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Literature summary extracted from
- Structure of the EntB multidomain nonribosomal peptide synthetase and functional analysis of its interaction with the EntE adenylation domain (2006), Chem. Biol., 13, 409-419.
Cloned(Commentary)
| EC Number | Cloned (Comment) | Organism |
|---|---|---|
| 3.3.2.1 | into the pGEM7z and the pET15bTEV vector for expression in Escherichia coli BL21DE3 cells | Escherichia coli |
| 6.2.1.71 | cloned in Escherichia coli BL21 | Escherichia coli |
Crystallization (Commentary)
| EC Number | Crystallization (Comment) | Organism |
|---|---|---|
| 3.3.2.1 | the crystal structure of the EntB protein is determined at a resolution of 2.3 A | Escherichia coli |
Protein Variants
| EC Number | Protein Variants | Comment | Organism |
|---|---|---|---|
| 3.3.2.1 | D227R | mutant constructed for functional analysis of EntB-EntE interaction | Escherichia coli |
| 3.3.2.1 | D240R | mutant constructed for functional analysis of EntB-EntE interaction | Escherichia coli |
| 3.3.2.1 | D244R | mutant constructed for functional analysis of EntB-EntE interaction | Escherichia coli |
| 3.3.2.1 | D263R | mutant constructed for functional analysis of EntB-EntE interaction | Escherichia coli |
| 3.3.2.1 | F264E | mutant constructed for functional analysis of EntB-EntE interaction | Escherichia coli |
| 3.3.2.1 | I239D | mutant constructed for functional analysis of EntB-EntE interaction | Escherichia coli |
| 3.3.2.1 | K269E | mutant constructed for functional analysis of EntB-EntE interaction | Escherichia coli |
| 3.3.2.1 | M249E | mutant constructed for functional analysis of EntB-EntE interaction | Escherichia coli |
| 3.3.2.1 | R247E | mutant constructed for functional analysis of EntB-EntE interaction | Escherichia coli |
| 3.3.2.1 | S245A | mutant constructed for functional analysis of EntB-EntE interaction | Escherichia coli |
| 6.2.1.71 | K473D | specific activity of mutant K473D by measuring AMP production with coupled assay= 0.0156 micromol/min/mg, by measuring diphosphate exchange= 182.7 micromol/min/mg | Escherichia coli |
| 6.2.1.71 | additional information | mutagenesis studies support hypothesis that members of adenylate -forming family of enzymes adopt two distinct conformations to catalyze the two-step mechanism | Escherichia coli |
| 6.2.1.71 | R437D | specific activity of mutant R437D by measuring AMP production with coupled assay= 0.018 micromol/min/mg, by measuring diphosphate exchange= 197.4 micromol/min/mg | Escherichia coli |
| 6.2.1.71 | R437D/K473D | specific activity of mutant R437D/K473D by measuring AMP production with coupled assay= 0.0054 micromol/min/mg, by measuring diphosphate exchange= 176.4 micromol/min/mg | Escherichia coli |
| 6.2.1.71 | R494D | specific activity of mutant R494D by measuring AMP production with coupled assay= 0.0165 micromol/min/mg, by measuring diphosphate exchange= 189 micromol/min/mg | Escherichia coli |
Metals/Ions
| EC Number | Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|---|
| 6.2.1.71 | Mg2+ | assay with 5mM MgCl2 | Escherichia coli |  |
Molecular Weight [Da]
| EC Number | Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
|---|---|---|---|---|
| 3.3.2.1 | 33090 | - |
calculated molecular mass | Escherichia coli |
| 3.3.2.1 | 33100 | - |
determinecd by MALDI-TOF spectrometry | Escherichia coli |
Organism
| EC Number | Organism | UniProt | Comment | Textmining |
|---|---|---|---|---|
| 3.3.2.1 | Escherichia coli | P0ADI4 | - |
- |
| 6.2.1.71 | Escherichia coli | - |
JM109 | - |
| 6.2.1.71 | Escherichia coli JM109 | - |
