Literature summary 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 .

Cloned(Commentary)

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

Crystallization (Commentary)

EC Number	Crystallization (Comment)	Organism
6.2.1.71	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

EC Number	Protein Variants	Comment	Organism
6.2.1.71	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
6.2.1.71	N235A	site-directed mutagenesis, altered substrate specificity and kinetics compared to wild-type	Escherichia coli
6.2.1.71	N340C	site-directed mutagenesis, altered substrate specificity and kinetics compared to wild-type	Escherichia coli
6.2.1.71	S240C	site-directed mutagenesis, altered substrate specificity and kinetics compared to wild-type	Escherichia coli
6.2.1.71	V339I	site-directed mutagenesis, altered substrate specificity and kinetics compared to wild-type	Escherichia coli
6.2.1.71	V339L	site-directed mutagenesis, altered substrate specificity and kinetics compared to wild-type	Escherichia coli
6.2.1.71	Y236F	site-directed mutagenesis, altered substrate specificity and kinetics compared to wild-type	Escherichia coli
6.2.1.71	Y236F/S240C	site-directed mutagenesis, altered substrate specificity and kinetics compared to wild-type	Escherichia coli
6.2.1.71	Y236F/S240C/N340C	site-directed mutagenesis, altered substrate specificity and kinetics compared to wild-type	Escherichia coli
6.2.1.71	Y236F/S240C/V339I/N340C	site-directed mutagenesis, altered substrate specificity and kinetics compared to wild-type	Escherichia coli
6.2.1.71	Y236F/S240C/V339L/N340C	site-directed mutagenesis, altered substrate specificity and kinetics compared to wild-type	Escherichia coli
6.2.1.71	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

EC Number	Inhibitors	Comment	Organism	Structure
6.2.1.71	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]

EC Number	KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
6.2.1.71	additional information	-	additional information	steady-state Michaelis-Menten kinetic analysis of wild-type and mutant enzymes, overview	Escherichia coli
6.2.1.71	0.002	-	2,3-Dihydroxybenzoate	pH 8.0, 37°C, wild-type enzyme	Escherichia coli
6.2.1.71	0.0027	-	2,3-Dihydroxybenzoate	pH 8.0, 37°C, mutant N340C	Escherichia coli
6.2.1.71	0.0049	-	2,3-Dihydroxybenzoate	pH 8.0, 37°C, mutant V339L	Escherichia coli
6.2.1.71	0.0052	-	2,3-Dihydroxybenzoate	pH 8.0, 37°C, mutant N235A	Escherichia coli
6.2.1.71	0.0055	-	salicylate	pH 8.0, 37°C, mutant V339I	Escherichia coli
6.2.1.71	0.0061	-	2,3-Dihydroxybenzoate	pH 8.0, 37°C, mutant V339I	Escherichia coli
6.2.1.71	0.0061	-	salicylate	pH 8.0, 37°C, mutant Y236F	Escherichia coli
6.2.1.71	0.0071	-	2,3-Dihydroxybenzoate	pH 8.0, 37°C, mutant Y236F	Escherichia coli
6.2.1.71	0.008	-	salicylate	pH 8.0, 37°C, mutant V339L	Escherichia coli
6.2.1.71	0.0089	-	salicylate	pH 8.0, 37°C, mutant N340C	Escherichia coli
6.2.1.71	0.024	-	salicylate	pH 8.0, 37°C, mutant Y236F/S240C/V339I/N340C	Escherichia coli
6.2.1.71	0.028	-	salicylate	pH 8.0, 37°C, wild-type enzyme	Escherichia coli
6.2.1.71	0.031	-	salicylate	pH 8.0, 37°C, mutant Y236F/S240C/N340C	Escherichia coli
6.2.1.71	0.039	-	salicylate	pH 8.0, 37°C, mutant Y236F/S240C	Escherichia coli
6.2.1.71	0.054	-	salicylate	pH 8.0, 37°C, mutant S240C	Escherichia coli
6.2.1.71	0.08	-	salicylate	pH 8.0, 37°C, mutant N235A	Escherichia coli
6.2.1.71	0.318	-	2,3-Dihydroxybenzoate	pH 8.0, 37°C, mutant Y236F/S240C/N340C	Escherichia coli
6.2.1.71	0.364	-	2,3-Dihydroxybenzoate	pH 8.0, 37°C, mutant S240C	Escherichia coli
6.2.1.71	0.408	-	2,3-Dihydroxybenzoate	pH 8.0, 37°C, mutant Y236F/S240C	Escherichia coli
6.2.1.71	0.417	-	2,3-Dihydroxybenzoate	pH 8.0, 37°C, mutant Y236F/S240C/V339I/N340C	Escherichia coli
6.2.1.71	0.533	-	salicylate	pH 8.0, 37°C, mutant Y236F/S240C/V339L/N340C	Escherichia coli

