Any feedback?
Please rate this page
(literature.php)
(0/150)

BRENDA support

Literature summary for 6.2.1.71 extracted from

  • Ishikawa, F.; Nohara, M.; Nakamura, S.; Nakanishi, I.; Tanabe, G.
    Precise probing of residue roles by NRPS code swapping mutation, enzymatic characterization, modeling, and substrate promiscuity of aryl acid adenylation domains (2020), Biochemistry, 59, 351-363 .
    View publication on PubMed
Search for more literature for 6.2.1.71

Cloned(Commentary)

Cloned (Comment) Organism
gene entE, recobinant expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) Escherichia coli

Crystallization (Commentary)

Crystallization (Comment) Organism
construction of a model for DHB binding to EntE (PDB ID 3RG2) through superposition of the DHB-DhbE complex structure (PDB entry 1MD9) Escherichia coli

Protein Variants

Protein Variants Comment Organism
additional information detection of the role of active-site residues in aryl acid adenylation domains (A-domains) by gradually grafting the NRPS codes used for salicylic acid (Sal) into an archetypal aryl acid A domain, EntE, that is specific for the substrate 2,3-dihydroxybenzoic acid (DHB). Enzyme kinetics and modeling studies of the EntE variants demonstrate that the NRPS code residues at positions 236, 240, and 339 collectively regulate the substrate specificity toward DHB and Sal. The EntE variants exhibit the ability to activate the non-native aryl acids 3-hydroxybenzoic acid, 3-aminobenzoic acid, 3-fluorobenzoic acid, and 3-chlorobenzoic acid. The didomain structure of the ArCP domain of EntB and EntE is also determined using mechanism-based inhibitors containing a vinylsulfonamide functional group appended to capture the terminal thiol group of the the 4'-phosphopantetheine (Ppant) arm of the ArCP domain Escherichia coli
N235A site-directed mutagenesis, altered substrate specificity and kinetics compared to wild-type Escherichia coli
N340C site-directed mutagenesis, altered substrate specificity and kinetics compared to wild-type Escherichia coli
S240C site-directed mutagenesis, altered substrate specificity and kinetics compared to wild-type Escherichia coli
V339I site-directed mutagenesis, altered substrate specificity and kinetics compared to wild-type Escherichia coli
V339L site-directed mutagenesis, altered substrate specificity and kinetics compared to wild-type Escherichia coli
Y236F site-directed mutagenesis, altered substrate specificity and kinetics compared to wild-type Escherichia coli
Y236F/S240C site-directed mutagenesis, altered substrate specificity and kinetics compared to wild-type Escherichia coli
Y236F/S240C/N340C site-directed mutagenesis, altered substrate specificity and kinetics compared to wild-type Escherichia coli
Y236F/S240C/V339I/N340C site-directed mutagenesis, altered substrate specificity and kinetics compared to wild-type Escherichia coli
Y236F/S240C/V339L/N340C site-directed mutagenesis, altered substrate specificity and kinetics compared to wild-type Escherichia coli
Y236F/S240C/V339L/N340C site-directed mutagenesis, altered substrate specificity and kinetics compared to wild-type, very poor activityy with 2,3-dihydroxybenzoate Escherichia coli

Inhibitors

Inhibitors Comment Organism Structure
additional information usage of mechanism-based inhibitors containing a vinylsulfonamide functional group appended to capture the terminal thiol group of the Ppant arm of the ArCP domain Escherichia coli

