase in enterobactin biosynthesis = EntH

EntH is a thioesterase that is involved in the biosynthesis of enterobactin [Leduc07]. It acts as a proofreading enzyme that hydrolyzes misacylated EntB aryl intermediates [Guo09, Chen09]. [Guo09] reports that the catalytic activity of EntH is sensitive to the pattern of hydroxyl groups on the aryl-CoA substrates [Guo09], while [Latham14] saw no such effect. Although EntH can utilize both CoA and holo-EntB as the substrate thioester, holo-ACP is a very poor substrate [Latham14].

While EntH is not required for enterobactin synthesis in vitro [Gehring98], it is required for optimal synthesis in vivo [Leduc07]. EntH interacts with phosphopantetheine-modified EntB in vivo [Leduc07].

Esterase activity of EntH was first discovered in a high-throughput screen of purified proteins [Kuznetsova05]. Benzoyl-CoA was found to be a good substrate, with a Km of 82 µM [Leduc07].

Crystal structures of EntH have been determined [Badger05, Wu14]. The quarternary structure is best described as a dimer of dimers [Wu14]. Enzymes with mutations in predicted active site residues have been purified and kinetically characterized [Guo09, Latham14, Wu14]. EntH belongs to the 4-hydroxybenzoyl-CoA thioesterase subfamily of the hot dog fold superfamily of proteins [Pidugu09]. Structural and functional comparisons between EntH and its paralog MenI provided insight into the substrate specificity and evolution of the hotdog fold enzymes [Latham14, Wu14].

EntH production is increased under iron starvation conditions; overproduction of EntH inhibits production of enterobactin [Leduc07].

EntH: "enterobactin" [Leduc07]

Gene Citations: [Nahlik89, Ozenberger89, Schultz91]

Locations: cytosol

Map Position: [628,523 -> 628,936] (13.55 centisomes, 49°)
Length: 414 bp / 137 aa

Molecular Weight of Polypeptide: 14.97 kD (from nucleotide sequence), 15 kD (experimental) [Nahlik87 ]

Molecular Weight of Multimer: 54.4 kD (experimental) [Guo09]

Unification Links: ASAP:ABE-0002057 , DIP:DIP-11343N , EchoBASE:EB1097 , EcoGene:EG11105 , EcoliWiki:b0597 , ModBase:P0A8Y8 , OU-Microarray:b0597 , PortEco:entH , Pride:P0A8Y8 , Protein Model Portal:P0A8Y8 , RefSeq:NP_415129 , RegulonDB:EG11105 , SMR:P0A8Y8 , String:511145.b0597 , Swiss-Model:P0A8Y8 , UniProt:P0A8Y8

Relationship Links: InterPro:IN-FAMILY:IPR003736 , InterPro:IN-FAMILY:IPR006683 , InterPro:IN-FAMILY:IPR026576 , InterPro:IN-FAMILY:IPR029069 , PDB:Structure:1VH9 , PDB:Structure:4K4C , PDB:Structure:4K4D , Pfam:IN-FAMILY:PF03061

Gene-Reaction Schematic: ?

Gene-Reaction Schematic

Genetic Regulation Schematic: ?

Genetic regulation schematic for entH

GO Terms:

Biological Process: GO:0009239 - enterobactin biosynthetic process Inferred from experiment Inferred by computational analysis [UniProtGOA12, GOA06, GOA01a, Leduc07]
Molecular Function: GO:0005515 - protein binding Inferred from experiment [Leduc07]
GO:0016289 - CoA hydrolase activity Inferred from experiment [Guo09]
GO:0016788 - hydrolase activity, acting on ester bonds Inferred from experiment [Kuznetsova05]
GO:0016790 - thiolester hydrolase activity Inferred from experiment Inferred by computational analysis [GOA06, GOA01a, Guo09]
GO:0042802 - identical protein binding Inferred from experiment [Guo09]
GO:0016787 - hydrolase activity Inferred by computational analysis [UniProtGOA11a]
Cellular Component: GO:0005829 - cytosol Inferred from experiment Inferred by computational analysis [DiazMejia09, Ishihama08]
GO:0005737 - cytoplasm Inferred by computational analysis [UniProtGOA11, UniProtGOA11a, GOA06]

MultiFun Terms: cell processes adaptations Fe aquisition
metabolism biosynthesis of building blocks cofactors, small molecule carriers enterochelin (enterobactin)

Essentiality data for entH knockouts: ?

