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(2,3-dihydroxybenzoyl)adenylate + holo-[aryl-carrier protein EntB]
AMP + 2,3-dihydroxybenzoyl-[aryl-carrier protein EntB]
-
-
-
?
ATP + 2,3,4-trihydroxybenzoic acid
diphosphate + (2,3,4-trihydroxybenzoyl)adenylate
-
-
-
-
?
ATP + 2,3-dihydroxybenzoate
diphosphate + (2,3-dihydroxybenzoyl)adenylate
ATP + 2,3-dihydroxybenzoate
diphosphate + 2,3-dihydroxybenzoyl adenylate
-
-
-
-
?
ATP + 2,3-dihydroxybenzoate + holo-[aryl-carrier protein EntB]
AMP + diphosphate + 2,3-dihydroxybenzoyl-[aryl-carrier protein EntB]
-
-
-
?
ATP + 2,3-dihydroxybenzoate + [aryl-carrier protein domain of EntB]
AMP + diphosphate + 2,3-dihydroxybenzoyl-[aryl-carrier protein domain of EntB]
-
-
-
?
ATP + 2,3-dihydroxybenzoate + [aryl-carrier protein EntB]
AMP + diphosphate + 2,3-dihydroxybenzoyl-[aryl-carrier protein EntB]
ATP + 2,4-dihydroxybenzoic acid
diphosphate + (2,4-dihydroxybenzoyl)adenylate
-
-
-
-
?
ATP + 2,5-dihydroxybenzoic acid
diphosphate + (2,5-dihydroxybenzoyl)adenylate
-
i.e. gentisic acid
-
-
?
ATP + 2-fluorobenzoate
diphosphate + (2-fluorobenzoyl)adenylate
low activity
-
-
?
ATP + 2-hydroxybenzoic acid
diphosphate + (2-hydroxybenzoyl)adenylate
-
i.e. salicylic acid, not 3- or 4-derivative
-
-
?
ATP + 3-aminobenzoate
diphosphate + (3-aminobenzoyl)adenylate
low activity
-
-
?
ATP + 3-bromobenzoate
diphosphate + (3-bromobenzoyl)adenylate
low activity
-
-
?
ATP + 3-chlorobenzoate
diphosphate + (3-chlorobenzoyl)adenylate
low activity
-
-
?
ATP + 3-ethynylbenzoate
diphosphate + (3-ethynylbenzoyl)adenylate
low activity
-
-
?
ATP + 3-fluorobenzoate
diphosphate + (3-fluorobenzoyl)adenylate
low activity
-
-
?
ATP + 3-hydroxybenzoate
diphosphate + (3-hydroxybenzoyl)adenylate
low activity
-
-
?
ATP + 3-hydroxybenzoate + [aryl-carrier protein EntB]
AMP + diphosphate + 3-hydroxybenzoyl-[aryl-carrier protein EntB]
-
-
-
?
ATP + 3-iodobenzoate
diphosphate + (3-iodobenzoyl)adenylate
low activity
-
-
?
ATP + 3-methoxybenzoate
diphosphate + (3-methoxybenzoyl)adenylate
low activity
-
-
?
ATP + 3-methylbenzoate
diphosphate + (3-methylbenzoyl)adenylate
low activity
-
-
?
ATP + 4-aminosalicylate + [aryl-carrier protein EntB]
AMP + diphosphate + 4-aminosalicyl-[aryl-carrier protein EntB]
-
-
-
?
ATP + anthranilate
diphosphate + anthranylyl-adenylate
low activity
-
-
?
ATP + salicylate
diphosphate + salicylyl-adenylate
low activity
-
-
r
ATP + salicylate + holo-[aryl-carrier protein domain of EntB]
AMP + diphosphate + salicyl-[aryl-carrier protein domain of EntB]
-
-
-
?
ATP + salicylate + holo-[aryl-carrier protein domain of SrfB1]
AMP + diphosphate + salicyl-[aryl-carrier protein domain of SrfB1]
SrfB1, i.e. peptidyl carrier protein of surfactin synthetase
-
-
?
ATP + salicylate + holo-[aryl-carrier protein EntB]
AMP + diphosphate + salicyl-[aryl-carrier protein EntB]
ATP + salicylate + [aryl-carrier protein EntB]
AMP + diphosphate + salicyl-[aryl-carrier protein EntB]
-
-
-
?
additional information
?
-
ATP + 2,3-dihydroxybenzoate
diphosphate + (2,3-dihydroxybenzoyl)adenylate
-
-
-
-
?