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
 additional information steady-state Michaelis-Menten kinetic analysis of wild-type and mutant enzymes, overview Escherichia coli
0.002
-
 2,3-Dihydroxybenzoate pH 8.0, 37°C, wild-type enzyme Escherichia coli
0.0027
-
 2,3-Dihydroxybenzoate pH 8.0, 37°C, mutant N340C Escherichia coli
0.0049
-
 2,3-Dihydroxybenzoate pH 8.0, 37°C, mutant V339L Escherichia coli
0.0052
-
 2,3-Dihydroxybenzoate pH 8.0, 37°C, mutant N235A Escherichia coli
0.0055
-
 salicylate pH 8.0, 37°C, mutant V339I Escherichia coli
0.0061
-
 2,3-Dihydroxybenzoate pH 8.0, 37°C, mutant V339I Escherichia coli
0.0061
-
 salicylate pH 8.0, 37°C, mutant Y236F Escherichia coli
0.0071
-
 2,3-Dihydroxybenzoate pH 8.0, 37°C, mutant Y236F Escherichia coli
0.008
-
 salicylate pH 8.0, 37°C, mutant V339L Escherichia coli
0.0089
-
 salicylate pH 8.0, 37°C, mutant N340C Escherichia coli
0.024
-
 salicylate pH 8.0, 37°C, mutant Y236F/S240C/V339I/N340C Escherichia coli
0.028
-
 salicylate pH 8.0, 37°C, wild-type enzyme Escherichia coli
0.031
-
 salicylate pH 8.0, 37°C, mutant Y236F/S240C/N340C Escherichia coli
0.039
-
 salicylate pH 8.0, 37°C, mutant Y236F/S240C Escherichia coli
0.054
-
 salicylate pH 8.0, 37°C, mutant S240C Escherichia coli
0.08
-
 salicylate pH 8.0, 37°C, mutant N235A Escherichia coli
0.318
-
 2,3-Dihydroxybenzoate pH 8.0, 37°C, mutant Y236F/S240C/N340C Escherichia coli
0.364
-
 2,3-Dihydroxybenzoate pH 8.0, 37°C, mutant S240C Escherichia coli
0.408
-
 2,3-Dihydroxybenzoate pH 8.0, 37°C, mutant Y236F/S240C Escherichia coli
0.417
-
 2,3-Dihydroxybenzoate pH 8.0, 37°C, mutant Y236F/S240C/V339I/N340C Escherichia coli
0.533
-
 salicylate pH 8.0, 37°C, mutant Y236F/S240C/V339L/N340C Escherichia coli

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+ required Escherichia coli

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
ATP + 2,3-dihydroxybenzoate Escherichia coli
-
 diphosphate + (2,3-dihydroxybenzoyl)adenylate
-
 r

Organism

Organism UniProt Comment Textmining
Escherichia coli P10378
-
-

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
ATP + 2,3-dihydroxybenzoate
-
 Escherichia coli diphosphate + (2,3-dihydroxybenzoyl)adenylate
-
 r
ATP + 2,3-dihydroxybenzoate highly preferred substrate Escherichia coli diphosphate + (2,3-dihydroxybenzoyl)adenylate
-
 r
ATP + 2-fluorobenzoate low activity Escherichia coli diphosphate + (2-fluorobenzoyl)adenylate
-
 ?
ATP + 3-aminobenzoate low activity Escherichia coli diphosphate + (3-aminobenzoyl)adenylate
-
 ?
ATP + 3-bromobenzoate low activity Escherichia coli diphosphate + (3-bromobenzoyl)adenylate
-
 ?
ATP + 3-chlorobenzoate low activity Escherichia coli diphosphate + (3-chlorobenzoyl)adenylate
-
 ?
ATP + 3-ethynylbenzoate low activity Escherichia coli diphosphate + (3-ethynylbenzoyl)adenylate
-
 ?
ATP + 3-fluorobenzoate low activity Escherichia coli diphosphate + (3-fluorobenzoyl)adenylate
-
 ?
ATP + 3-hydroxybenzoate low activity Escherichia coli diphosphate + (3-hydroxybenzoyl)adenylate
-
 ?
ATP + 3-iodobenzoate low activity Escherichia coli diphosphate + (3-iodobenzoyl)adenylate
-
 ?
ATP + 3-methoxybenzoate low activity Escherichia coli diphosphate + (3-methoxybenzoyl)adenylate
-
 ?
ATP + 3-methylbenzoate low activity Escherichia coli diphosphate + (3-methylbenzoyl)adenylate
-
 ?
ATP + anthranilate low activity Escherichia coli diphosphate + anthranylyl-adenylate
-
 ?
ATP + salicylate low activity Escherichia coli diphosphate + salicylyl-adenylate
-
 r
additional information substrate specificity analysis of wild-type and mutant enzymes, overview. veryl low activity of wild-type enzyme with 3-nitrobenzoate, 3-cyanobenzoate, 2-nitrobenzoate, 2-methylbenzoate, 2-trifluoromethylbenzoate, 2-ethynylbenzoate, 2-azidobenzoate, 2-chlorobenzoate, 2-iodobenzoate, and 2-bromobenzoate Escherichia coli ?
-
-

