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

BRENDA support

Literature summary 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 .
    View publication on PubMed

Cloned(Commentary)

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

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
6.2.1.25 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

EC Number Protein Variants Comment Organism
6.2.1.25 A227G site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme, overview Rhodopseudomonas palustris
6.2.1.25 H334A site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme, overview Rhodopseudomonas palustris
6.2.1.25 I335A site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme, overview Rhodopseudomonas palustris
6.2.1.25 K427A site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme, overview Rhodopseudomonas palustris
6.2.1.25 K427A site-directed mutagenesis, the mutant shows substrate specificity compared to the wild-type enzyme, overview Rhodopseudomonas palustris
6.2.1.25 L333A site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme, overview Rhodopseudomonas palustris
6.2.1.25 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]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
6.2.1.25 additional information
-
 additional information Michaelis-Menten steady-state kinetic analysis of wild-type and mutant enzymes, overview Rhodopseudomonas palustris
6.2.1.25 0.0016
-
 2-Hydroxybenzoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 0.0044
-
 benzoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 0.0066
-
 4-Fluorobenzoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 0.0081
-
 2-Fluorobenzoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 0.021
-
 2-Aminobenzoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 0.025
-
 4-Aminobenzoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 0.037
-
 Thiophene-2-carboxylate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 0.044
-
 2-Methylbenzoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 0.05
-
 1-cyclohexene-carboxylate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 0.056
-
 3-cyclohexene-carboxylate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 0.071
-
 3-furoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 0.073
-
 3-Fluorobenzoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 0.083
-
 4-Chlorobenzoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 0.13
-
 2-Chlorobenzoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 0.158
-
 4-hydroxybenzoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 0.174
-
 3-aminobenzoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 0.213
-
 2-cyanobenzoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 0.215
-
 3-methylbenzoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 0.229
-
 3-hydroxybenzoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 0.29
-
 3-chlorobenzoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 0.59
-
 4-Methylbenzoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 0.789
-
 2-Methoxybenzoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 1.47
-
 cyclohexane carboxylate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 2.015
-
 2-nitrobenzoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
6.2.1.25 Mg2+ required Rhodopseudomonas palustris

Natural Substrates/ Products (Substrates)

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

Organism

EC Number Organism UniProt Comment Textmining
6.2.1.25 Rhodopseudomonas palustris Q3LB11
-
-

Purification (Commentary)

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

Reaction

EC Number Reaction Comment Organism Reaction ID
6.2.1.25 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)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
6.2.1.25 ATP + 1-cyclohexene-carboxylate + CoA
-
 Rhodopseudomonas palustris AMP + diphosphate + 1-cyclohexene-carboxyl-CoA
-
 ?
6.2.1.25 ATP + 2-aminobenzoate + CoA
-
 Rhodopseudomonas palustris AMP + diphosphate + 2-aminobenzoyl-CoA
-
 ?
6.2.1.25 ATP + 2-chlorobenzoate + CoA
-
 Rhodopseudomonas palustris AMP + diphosphate + 2-chlorobenzoyl-CoA
-
 ?
6.2.1.25 ATP + 2-cyanobenzoate + CoA
-
 Rhodopseudomonas palustris AMP + diphosphate + 2-cyanobenzoyl-CoA
-
 ?
6.2.1.25 ATP + 2-fluorobenzoate + CoA
-
 Rhodopseudomonas palustris AMP + diphosphate + 2-fluorobenzoyl-CoA
-
 ?
6.2.1.25 ATP + 2-hydroxybenzoate + CoA
-
 Rhodopseudomonas palustris AMP + diphosphate + 2-hydroxybenzoyl-CoA
-
 ?
6.2.1.25 ATP + 2-methoxybenzoate + CoA
-
 Rhodopseudomonas palustris AMP + diphosphate + 2-methoxybenzoyl-CoA
-
 ?
6.2.1.25 ATP + 2-methylbenzoate + CoA
-
 Rhodopseudomonas palustris AMP + diphosphate + 2-methylbenzoyl-CoA
-
 ?
6.2.1.25 ATP + 2-nitrobenzoate + CoA
-
 Rhodopseudomonas palustris AMP + diphosphate + 2-nitrobenzoyl-CoA
-
 ?
6.2.1.25 ATP + 3-aminobenzoate + CoA
-
 Rhodopseudomonas palustris AMP + diphosphate + 3-aminobenzoyl-CoA
-
 ?
6.2.1.25 ATP + 3-chlorobenzoate + CoA
-
 Rhodopseudomonas palustris AMP + diphosphate + 3-chlorobenzoyl-CoA
-
 ?
6.2.1.25 ATP + 3-cyanobenzoate + CoA
-
 Rhodopseudomonas palustris AMP + diphosphate + 3-chlorobenzoyl-CoA
-
 ?
6.2.1.25 ATP + 3-cyclohexene-carboxylate + CoA
-
 Rhodopseudomonas palustris AMP + diphosphate + 3-cyclohexene-carboxyl-CoA
-
 ?
6.2.1.25 ATP + 3-fluorobenzoate + CoA
-
 Rhodopseudomonas palustris AMP + diphosphate + 3-fluorobenzoyl-CoA
-
 ?
6.2.1.25 ATP + 3-furoate + CoA
-
 Rhodopseudomonas palustris AMP + diphosphate + 3-furoyl-CoA
-
 ?
6.2.1.25 ATP + 3-hydroxybenzoate + CoA
-
 Rhodopseudomonas palustris AMP + diphosphate + 3-hydroxybenzoyl-CoA
-
 ?
6.2.1.25 ATP + 3-methylbenzoate + CoA
-
 Rhodopseudomonas palustris AMP + diphosphate + 3-methylbenzoyl-CoA
-
 ?
6.2.1.25 ATP + 4-aminobenzoate + CoA
-
 Rhodopseudomonas palustris AMP + diphosphate + 4-aminobenzoyl-CoA
-
 ?
6.2.1.25 ATP + 4-chlorobenzoate + CoA
-
 Rhodopseudomonas palustris AMP + diphosphate + 4-chlorobenzoyl-CoA
-
 ?
6.2.1.25 ATP + 4-fluorobenzoate + CoA
-
 Rhodopseudomonas palustris AMP + diphosphate + 4-fluorobenzoyl-CoA
-
 ?
6.2.1.25 ATP + 4-hydroxybenzoate + CoA
-
 Rhodopseudomonas palustris AMP + diphosphate + 4-hydroxybenzoyl-CoA
-
 ?
6.2.1.25 ATP + 4-methylbenzoate + CoA
-
 Rhodopseudomonas palustris AMP + diphosphate + 4-methylbenzoyl-CoA
-
 ?
6.2.1.25 ATP + benzoate + CoA
-
 Rhodopseudomonas palustris AMP + diphosphate + benzoyl-CoA
-
 ?
6.2.1.25 ATP + cyclohexane carboxylate + CoA
-
 Rhodopseudomonas palustris AMP + diphosphate + cyclohexane carboxyl-CoA
-
 ?
6.2.1.25 ATP + thiophene-2-carboxylate + CoA
-
 Rhodopseudomonas palustris AMP + diphosphate + thiophene-2-carboxyl-CoA
-
 ?
6.2.1.25 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