ATP + 2,3-dihydroxybenzoate
diphosphate + (2,3-dihydroxybenzoyl)adenylate
-
-
-
?
ATP + 2,3-dihydroxybenzoate
diphosphate + (2,3-dihydroxybenzoyl)adenylate
-
-
-
r
ATP + 2,3-dihydroxybenzoate
diphosphate + (2,3-dihydroxybenzoyl)adenylate
-
-
product remains enzyme-bound for further reaction in overall biosynthesis of enterobactin
?
ATP + 2,3-dihydroxybenzoate
diphosphate + (2,3-dihydroxybenzoyl)adenylate
highly preferred substrate
-
-
r
ATP + 2,3-dihydroxybenzoate
diphosphate + (2,3-dihydroxybenzoyl)adenylate
-
activation of 2,3-dihydroxybenzoic acid in the biosynthesis of siderophore enterobactin
-
-
?
ATP + 2,3-dihydroxybenzoate
diphosphate + (2,3-dihydroxybenzoyl)adenylate
-
two-step reaction catalyzed by EnTE of Escherichia coli: enzyme first catalyzes the formation of the DHB adenylate
-
-
r
ATP + 2,3-dihydroxybenzoate
diphosphate + (2,3-dihydroxybenzoyl)adenylate
-
two-step reaction catalyzed by EnTE of Escherichia coli: enzyme first catalyzes the formation of the DHB adenylate
-
-
r
ATP + 2,3-dihydroxybenzoate
diphosphate + (2,3-dihydroxybenzoyl)adenylate
-
-
-
-
?
ATP + 2,3-dihydroxybenzoate
diphosphate + (2,3-dihydroxybenzoyl)adenylate
-
involved in the biosynthesis of anguibactin
-
-
?
ATP + 2,3-dihydroxybenzoate + [aryl-carrier protein EntB]
AMP + diphosphate + 2,3-dihydroxybenzoyl-[aryl-carrier protein EntB]
-
-
-
?
ATP + 2,3-dihydroxybenzoate + [aryl-carrier protein EntB]
AMP + diphosphate + 2,3-dihydroxybenzoyl-[aryl-carrier protein EntB]
2,3-dihydroxybenzoate binds to EntE with a 1:1 stoichiometry and a KD of 7.4 microM. The EC50 value of EntE-EntB interaction is approximately 1.5 microM
-
-
?
ATP + 2,3-dihydroxybenzoate + [aryl-carrier protein EntB]
AMP + diphosphate + 2,3-dihydroxybenzoyl-[aryl-carrier protein EntB]
-
-
-
?
ATP + salicylate + holo-[aryl-carrier protein EntB]
AMP + diphosphate + salicyl-[aryl-carrier protein EntB]
-
-
-
?
ATP + salicylate + holo-[aryl-carrier protein EntB]
AMP + diphosphate + salicyl-[aryl-carrier protein EntB]
-
-
-
?
additional information
?
-
-
no substrates are benzoic acid, 2,6-dihydroxybenzoic acid, 2,4,6-trihydroxybenzoic acid, anthranilic acid, thiosalicylic acid
-
-
?
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
-
-
?
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
-
-
-
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
-
-
?
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(2,3-dihydroxybenzoyl)adenylate + holo-[aryl-carrier protein EntB]
AMP + 2,3-dihydroxybenzoyl-[aryl-carrier protein EntB]
-
-
-
?
ATP + 2,3-dihydroxybenzoate
diphosphate + (2,3-dihydroxybenzoyl)adenylate
ATP + 2,3-dihydroxybenzoate + holo-[aryl-carrier protein EntB]
AMP + diphosphate + 2,3-dihydroxybenzoyl-[aryl-carrier protein EntB]
-
-
-
?
ATP + 2,3-dihydroxybenzoate
diphosphate + (2,3-dihydroxybenzoyl)adenylate
-
-
-
?
ATP + 2,3-dihydroxybenzoate
diphosphate + (2,3-dihydroxybenzoyl)adenylate
-
-
-
r
ATP + 2,3-dihydroxybenzoate
diphosphate + (2,3-dihydroxybenzoyl)adenylate
-
activation of 2,3-dihydroxybenzoic acid in the biosynthesis of siderophore enterobactin
-
-
?
ATP + 2,3-dihydroxybenzoate
diphosphate + (2,3-dihydroxybenzoyl)adenylate
-
involved in the biosynthesis of anguibactin
-
-
?