Synonyms

Synonyms Comment Organism
DHB-activating A-domain
-
 Escherichia coli
EntE
-
 Escherichia coli
EntE (DHB-specific)
-
 Escherichia coli

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
37
-
 assay at Escherichia coli

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.016
-
 2,3-Dihydroxybenzoate pH 8.0, 37°C, mutant N340C Escherichia coli
0.017
-
 2,3-Dihydroxybenzoate pH 8.0, 37°C, mutant N235A Escherichia coli
0.025
-
 2,3-Dihydroxybenzoate pH 8.0, 37°C, mutant V339L Escherichia coli
0.027
-
 2,3-Dihydroxybenzoate pH 8.0, 37°C, mutant Y236F Escherichia coli
0.03
-
 2,3-Dihydroxybenzoate pH 8.0, 37°C, mutant V339I Escherichia coli
0.037
-
 salicylate pH 8.0, 37°C, mutant N235A Escherichia coli
0.043
-
 2,3-Dihydroxybenzoate pH 8.0, 37°C, wild-type enzyme Escherichia coli
0.082
-
 2,3-Dihydroxybenzoate pH 8.0, 37°C, mutant Y236F/S240C Escherichia coli
0.092
-
 salicylate pH 8.0, 37°C, mutant Y236F/S240C/N340C Escherichia coli
0.102
-
 salicylate pH 8.0, 37°C, mutant Y236F/S240C/V339L/N340C Escherichia coli
0.115
-
 salicylate pH 8.0, 37°C, mutant Y236F Escherichia coli
0.117
-
 salicylate pH 8.0, 37°C, mutant Y236F/S240C/V339I/N340C Escherichia coli
0.13
-
 salicylate pH 8.0, 37°C, mutant N340C Escherichia coli
0.133
-
 2,3-Dihydroxybenzoate pH 8.0, 37°C, mutant Y236F/S240C/N340C Escherichia coli
0.138
-
 salicylate pH 8.0, 37°C, mutant Y236F/S240C Escherichia coli
0.147
-
 2,3-Dihydroxybenzoate pH 8.0, 37°C, mutant Y236F/S240C/V339I/N340C Escherichia coli
0.148
-
 salicylate pH 8.0, 37°C, mutant V339I Escherichia coli
0.155
-
 salicylate pH 8.0, 37°C, mutant V339L Escherichia coli
0.167
-
 2,3-Dihydroxybenzoate pH 8.0, 37°C, mutant S240C Escherichia coli
0.183
-
 salicylate pH 8.0, 37°C, wild-type enzyme Escherichia coli
0.2
-
 salicylate pH 8.0, 37°C, mutant S240C Escherichia coli

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5 8 assay at Escherichia coli