Growth Medium Growth? T (°C) O2 pH Osm/L Growth Observations
LB enriched Yes 37 Aerobic 6.95   Yes [Gerdes03, Comment 1]
LB Lennox Yes 37 Aerobic 7   Yes [Baba06, Comment 2]
M9 medium with 1% glycerol Yes 37 Aerobic 7.2 0.35 Yes [Joyce06, Comment 3]
MOPS medium with 0.4% glucose Yes 37 Aerobic 7.2 0.22 Yes [Baba06, Comment 2]

Created 10-Mar-2009 by Keseler I , SRI International
Last-Curated ? 15-Jul-2014 by Keseler I , SRI International

Enzymatic reaction of: proofreading thioesterase

2,3-dihydroxybenzoyl-EntB + H2O <=> holo-EntB dimer + 2,3-dihydroxybenzoate

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction of enzyme catalysis.

The reaction is physiologically favored in the direction shown.

Both the ring hydroxyl positional isomer 2,4-DHB-EntB and the pathway intermediate 2,3-DHB-EntB are substrates of the enzyme [Chen09].

Enzymatic reaction of: 3-hydroxybenzoyl-CoA thioesterase (proofreading thioesterase in enterobactin biosynthesis)

EC Number: 3.1.2.-

3-hydroxybenzoyl-CoA + H2O <=> 3-hydroxybenzoate + coenzyme A + 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.

Alternative Substrates for 3-hydroxybenzoyl-CoA: 3,5-dihydroxybenzoyl-CoA [Guo09 ] , benzoyl-CoA [Latham14 , Guo09 , Leduc07 ] , 4-hydroxybenzoyl-CoA [Latham14 , Guo09 , Chen09 ] , 3-hydroxyphenylacetyl-CoA [Latham14 , Chen09 ] , palmitoyl-CoA [Guo09 ] , 1,4-dihydroxy-2-naphthoyl-CoA [Chen13 ]

The enzyme has significant hydrolytic activity towards aryl-CoA thioester substrates, but little activity towards acyl-CoA thioesters [Chen09, Guo09]. The substrate range was further investigated in [Latham14].

Kinetic Parameters:

Km (μM)
kcat (sec-1)
kcat/Km (sec-1 μM-1)

Sequence Features

Protein sequence of proofreading thioesterase in enterobactin biosynthesis with features indicated

Feature Class Location Citations Comment
Mutagenesis-Variant 48
[Guo09, UniProt12a]
[Guo09, Wu14, UniProt15]
Q → N: 290-fold decrease in activity toward salicylyl-CoA.
Q → A: Loss of activity.
Amino-Acid-Sites-That-Bind 48
[Wu14, UniProt15]
UniProt: Substrate.
Mutagenesis-Variant 54
[Guo09, Wu14, UniProt15]
UniProt: 229-fold decrease in activity toward salicylyl-CoA. Loss of activity toward benzoyl-CoA.
Protein-Segment 54 -> 55
[Wu14, UniProt15]
UniProt: Substrate binding; Sequence Annotation Type: region of interest.
Mutagenesis-Variant 63
[Guo09, Wu14, UniProt15]
E → A, D or Q: Loss of activity.
Active-Site 63
[Guo09, Wu14, UniProt15]
UniProt: Nucleophile or proton acceptor.
Mutagenesis-Variant 64
[Guo09, UniProt12a]
UniProt: 13-fold decrease in activity toward salicylyl-CoA.
Mutagenesis-Variant 67
[Guo09, UniProt12a]
[Guo09, UniProt12a]
S → C: 104-fold decrease in activity toward salicylyl-CoA.
S → A: 140-fold decrease in activity toward salicylyl-CoA.
Mutagenesis-Variant 68
[Guo09, UniProt12a]
[Wu14, Latham14, UniProt15]
M → A: 130-fold decrease in activity toward salicylyl-CoA.
M → V: 47-fold increase in catalytic efficiency toward 1,4-dihydroxy-2-naphthoyl-CoA and 10-fold increase in catalytic efficiency toward lauroyl-CoA. Does not affect catalytic efficiency toward benzoyl-CoA.
Amino-Acid-Sites-That-Bind 82
[Wu14, UniProt15]
UniProt: Substrate; via carbonyl oxygen.
Protein-Segment 89 -> 92
[Wu14, UniProt15]
UniProt: Substrate binding; Sequence Annotation Type: region of interest.