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0.093
2,3,4-trihydroxybenzoic acid
-
pH 9.0, 37°C
0.00046 - 0.417
2,3-Dihydroxybenzoate
0.0027
2,3-Dihydroxybenzoic acid
-
pH 9.0, 37°C
0.242
2,4-dihydroxybenzoic acid
-
pH 9.0, 37°C
0.552
2,5-dihydroxybenzoic acid
-
pH 9.0, 37°C
0.091
2-hydroxybenzoic acid
-
pH 9.0, 37°C
0.07
3-hydroxybenzoate
pH 7.8, 25°C
3.1
4-Aminosalicylate
pH 7.8, 25°C
0.0004
holo-[aryl-carrier protein EntB]
pH 7.5, 37°C
-
0.0055 - 0.533
salicylate
0.0029
[aryl-carrier protein EntB]
pH 7.8, 25°C
-
additional information
additional information
steady-state Michaelis-Menten kinetic analysis of wild-type and mutant enzymes, overview
-
0.00046
2,3-Dihydroxybenzoate
pH 7.5, 30°C
0.002
2,3-Dihydroxybenzoate
pH 8.0, 37°C, wild-type enzyme
0.0025
2,3-Dihydroxybenzoate
pH 7.8, 25°C
0.0027
2,3-Dihydroxybenzoate
pH 8.0, 37°C, mutant N340C
0.0049
2,3-Dihydroxybenzoate
pH 8.0, 37°C, mutant V339L
0.0052
2,3-Dihydroxybenzoate
pH 8.0, 37°C, mutant N235A
0.0061
2,3-Dihydroxybenzoate
pH 8.0, 37°C, mutant V339I
0.0071
2,3-Dihydroxybenzoate
pH 8.0, 37°C, mutant Y236F
0.318
2,3-Dihydroxybenzoate
pH 8.0, 37°C, mutant Y236F/S240C/N340C
0.364
2,3-Dihydroxybenzoate
pH 8.0, 37°C, mutant S240C
0.408
2,3-Dihydroxybenzoate
pH 8.0, 37°C, mutant Y236F/S240C
0.417
2,3-Dihydroxybenzoate
pH 8.0, 37°C, mutant Y236F/S240C/V339I/N340C
0.43
ATP
pH 7.8, 25°C
0.0055
salicylate
pH 8.0, 37°C, mutant V339I
0.0061
salicylate
pH 8.0, 37°C, mutant Y236F
0.008
salicylate
pH 8.0, 37°C, mutant V339L
0.0089
salicylate
pH 8.0, 37°C, mutant N340C
0.016
salicylate
pH 7.5, 30°C
0.024
salicylate
pH 8.0, 37°C, mutant Y236F/S240C/V339I/N340C
0.028
salicylate
pH 8.0, 37°C, wild-type enzyme
0.031
salicylate
pH 8.0, 37°C, mutant Y236F/S240C/N340C
0.039
salicylate
pH 8.0, 37°C, mutant Y236F/S240C
0.054
salicylate
pH 8.0, 37°C, mutant S240C
0.07
salicylate
pH 7.8, 25°C
0.08
salicylate
pH 8.0, 37°C, mutant N235A
0.533
salicylate
pH 8.0, 37°C, mutant Y236F/S240C/V339L/N340C
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0.45
2,3,4-Trihydroxybenzoate
-
pH 9.0, 37°C
0.016 - 5.5
2,3-Dihydroxybenzoate
3.23
2,4-Dihydroxybenzoate
-
pH 9.0, 37°C
3.15
2,5-dihydroxybenzoic acid
-
pH 9.0, 37°C
2.5
2-hydroxybenzoic acid
-
pH 9.0, 37°C
0.3
3-hydroxybenzoate
pH 7.8, 25°C
4.4
4-Aminosalicylate
pH 7.8, 25°C
1.67
holo-[aryl-carrier protein EntB]
pH 7.5, 37°C
-
2.8
[aryl-carrier protein EntB]
pH 7.8, 25°C
-
0.016
2,3-Dihydroxybenzoate
pH 8.0, 37°C, mutant N340C
0.017
2,3-Dihydroxybenzoate
pH 8.0, 37°C, mutant N235A
0.025
2,3-Dihydroxybenzoate
pH 8.0, 37°C, mutant V339L
0.027
2,3-Dihydroxybenzoate
pH 8.0, 37°C, mutant Y236F
0.03
2,3-Dihydroxybenzoate
pH 8.0, 37°C, mutant V339I
0.043
2,3-Dihydroxybenzoate
pH 8.0, 37°C, wild-type enzyme
0.082
2,3-Dihydroxybenzoate
pH 8.0, 37°C, mutant Y236F/S240C
0.133
2,3-Dihydroxybenzoate
pH 8.0, 37°C, mutant Y236F/S240C/N340C
0.147
2,3-Dihydroxybenzoate
pH 8.0, 37°C, mutant Y236F/S240C/V339I/N340C
0.167
2,3-Dihydroxybenzoate
pH 8.0, 37°C, mutant S240C
2.3
2,3-Dihydroxybenzoate
pH 7.5, 30°C
2.8
2,3-Dihydroxybenzoate
pH 7.8, 25°C
5.5
2,3-Dihydroxybenzoate
-
pH 9.0, 37°C
2.8
ATP
pH 7.8, 25°C
0.008
salicylate
acceptor aryl-carrier protein domain of SrfB1, pH 7.5, 37°C
0.037
salicylate
pH 8.0, 37°C, mutant N235A
0.092
salicylate
pH 8.0, 37°C, mutant Y236F/S240C/N340C
0.102
salicylate
pH 8.0, 37°C, mutant Y236F/S240C/V339L/N340C
0.115
salicylate
pH 8.0, 37°C, mutant Y236F
0.117
salicylate
pH 8.0, 37°C, mutant Y236F/S240C/V339I/N340C
0.13
salicylate
pH 8.0, 37°C, mutant N340C
0.138
salicylate
pH 8.0, 37°C, mutant Y236F/S240C
0.148
salicylate
pH 8.0, 37°C, mutant V339I
0.155
salicylate
pH 8.0, 37°C, mutant V339L
0.183
salicylate
pH 8.0, 37°C, wild-type enzyme
0.2
salicylate
