Literature summary for 6.2.1.25 extracted from

Thornburg, C.K.; Wortas-Strom, S.; Nosrati, M.; Geiger, J.H.; Walker, K.D.
Kinetically and crystallographically guided mutations of a benzoate CoA ligase (BadA) elucidate mechanism and expand substrate permissivity (2015), Biochemistry, 54, 6230-6242 .

Cloned(Commentary)

Cloned (Comment)	Organism
gene badA, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli BL21(DE3)	Rhodopseudomonas palustris

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant His-tagged wild-type enzyme, hanging drop vapor diffusion method, mixing of 0.002 ml of 17.5 mg/ml protein in 20 mM Tris, pH 8.0, with or without 4 equiv carboxylic acid ligand, e.g. 2-fluorobenzoate, 2-methylbenzoate, 3-furoate, and thiophene-2-carboxylate, with 0.002 ml of reservoir solution containing 0.1 M Tris-HCl, pH 7.0, and 15% w/v PEG 3350, best crystals grew in a pH range from pH 6.5-7.5, X-ray diffraction structure determination and analysis	Rhodopseudomonas palustris

Crystallization (Comment)

Organism

purified recombinant His-tagged wild-type enzyme, hanging drop vapor diffusion method, mixing of 0.002 ml of 17.5 mg/ml protein in 20 mM Tris, pH 8.0, with or without 4 equiv carboxylic acid ligand, e.g. 2-fluorobenzoate, 2-methylbenzoate, 3-furoate, and thiophene-2-carboxylate, with 0.002 ml of reservoir solution containing 0.1 M Tris-HCl, pH 7.0, and 15% w/v PEG 3350, best crystals grew in a pH range from pH 6.5-7.5, X-ray diffraction structure determination and analysis

Rhodopseudomonas palustris

Protein Variants

Protein Variants	Comment	Organism
A227G	site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme, overview	Rhodopseudomonas palustris
H334A	site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme, overview	Rhodopseudomonas palustris
I335A	site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme, overview	Rhodopseudomonas palustris
K427A	site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme, overview	Rhodopseudomonas palustris
K427A	site-directed mutagenesis, the mutant shows substrate specificity compared to the wild-type enzyme, overview	Rhodopseudomonas palustris
L333A	site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme, overview	Rhodopseudomonas palustris
additional information	mutational analysis and modification to expand the substrate specificity substantially compared to that of wild-type BadA, overview. Activities are achieved for substrates with larger substituents, including phenyl acetate. The Lys427 nonconserved residue is essential for the thiolation step of BadA, but not adenylation. Variously acylated CoAs can serve as substrates of acyl CoA-dependent acyltransferases in coupled enzyme assays to produce analogues of bioactive natural products	Rhodopseudomonas palustris

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
additional information	-	additional information	Michaelis-Menten steady-state kinetic analysis of wild-type and mutant enzymes, overview	Rhodopseudomonas palustris
0.0016	-	2-Hydroxybenzoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
0.0044	-	benzoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
0.0066	-	4-Fluorobenzoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
0.0081	-	2-Fluorobenzoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
0.021	-	2-Aminobenzoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
0.025	-	4-Aminobenzoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
0.037	-	Thiophene-2-carboxylate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
0.044	-	2-Methylbenzoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
0.05	-	1-cyclohexene-carboxylate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
0.056	-	3-cyclohexene-carboxylate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
0.071	-	3-furoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
0.073	-	3-Fluorobenzoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
0.083	-	4-Chlorobenzoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
0.13	-	2-Chlorobenzoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
0.158	-	4-hydroxybenzoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
0.174	-	3-aminobenzoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
0.213	-	2-cyanobenzoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
0.215	-	3-methylbenzoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
0.229	-	3-hydroxybenzoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
0.29	-	3-chlorobenzoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
0.59	-	4-Methylbenzoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
0.789	-	2-Methoxybenzoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
1.47	-	cyclohexane carboxylate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
2.015	-	2-nitrobenzoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	required	Rhodopseudomonas palustris

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
ATP + benzoate + CoA	Rhodopseudomonas palustris	-	AMP + diphosphate + benzoyl-CoA	-	?