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

Synonyms

EC Number Synonyms Comment Organism
6.2.1.25 BadA
-
 Rhodopseudomonas palustris
6.2.1.25 benzoate CoA ligase
-
 Rhodopseudomonas palustris

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
6.2.1.25 31
-
 assay at Rhodopseudomonas palustris

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
6.2.1.25 0.011
-
 2-Methoxybenzoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 0.11
-
 2-cyanobenzoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 0.18
-
 4-Aminobenzoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 0.27
-
 2-nitrobenzoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 0.29
-
 4-Chlorobenzoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 0.33
-
 3-chlorobenzoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 0.54
-
 3-methylbenzoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 0.63
-
 2-Hydroxybenzoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 0.7
-
 4-Methylbenzoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 0.93
-
 4-hydroxybenzoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 1.2
-
 2-Methylbenzoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 1.8
-
 3-hydroxybenzoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 1.8
-
 3-aminobenzoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 2 8 benzoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 2.2
-
 2-Chlorobenzoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 3.9
-
 2-Aminobenzoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 4.8
-
 Thiophene-2-carboxylate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 5.6
-
 1-cyclohexene-carboxylate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 6.7
-
 3-furoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 13.5
-
 cyclohexane carboxylate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 16
-
 3-cyclohexene-carboxylate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 22
-
 4-Fluorobenzoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 34
-
 2-Fluorobenzoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 35
-
 3-Fluorobenzoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
6.2.1.25 8
-
 assay at Rhodopseudomonas palustris

Cofactor

EC Number Cofactor Comment Organism Structure
6.2.1.25 ATP
-
 Rhodopseudomonas palustris

General Information

EC Number General Information Comment Organism
6.2.1.25 additional information Lys427 nonconserved residue is essential for the thiolation step of BadA, active site structure of BadA Rhodopseudomonas palustris
6.2.1.25 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]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
6.2.1.25 0.014
-
 2-Methoxybenzoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 0.13
-
 2-nitrobenzoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 0.52
-
 2-cyanobenzoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 1.1
-
 3-chlorobenzoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 1.2
-
 4-Methylbenzoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 2.5
-
 3-methylbenzoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 3.5
-
 4-Chlorobenzoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 5.9
-
 4-hydroxybenzoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 7.2
-
 4-Aminobenzoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 7.9
-
 3-hydroxybenzoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 9.2
-
 cyclohexane carboxylate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 10
-
 3-aminobenzoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 17
-
 2-Chlorobenzoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 27
-
 2-Methylbenzoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 94
-
 3-furoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 110
-
 1-cyclohexene-carboxylate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 130
-
 Thiophene-2-carboxylate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 190
-
 2-Aminobenzoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 290
-
 3-cyclohexene-carboxylate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 390
-
 2-Hydroxybenzoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 480
-
 3-Fluorobenzoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 3300
-
 4-Fluorobenzoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 4200
-
 2-Fluorobenzoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris
6.2.1.25 6400
-
 benzoate pH 8.0, 31°C, recombinant wild-type enzyme Rhodopseudomonas palustris