Metals/Ions

EC Number	Metals/Ions	Comment	Organism	Structure
6.2.1.71	Mg2+	required	Escherichia coli

Natural Substrates/ Products (Substrates)

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

Organism

EC Number	Organism	UniProt	Comment	Textmining
6.2.1.71	Escherichia coli	P10378	-	-

Substrates and Products (Substrate)

EC Number	Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
6.2.1.71	ATP + 2,3-dihydroxybenzoate	-	Escherichia coli	diphosphate + (2,3-dihydroxybenzoyl)adenylate	-	r
6.2.1.71	ATP + 2,3-dihydroxybenzoate	highly preferred substrate	Escherichia coli	diphosphate + (2,3-dihydroxybenzoyl)adenylate	-	r
6.2.1.71	ATP + 2-fluorobenzoate	low activity	Escherichia coli	diphosphate + (2-fluorobenzoyl)adenylate	-	?
6.2.1.71	ATP + 3-aminobenzoate	low activity	Escherichia coli	diphosphate + (3-aminobenzoyl)adenylate	-	?
6.2.1.71	ATP + 3-bromobenzoate	low activity	Escherichia coli	diphosphate + (3-bromobenzoyl)adenylate	-	?
6.2.1.71	ATP + 3-chlorobenzoate	low activity	Escherichia coli	diphosphate + (3-chlorobenzoyl)adenylate	-	?
6.2.1.71	ATP + 3-ethynylbenzoate	low activity	Escherichia coli	diphosphate + (3-ethynylbenzoyl)adenylate	-	?
6.2.1.71	ATP + 3-fluorobenzoate	low activity	Escherichia coli	diphosphate + (3-fluorobenzoyl)adenylate	-	?
6.2.1.71	ATP + 3-hydroxybenzoate	low activity	Escherichia coli	diphosphate + (3-hydroxybenzoyl)adenylate	-	?
6.2.1.71	ATP + 3-iodobenzoate	low activity	Escherichia coli	diphosphate + (3-iodobenzoyl)adenylate	-	?
6.2.1.71	ATP + 3-methoxybenzoate	low activity	Escherichia coli	diphosphate + (3-methoxybenzoyl)adenylate	-	?
6.2.1.71	ATP + 3-methylbenzoate	low activity	Escherichia coli	diphosphate + (3-methylbenzoyl)adenylate	-	?
6.2.1.71	ATP + anthranilate	low activity	Escherichia coli	diphosphate + anthranylyl-adenylate	-	?
6.2.1.71	ATP + salicylate	low activity	Escherichia coli	diphosphate + salicylyl-adenylate	-	r
6.2.1.71	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

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

Temperature Optimum [°C]

EC Number	Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
6.2.1.71	37	-	assay at	Escherichia coli

Turnover Number [1/s]

EC Number	Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
6.2.1.71	0.016	-	2,3-Dihydroxybenzoate	pH 8.0, 37°C, mutant N340C	Escherichia coli
6.2.1.71	0.017	-	2,3-Dihydroxybenzoate	pH 8.0, 37°C, mutant N235A	Escherichia coli
6.2.1.71	0.025	-	2,3-Dihydroxybenzoate	pH 8.0, 37°C, mutant V339L	Escherichia coli
6.2.1.71	0.027	-	2,3-Dihydroxybenzoate	pH 8.0, 37°C, mutant Y236F	Escherichia coli
6.2.1.71	0.03	-	2,3-Dihydroxybenzoate	pH 8.0, 37°C, mutant V339I	Escherichia coli
6.2.1.71	0.037	-	salicylate	pH 8.0, 37°C, mutant N235A	Escherichia coli
6.2.1.71	0.043	-	2,3-Dihydroxybenzoate	pH 8.0, 37°C, wild-type enzyme	Escherichia coli
6.2.1.71	0.082	-	2,3-Dihydroxybenzoate	pH 8.0, 37°C, mutant Y236F/S240C	Escherichia coli
6.2.1.71	0.092	-	salicylate	pH 8.0, 37°C, mutant Y236F/S240C/N340C	Escherichia coli
6.2.1.71	0.102	-	salicylate	pH 8.0, 37°C, mutant Y236F/S240C/V339L/N340C	Escherichia coli
6.2.1.71	0.115	-	salicylate	pH 8.0, 37°C, mutant Y236F	Escherichia coli
6.2.1.71	0.117	-	salicylate	pH 8.0, 37°C, mutant Y236F/S240C/V339I/N340C	Escherichia coli
6.2.1.71	0.13	-	salicylate	pH 8.0, 37°C, mutant N340C	Escherichia coli
6.2.1.71	0.133	-	2,3-Dihydroxybenzoate	pH 8.0, 37°C, mutant Y236F/S240C/N340C	Escherichia coli
6.2.1.71	0.138	-	salicylate	pH 8.0, 37°C, mutant Y236F/S240C	Escherichia coli
6.2.1.71	0.147	-	2,3-Dihydroxybenzoate	pH 8.0, 37°C, mutant Y236F/S240C/V339I/N340C	Escherichia coli
6.2.1.71	0.148	-	salicylate	pH 8.0, 37°C, mutant V339I	Escherichia coli
6.2.1.71	0.155	-	salicylate	pH 8.0, 37°C, mutant V339L	Escherichia coli
6.2.1.71	0.167	-	2,3-Dihydroxybenzoate	pH 8.0, 37°C, mutant S240C	Escherichia coli
6.2.1.71	0.183	-	salicylate	pH 8.0, 37°C, wild-type enzyme	Escherichia coli
6.2.1.71	0.2	-	salicylate	pH 8.0, 37°C, mutant S240C	Escherichia coli