Organism

Organism	UniProt	Comment	Textmining
Rhodopseudomonas palustris	Q3LB11	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli BL21(DE3) by nickel affinity chromatography, dialysis, and ultrafiltration	Rhodopseudomonas palustris

Reaction

Reaction	Comment	Organism	Reaction ID
ATP + benzoate + CoA = AMP + diphosphate + benzoyl-CoA	ATP-dependent, two-step mechanism of a benzoate:CoA ligase in the presence of magnesium, overview. The enzyme follows a mechanism that involves substrate binding in the thiolation conformation, followed by substrate rotation to an active orientation upon the transition to the adenylation conformation	Rhodopseudomonas palustris	a

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
ATP + 1-cyclohexene-carboxylate + CoA	-	Rhodopseudomonas palustris	AMP + diphosphate + 1-cyclohexene-carboxyl-CoA	-	?
ATP + 2-aminobenzoate + CoA	-	Rhodopseudomonas palustris	AMP + diphosphate + 2-aminobenzoyl-CoA	-	?
ATP + 2-chlorobenzoate + CoA	-	Rhodopseudomonas palustris	AMP + diphosphate + 2-chlorobenzoyl-CoA	-	?
ATP + 2-cyanobenzoate + CoA	-	Rhodopseudomonas palustris	AMP + diphosphate + 2-cyanobenzoyl-CoA	-	?
ATP + 2-fluorobenzoate + CoA	-	Rhodopseudomonas palustris	AMP + diphosphate + 2-fluorobenzoyl-CoA	-	?
ATP + 2-hydroxybenzoate + CoA	-	Rhodopseudomonas palustris	AMP + diphosphate + 2-hydroxybenzoyl-CoA	-	?
ATP + 2-methoxybenzoate + CoA	-	Rhodopseudomonas palustris	AMP + diphosphate + 2-methoxybenzoyl-CoA	-	?
ATP + 2-methylbenzoate + CoA	-	Rhodopseudomonas palustris	AMP + diphosphate + 2-methylbenzoyl-CoA	-	?
ATP + 2-nitrobenzoate + CoA	-	Rhodopseudomonas palustris	AMP + diphosphate + 2-nitrobenzoyl-CoA	-	?
ATP + 3-aminobenzoate + CoA	-	Rhodopseudomonas palustris	AMP + diphosphate + 3-aminobenzoyl-CoA	-	?
ATP + 3-chlorobenzoate + CoA	-	Rhodopseudomonas palustris	AMP + diphosphate + 3-chlorobenzoyl-CoA	-	?
ATP + 3-cyanobenzoate + CoA	-	Rhodopseudomonas palustris	AMP + diphosphate + 3-chlorobenzoyl-CoA	-	?
ATP + 3-cyclohexene-carboxylate + CoA	-	Rhodopseudomonas palustris	AMP + diphosphate + 3-cyclohexene-carboxyl-CoA	-	?
ATP + 3-fluorobenzoate + CoA	-	Rhodopseudomonas palustris	AMP + diphosphate + 3-fluorobenzoyl-CoA	-	?
ATP + 3-furoate + CoA	-	Rhodopseudomonas palustris	AMP + diphosphate + 3-furoyl-CoA	-	?
ATP + 3-hydroxybenzoate + CoA	-	Rhodopseudomonas palustris	AMP + diphosphate + 3-hydroxybenzoyl-CoA	-	?
ATP + 3-methylbenzoate + CoA	-	Rhodopseudomonas palustris	AMP + diphosphate + 3-methylbenzoyl-CoA	-	?
ATP + 4-aminobenzoate + CoA	-	Rhodopseudomonas palustris	AMP + diphosphate + 4-aminobenzoyl-CoA	-	?
ATP + 4-chlorobenzoate + CoA	-	Rhodopseudomonas palustris	AMP + diphosphate + 4-chlorobenzoyl-CoA	-	?
ATP + 4-fluorobenzoate + CoA	-	Rhodopseudomonas palustris	AMP + diphosphate + 4-fluorobenzoyl-CoA	-	?
ATP + 4-hydroxybenzoate + CoA	-	Rhodopseudomonas palustris	AMP + diphosphate + 4-hydroxybenzoyl-CoA	-	?
ATP + 4-methylbenzoate + CoA	-	Rhodopseudomonas palustris	AMP + diphosphate + 4-methylbenzoyl-CoA	-	?
ATP + benzoate + CoA	-	Rhodopseudomonas palustris	AMP + diphosphate + benzoyl-CoA	-	?
ATP + cyclohexane carboxylate + CoA	-	Rhodopseudomonas palustris	AMP + diphosphate + cyclohexane carboxyl-CoA	-	?
ATP + thiophene-2-carboxylate + CoA	-	Rhodopseudomonas palustris	AMP + diphosphate + thiophene-2-carboxyl-CoA	-	?
additional information	substrate specificity analysis using 31 additional potential substrates, analysis of active site architecture, overview. BadA converts ortho-substituted substrates better than the corresponding meta and para regioisomers, and the turnover number is more affected by steric rather than electronic effects, all the aryl carboxylates are uniquely oriented within the active site, relative to other structures. No or poor activity with 4-nitrobenzoate, 4-methoxybenzoate, 4-cyanobenzoate, 3-nitrobenzoate, 3-methoxybenzoate, 3-cyanobenzoate, cinnamate, and phenylacetate	Rhodopseudomonas palustris	?	-	?