pH 8.0, 37°C, mutant S240C
0.49
salicylate
pH 7.5, 30°C
0.8
salicylate
pH 7.8, 25°C
2.15
salicylate
acceptor aryl-carrier protein domain of EntB, pH 7.5, 37°C
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0.201 - 5000
2,3-Dihydroxybenzoate
4.6
3-hydroxybenzoate
pH 7.8, 25°C
15
4-Aminosalicylate
pH 7.8, 25°C
4167
holo-[aryl-carrier protein EntB]
pH 7.5, 37°C
-
980
[aryl-carrier protein EntB]
pH 7.8, 25°C
-
0.201
2,3-Dihydroxybenzoate
pH 8.0, 37°C, mutant Y236F/S240C
0.353
2,3-Dihydroxybenzoate
pH 8.0, 37°C, mutant Y236F/S240C/V339I/N340C
0.42
2,3-Dihydroxybenzoate
pH 8.0, 37°C, mutant Y236F/S240C/N340C
0.459
2,3-Dihydroxybenzoate
pH 8.0, 37°C, mutant S240C
3.27
2,3-Dihydroxybenzoate
pH 8.0, 37°C, mutant N235A
3.8
2,3-Dihydroxybenzoate
pH 8.0, 37°C, mutant Y236F
4.92
2,3-Dihydroxybenzoate
pH 8.0, 37°C, mutant V339I
5.102
2,3-Dihydroxybenzoate
pH 8.0, 37°C, mutant V339L
5.93
2,3-Dihydroxybenzoate
pH 8.0, 37°C, mutant N340C
21.5
2,3-Dihydroxybenzoate
pH 8.0, 37°C, wild-type enzyme
880
2,3-Dihydroxybenzoate
pH 7.8, 25°C
5000
2,3-Dihydroxybenzoate
pH 7.5, 30°C
0.191
salicylate
pH 8.0, 37°C, mutant Y236F/S240C/V339L/N340C
0.463
salicylate
pH 8.0, 37°C, mutant N235A
2.967
salicylate
pH 8.0, 37°C, mutant Y236F/S240C/N340C
3.54
salicylate
pH 8.0, 37°C, mutant Y236F/S240C
3.704
salicylate
pH 8.0, 37°C, mutant S240C
4.875
salicylate
pH 8.0, 37°C, mutant Y236F/S240C/V339I/N340C
6.54
salicylate
pH 8.0, 37°C, wild-type enzyme
11
salicylate
pH 7.8, 25°C
14.61
salicylate
pH 8.0, 37°C, mutant N340C
18.85
salicylate
pH 8.0, 37°C, mutant Y236F
19.34
salicylate
pH 8.0, 37°C, mutant V339L
26.9
salicylate
pH 8.0, 37°C, mutant V339I
30
salicylate
pH 7.5, 30°C
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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
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
metabolism
EntE only interacts efficiently with EntB in the presence of 2,3-dihydroxybenzoate. The transient complex is formed to facilitate channeling of 2,3-dihydroxybenzoate-AMP
metabolism
EntE proceeds via a Bi Uni Uni Bi ping-pong kinetic mechanism. During the first half-reaction (adenylation), dihydroxybenzoate binds first to the free enzyme, followed by ATP and the release of diphosphate to form the adenylate intermediate. During the second half-reaction (ligation), phosphopantetheinylated EntB binds to the enzyme followed by the release of products, AMP and arylated EntB
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
physiological function
during biosynthesis of Vibrio cholerae siderophore vibriobactin, VibE activates 2,3-dihydroxybenzoate as the acyl adenylate and then transfers it to the free thiol of the phosphopantetheine arm of VibB's aryl carrier domain
physiological function
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
physiological function
The Escherichia coli gene cluster for the synthesis and activation of 2,3-dihydroxybenzoic acid in the formation of the catechol siderophore enterobactin shows the gene order entEBG(AC). The genes are organized as three independent transcriptional units, composed of entE, entBG, and entAC, with the entBG mRNA transcribed in a clockwise direction
physiological function
the holo-EntB ArCP domain is acylated with 2,3-dihydroxybenzoate in a reaction in which EntE catalyzes the activation of 2,3-dihydroxybenzoate by adenylation as well as the transfer of 2,3-dihydroxybenzoate to the phosphopantetheine cofactor of holo-EntB
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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
N235A
site-directed mutagenesis, altered substrate specificity and kinetics compared to wild-type
N340C
site-directed mutagenesis, altered substrate specificity and kinetics compared to wild-type
