Cloned (Comment) | Organism |
---|---|
gene badA, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli BL21(DE3) | 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 | 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 Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
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 | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | required | Rhodopseudomonas palustris |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + benzoate + CoA | Rhodopseudomonas palustris | - |
AMP + diphosphate + benzoyl-CoA | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Rhodopseudomonas palustris | Q3LB11 | - |
- |
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 | 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 |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
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 | Comment | Organism |
---|---|---|
? | x * 58900, about, recombinant wild-type enzyme, gel filtration and mass spectrometry | Rhodopseudomonas palustris |
Synonyms | Comment | Organism |
---|---|---|
BadA | - |
Rhodopseudomonas palustris |
benzoate CoA ligase | - |
Rhodopseudomonas palustris |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
31 | - |
assay at | Rhodopseudomonas palustris |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
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 Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8 | - |
assay at | Rhodopseudomonas palustris |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
ATP | - |
Rhodopseudomonas palustris |
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 Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
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 |