Subunits

Subunits	Comment	Organism
?	x * 58900, about, recombinant wild-type enzyme, gel filtration and mass spectrometry	Rhodopseudomonas palustris

Synonyms

Synonyms	Comment	Organism
BadA	-	Rhodopseudomonas palustris
benzoate CoA ligase	-	Rhodopseudomonas palustris

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
31	-	assay at	Rhodopseudomonas palustris

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
0.011	-	2-Methoxybenzoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
0.11	-	2-cyanobenzoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
0.18	-	4-Aminobenzoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
0.27	-	2-nitrobenzoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
0.29	-	4-Chlorobenzoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
0.33	-	3-chlorobenzoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
0.54	-	3-methylbenzoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
0.63	-	2-Hydroxybenzoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
0.7	-	4-Methylbenzoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
0.93	-	4-hydroxybenzoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
1.2	-	2-Methylbenzoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
1.8	-	3-hydroxybenzoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
1.8	-	3-aminobenzoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
2	8	benzoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
2.2	-	2-Chlorobenzoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
3.9	-	2-Aminobenzoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
4.8	-	Thiophene-2-carboxylate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
5.6	-	1-cyclohexene-carboxylate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
6.7	-	3-furoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
13.5	-	cyclohexane carboxylate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
16	-	3-cyclohexene-carboxylate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
22	-	4-Fluorobenzoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
34	-	2-Fluorobenzoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
35	-	3-Fluorobenzoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
8	-	assay at	Rhodopseudomonas palustris

Cofactor

Cofactor	Comment	Organism	Structure
ATP	-	Rhodopseudomonas palustris

General Information

General Information	Comment	Organism
additional information	Lys427 nonconserved residue is essential for the thiolation step of BadA, active site structure of BadA	Rhodopseudomonas palustris
physiological function	benzoate CoA ligase (BadA) catalyzes the conversion of benzoate to benzoyl CoA on the catabolic pathway of aromatic carboxylic acids	Rhodopseudomonas palustris

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
0.014	-	2-Methoxybenzoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
0.13	-	2-nitrobenzoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
0.52	-	2-cyanobenzoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
1.1	-	3-chlorobenzoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
1.2	-	4-Methylbenzoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
2.5	-	3-methylbenzoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
3.5	-	4-Chlorobenzoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
5.9	-	4-hydroxybenzoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
7.2	-	4-Aminobenzoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
7.9	-	3-hydroxybenzoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
9.2	-	cyclohexane carboxylate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
10	-	3-aminobenzoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
17	-	2-Chlorobenzoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
27	-	2-Methylbenzoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
94	-	3-furoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
110	-	1-cyclohexene-carboxylate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
130	-	Thiophene-2-carboxylate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
190	-	2-Aminobenzoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
290	-	3-cyclohexene-carboxylate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
390	-	2-Hydroxybenzoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
480	-	3-Fluorobenzoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
3300	-	4-Fluorobenzoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
4200	-	2-Fluorobenzoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris
6400	-	benzoate	pH 8.0, 31°C, recombinant wild-type enzyme	Rhodopseudomonas palustris