Gene Local Context (not to scale): ?

Gene local context diagram

Transcription Units:

Transcription-unit diagram

Transcription-unit diagram


Peter D. Karp on Thu Jan 16, 2003:
Predicted gene function revised as a result of E. coli genome reannotation by Serres et al. [Serres01 ].
10/20/97 Gene b0597 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG11105; confirmed by SwissProt match.


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

Badger05: Badger J, Sauder JM, Adams JM, Antonysamy S, Bain K, Bergseid MG, Buchanan SG, Buchanan MD, Batiyenko Y, Christopher JA, Emtage S, Eroshkina A, Feil I, Furlong EB, Gajiwala KS, Gao X, He D, Hendle J, Huber A, Hoda K, Kearins P, Kissinger C, Laubert B, Lewis HA, Lin J, Loomis K, Lorimer D, Louie G, Maletic M, Marsh CD, Miller I, Molinari J, Muller-Dieckmann HJ, Newman JM, Noland BW, Pagarigan B, Park F, Peat TS, Post KW, Radojicic S, Ramos A, Romero R, Rutter ME, Sanderson WE, Schwinn KD, Tresser J, Winhoven J, Wright TA, Wu L, Xu J, Harris TJ (2005). "Structural analysis of a set of proteins resulting from a bacterial genomics project." Proteins 60(4);787-96. PMID: 16021622

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

Chen13: Chen M, Ma X, Chen X, Jiang M, Song H, Guo Z (2013). "Identification of a Hotdog Fold Thioesterase Involved in the Biosynthesis of Menaquinone in Escherichia coli." J Bacteriol 195(12);2768-75. PMID: 23564174

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

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

GOA01a: GOA, DDB, FB, MGI, ZFIN (2001). "Gene Ontology annotation through association of InterPro records with GO terms."

GOA06: GOA, SIB (2006). "Electronic Gene Ontology annotations created by transferring manual GO annotations between orthologous microbial proteins."

Guo09: 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

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

Kuznetsova05: Kuznetsova E, Proudfoot M, Sanders SA, Reinking J, Savchenko A, Arrowsmith CH, Edwards AM, Yakunin AF (2005). "Enzyme genomics: Application of general enzymatic screens to discover new enzymes." FEMS Microbiol Rev 29(2);263-79. PMID: 15808744

Latham14: Latham JA, Chen D, Allen KN, Dunaway-Mariano D (2014). "Divergence of Substrate Specificity and Function in the Escherichia coli Hotdog-fold Thioesterase Paralogs YdiI and YbdB." Biochemistry. PMID: 24992697

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

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

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

Pidugu09: Pidugu LS, Maity K, Ramaswamy K, Surolia N, Suguna K (2009). "Analysis of proteins with the 'hot dog' fold: prediction of function and identification of catalytic residues of hypothetical proteins." BMC Struct Biol 9;37. PMID: 19473548

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

Serres01: Serres MH, Gopal S, Nahum LA, Liang P, Gaasterland T, Riley M (2001). "A functional update of the Escherichia coli K-12 genome." Genome Biol 2(9);RESEARCH0035. PMID: 11574054

UniProt12a: UniProt Consortium (2012). "UniProt version 2012-02 released on 2012-02-29 00:00:00." Database.

UniProt15: UniProt Consortium (2015). "UniProt version 2015-01 released on 2015-01-16 00:00:00." 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."

Wu14: Wu R, Latham JA, Chen D, Farelli JD, Zhao H, Matthews K, Allen KN, Dunaway-Mariano D (2014). "Structure and Catalysis in the Escherichia coli Hotdog-fold Thioesterase Paralogs YdiI and YbdB." Biochemistry. PMID: 25010423

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

Chen07a: 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

MendozaVargas09: Mendoza-Vargas A, Olvera L, Olvera M, Grande R, Vega-Alvarado L, Taboada B, Jimenez-Jacinto V, Salgado H, Juarez K, Contreras-Moreira B, Huerta AM, Collado-Vides J, Morett E (2009). "Genome-wide identification of transcription start sites, promoters and transcription factor binding sites in E. coli." PLoS One 4(10);e7526. PMID: 19838305

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

Zhang05: 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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