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
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
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
S240C
site-directed mutagenesis, altered substrate specificity and kinetics compared to wild-type
V339I
site-directed mutagenesis, altered substrate specificity and kinetics compared to wild-type
V339L
site-directed mutagenesis, altered substrate specificity and kinetics compared to wild-type
Y236F
site-directed mutagenesis, altered substrate specificity and kinetics compared to wild-type
Y236F/S240C
site-directed mutagenesis, altered substrate specificity and kinetics compared to wild-type
Y236F/S240C/N340C
site-directed mutagenesis, altered substrate specificity and kinetics compared to wild-type
Y236F/S240C/V339I/N340C
site-directed mutagenesis, altered substrate specificity and kinetics compared to wild-type
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
-
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
-
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
-
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
-
Y236F/S240C/V339L/N340C
site-directed mutagenesis, altered substrate specificity and kinetics compared to wild-type
Y236F/S240C/V339L/N340C
site-directed mutagenesis, altered substrate specificity and kinetics compared to wild-type, very poor activityy with 2,3-dihydroxybenzoate
additional information
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mutagenesis studies support hypothesis that members of adenylate -forming family of enzymes adopt two distinct conformations to catalyze the two-step mechanism
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
additional information
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mutagenesis studies support hypothesis that members of adenylate -forming family of enzymes adopt two distinct conformations to catalyze the two-step mechanism
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2011
Escherichia coli
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1997
Escherichia coli (P10378)
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2000
Vibrio cholerae (Q9FDB2)
brenda
Sikora, A.; Wilson, D.; Aldrich, C.; Blanchard, J.
Kinetic and inhibition studies of dihydroxybenzoate-AMP ligase from Escherichia coli
Biochemistry
49
3648-3657
2010
Escherichia coli (P10378)
brenda
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
Biochemistry
59
351-363
2020
Escherichia coli (P10378)
brenda
Nahlik, M.S.; Fleming, T.P.; McIntosh, M.A.
Cluster of genes controlling synthesis and activation of 2,3-dihydroxybenzoic acid in production of enterobactin in Escherichia coli
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1987
Escherichia coli (P10378)
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Wyckoff, E.; Stoebner, J.; Reed, K.; Payne, S.
Cloning of a Vibrio cholerae vibriobactin gene cluster Identification of genes required for early steps in siderophore biosynthesis
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7055-7062
1997
Vibrio cholerae serotype O1 (O07899), Vibrio cholerae serotype O1 ATCC 39315 (O07899)
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Khalil, S.; Pawelek, P.D.
Ligand-induced conformational rearrangements promote interaction between the Escherichia coli enterobactin biosynthetic proteins EntE and EntB
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393
658-671
2009
Escherichia coli (P10378)
brenda
Ehmann, D.; Shaw-Reid, C.; Losey, H.; Walsh, C.
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. USA
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2000
Escherichia coli (P10378)
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