Cofactor

Cofactor Comment Organism Structure
ATP
-
 Escherichia coli

General Information

General Information Comment Organism
evolution the NRPS codes of DHB- and Sal-activating A-domains are about 60% similar. The notable differences involve amino acid residues at positions 236, 240, 339, and 340. Tyr236, which is positioned near the aryl group of DHB in EntE, tends to be a Phe in Sal-activating A-domains. The hydroxyl group of active-site Ser240 of EntE engages in effective hydrogen-bonding interaction with the 3-hydroxyl moiety of the DHB substrate. Consequently, the Ser residue is highly conserved in DHB-activating A-domains (EntE, BasE, and VibE). Sequence alignment reveals that this Ser is replaced with a Cys residue in Sal-activating A-domains (YbtE, MbtA, and PchD). Val339 of DhbE is conserved in the DHB-activating A-domains EntE and BasE, but not VibE, whereas the corresponding residue is replaced with a relatively more sterically demanding Leu (MbtA and YbtE) or Ile (PchD) in Sal-activating A-domains. In addition to Ser240, which functions as the hydrogen-bonding donor, Val339 is likely to act as a key residue for the selective recognition of the DHB substrate in DHB-activating A-domains. Asn340 in the active site of DhbE is strictly conserved in the DHB-activating A-domains EntE, BasE, and VibE. By contrast, the residue corresponding to Asn340 is a Cys in the Sal-activating A-domains YbtE and PchD, but not MbtA Escherichia coli
malfunction substituting Val339 with a bulky Leu or Ile residue reduces the size of the active-site cavity, allowing for the selective activation of the Sal substrate in the active site Escherichia coli
metabolism enzyme EntE catalyzes the adenylation of DHB with ATP to form DHB-AMP. The activated DHB is transferred to the ArCP domain of EntB to generate a thioester conjugate of DHB harboring the 4'-phosphopantetheine (Ppant) functionality of the ArCP domain of EntB Escherichia coli

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.191
-
 salicylate pH 8.0, 37°C, mutant Y236F/S240C/V339L/N340C Escherichia coli
0.201
-
 2,3-Dihydroxybenzoate pH 8.0, 37°C, mutant Y236F/S240C Escherichia coli
0.353
-
 2,3-Dihydroxybenzoate pH 8.0, 37°C, mutant Y236F/S240C/V339I/N340C Escherichia coli
0.42
-
 2,3-Dihydroxybenzoate pH 8.0, 37°C, mutant Y236F/S240C/N340C Escherichia coli
0.459
-
 2,3-Dihydroxybenzoate pH 8.0, 37°C, mutant S240C Escherichia coli
0.463
-
 salicylate pH 8.0, 37°C, mutant N235A Escherichia coli
2.967
-
 salicylate pH 8.0, 37°C, mutant Y236F/S240C/N340C Escherichia coli
3.27
-
 2,3-Dihydroxybenzoate pH 8.0, 37°C, mutant N235A Escherichia coli
3.54
-
 salicylate pH 8.0, 37°C, mutant Y236F/S240C Escherichia coli
3.704
-
 salicylate pH 8.0, 37°C, mutant S240C Escherichia coli
3.8
-
 2,3-Dihydroxybenzoate pH 8.0, 37°C, mutant Y236F Escherichia coli
4.875
-
 salicylate pH 8.0, 37°C, mutant Y236F/S240C/V339I/N340C Escherichia coli
4.92
-
 2,3-Dihydroxybenzoate pH 8.0, 37°C, mutant V339I Escherichia coli
5.102
-
 2,3-Dihydroxybenzoate pH 8.0, 37°C, mutant V339L Escherichia coli
5.93
-
 2,3-Dihydroxybenzoate pH 8.0, 37°C, mutant N340C Escherichia coli
6.54
-
 salicylate pH 8.0, 37°C, wild-type enzyme Escherichia coli
14.61
-
 salicylate pH 8.0, 37°C, mutant N340C Escherichia coli
18.85
-
 salicylate pH 8.0, 37°C, mutant Y236F Escherichia coli
19.34
-
 salicylate pH 8.0, 37°C, mutant V339L Escherichia coli
21.5
-
 2,3-Dihydroxybenzoate pH 8.0, 37°C, wild-type enzyme Escherichia coli
26.9
-
 salicylate pH 8.0, 37°C, mutant V339I Escherichia coli