pH Optimum

EC Number	pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
6.2.1.71	7.5	8	assay at	Escherichia coli

Cofactor

EC Number	Cofactor	Comment	Organism	Structure
6.2.1.71	ATP	-	Escherichia coli

General Information

EC Number	General Information	Comment	Organism
6.2.1.71	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
6.2.1.71	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
6.2.1.71	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]

EC Number	kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
6.2.1.71	0.191	-	salicylate	pH 8.0, 37°C, mutant Y236F/S240C/V339L/N340C	Escherichia coli
6.2.1.71	0.201	-	2,3-Dihydroxybenzoate	pH 8.0, 37°C, mutant Y236F/S240C	Escherichia coli
6.2.1.71	0.353	-	2,3-Dihydroxybenzoate	pH 8.0, 37°C, mutant Y236F/S240C/V339I/N340C	Escherichia coli
6.2.1.71	0.42	-	2,3-Dihydroxybenzoate	pH 8.0, 37°C, mutant Y236F/S240C/N340C	Escherichia coli
6.2.1.71	0.459	-	2,3-Dihydroxybenzoate	pH 8.0, 37°C, mutant S240C	Escherichia coli
6.2.1.71	0.463	-	salicylate	pH 8.0, 37°C, mutant N235A	Escherichia coli
6.2.1.71	2.967	-	salicylate	pH 8.0, 37°C, mutant Y236F/S240C/N340C	Escherichia coli
6.2.1.71	3.27	-	2,3-Dihydroxybenzoate	pH 8.0, 37°C, mutant N235A	Escherichia coli
6.2.1.71	3.54	-	salicylate	pH 8.0, 37°C, mutant Y236F/S240C	Escherichia coli
6.2.1.71	3.704	-	salicylate	pH 8.0, 37°C, mutant S240C	Escherichia coli
6.2.1.71	3.8	-	2,3-Dihydroxybenzoate	pH 8.0, 37°C, mutant Y236F	Escherichia coli
6.2.1.71	4.875	-	salicylate	pH 8.0, 37°C, mutant Y236F/S240C/V339I/N340C	Escherichia coli
6.2.1.71	4.92	-	2,3-Dihydroxybenzoate	pH 8.0, 37°C, mutant V339I	Escherichia coli
6.2.1.71	5.102	-	2,3-Dihydroxybenzoate	pH 8.0, 37°C, mutant V339L	Escherichia coli
6.2.1.71	5.93	-	2,3-Dihydroxybenzoate	pH 8.0, 37°C, mutant N340C	Escherichia coli
6.2.1.71	6.54	-	salicylate	pH 8.0, 37°C, wild-type enzyme	Escherichia coli
6.2.1.71	14.61	-	salicylate	pH 8.0, 37°C, mutant N340C	Escherichia coli
6.2.1.71	18.85	-	salicylate	pH 8.0, 37°C, mutant Y236F	Escherichia coli
6.2.1.71	19.34	-	salicylate	pH 8.0, 37°C, mutant V339L	Escherichia coli
6.2.1.71	21.5	-	2,3-Dihydroxybenzoate	pH 8.0, 37°C, wild-type enzyme	Escherichia coli
6.2.1.71	26.9	-	salicylate	pH 8.0, 37°C, mutant V339I	Escherichia coli