JM109 | - |
Purification (Commentary)
| EC Number | Purification (Comment) | Organism |
|---|---|---|
| 3.3.2.1 | purification is obtained by two metal chelate chromatography steps seperated by emoval of the His tag with TEV protease | Escherichia coli |
| 6.2.1.71 | protein is purified by metal chelate chromatography | Escherichia coli |
Specific Activity [micromol/min/mg]
| EC Number | Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
|---|---|---|---|---|
| 6.2.1.71 | 0.0054 | - |
specific activity of mutant R437D/K473D by using complete reaction, measuring AMP production with coupled assay | Escherichia coli |
| 6.2.1.71 | 0.018 | - |
specific activity of mutant R437D by using complete reaction, measuring AMP production with coupled assay | Escherichia coli |
| 6.2.1.71 | 0.156 | - |
specific activity of mutant K473D by using complete reaction, measuring AMP production with coupled assay | Escherichia coli |
| 6.2.1.71 | 0.165 | - |
specific activity of mutant R494D by using complete reaction, measuring AMP production with coupled assay | Escherichia coli |
| 6.2.1.71 | 0.18 | - |
specific activity of wild-type EntE by using complete reaction, measuring AMP production with coupled assay | Escherichia coli |
| 6.2.1.71 | 176.4 | - |
specific activity of mutant R437D/K473D by measuring diphosphate exchange | Escherichia coli |
| 6.2.1.71 | 182.7 | - |
specific activity of mutant K473D by measuring diphosphate exchange | Escherichia coli |
| 6.2.1.71 | 189 | - |
specific activity of mutant R494D by measuring diphosphate exchange | Escherichia coli |
| 6.2.1.71 | 197.4 | - |
specific activity of mutant R437D by measuring diphosphate exchange | Escherichia coli |
| 6.2.1.71 | 210 | - |
specific activity of wild-type EntE by measuring diphosphate exchange | Escherichia coli |
Substrates and Products (Substrate)
| EC Number | Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|---|
| 6.2.1.71 | ATP + 2,3-dihydroxybenzoate | two-step reaction catalyzed by EnTE of Escherichia coli: enzyme first catalyzes the formation of the DHB adenylate | Escherichia coli | diphosphate + (2,3-dihydroxybenzoyl)adenylate | - |
r | |
| 6.2.1.71 | ATP + 2,3-dihydroxybenzoate | two-step reaction catalyzed by EnTE of Escherichia coli: enzyme first catalyzes the formation of the DHB adenylate | Escherichia coli JM109 | diphosphate + (2,3-dihydroxybenzoyl)adenylate | - |
r | |
| 6.2.1.71 | additional information | in a second half reaction, EnTE transfers the DHB moiety to the phosphopantetheine (PPant) cofactor of the EntB aryl carrier protein (ArCP) domain, corresponding to the reaction (2,3-dihydroxybenzoyl)adenylate + phosphopantetheine cofactor-ENTB = 2,3-DHB-S-EntB + AMP | Escherichia coli | ? | - |
? | |
| 6.2.1.71 | additional information | in a second half reaction, EnTE transfers the DHB moiety to the phosphopantetheine (PPant) cofactor of the EntB aryl carrier protein (ArCP) domain, corresponding to the reaction (2,3-dihydroxybenzoyl)adenylate + phosphopantetheine cofactor-ENTB = 2,3-DHB-S-EntB + AMP | Escherichia coli JM109 | ? | - |
? | |
Subunits
| EC Number | Subunits | Comment | Organism |
|---|---|---|---|
| 3.3.2.1 | dimer | - |
Escherichia coli |
Synonyms
| EC Number | Synonyms | Comment | Organism |
|---|---|---|---|
| 3.3.2.1 | EntB | contains an N-terminal isochorismate lyase domain | Escherichia coli |
| 6.2.1.71 | EntE | - |
Escherichia coli |
pH Optimum
| EC Number | pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|---|
| 6.2.1.71 | 7.5 | - |
assay at | Escherichia coli |
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