Crystallization (Comment) | Organism |
---|---|
wild-type and mutant structures in complex with ligands 4-hydroxybenzoyl-CoA or CoA | Arthrobacter sp. |
Protein Variants | Comment | Organism |
---|---|---|
D31A | steady-state kinetic analysis | Arthrobacter sp. |
D31N | steady-state kinetic analysis | Arthrobacter sp. |
E73A | steady-state kinetic analysis, crystallization data | Arthrobacter sp. |
E73D | mutation switches the function of the carboxylate residue from nucleophilic catalysis to base catalysis and thus, the reaction from a two-step process involving a covalent enzyme intermediate to a single-step hydrolysis reaction | Arthrobacter sp. |
E73D | steady-state kinetic analysis | Arthrobacter sp. |
E73D/T77A | mutant regains most of the catalytic efficiency lost in the E73D single mutant | Arthrobacter sp. |
E73D/T77S | steady-state kinetic analysis | Arthrobacter sp. |
E73Q | steady-state kinetic analysis, crystallization data | Arthrobacter sp. |
E78A | steady-state kinetic analysis | Arthrobacter sp. |
H64A | steady-state kinetic analysis, crystallization data | Arthrobacter sp. |
H64Q | steady-state kinetic analysis | Arthrobacter sp. |
Q58A | steady-state kinetic analysis, crystallization data | Arthrobacter sp. |
Q58D | steady-state kinetic analysis | Arthrobacter sp. |
Q58D/E73D NA | complete loss of activity | Arthrobacter sp. |
Q58E | steady-state kinetic analysis | Arthrobacter sp. |
Q58E/E73A NA | complete loss of activity | Arthrobacter sp. |
Q58E/E73Q NA | complete loss of activity | Arthrobacter sp. |
R102A | steady-state kinetic analysis | Arthrobacter sp. |
R150A | steady-state kinetic analysis | Arthrobacter sp. |
S120A | steady-state kinetic analysis | Arthrobacter sp. |
T121A | steady-state kinetic analysis | Arthrobacter sp. |
T77A | configuration of the active site residues is unchanged. The Thr77 side chain is positioned to bind and orient a water molecule for attack at the benzoyl carbonyl carbon of the putative anhydride intermediate | Arthrobacter sp. |
T77A | steady-state kinetic analysis, crystallization data | Arthrobacter sp. |
T77D | steady-state kinetic analysis | Arthrobacter sp. |
T77S | Ser77 is more effective than is the Thr77 at positioning the water for attack at the 4-hydroxybenzoyl carbonyl carbon of the anhydride intermediate | Arthrobacter sp. |
T77S | steady-state kinetic analysis, crystallization data | Arthrobacter sp. |
T77V | steady-state kinetic analysis | Arthrobacter sp. |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.00038 | - |
4-hydroxybenzoyl-CoA | mutant T77D, pH 7.5, 25°C | Arthrobacter sp. | |
0.00077 | - |
4-hydroxybenzoyl-CoA | mutant T77S, pH 7.5, 25°C | Arthrobacter sp. | |
0.0012 | - |
4-hydroxybenzoyl-CoA | wild-type, pH 7.5, 25°C | Arthrobacter sp. | |
0.0012 | - |
4-hydroxybenzoyl-CoA | mutant T77A, pH 7.5, 25°C | Arthrobacter sp. | |
0.0016 | - |
4-hydroxybenzoyl-CoA | mutant Q58A, pH 7.5, 25°C | Arthrobacter sp. | |
0.0016 | - |
4-hydroxybenzoyl-CoA | mutant S120A, pH 7.5, 25°C | Arthrobacter sp. | |
0.004 | - |
4-hydroxybenzoyl-CoA | mutant D31A, pH 7.5, 25°C | Arthrobacter sp. | |
0.004 | - |
4-hydroxybenzoyl-CoA | mutant Q58E, pH 7.5, 25°C | Arthrobacter sp. | |
0.0046 | - |
4-hydroxybenzoyl-CoA | mutant E73D/T77A, pH 7.5, 25°C | Arthrobacter sp. | |
0.0055 | - |
4-hydroxybenzoyl-CoA | mutant T77V, pH 7.5, 25°C | Arthrobacter sp. | |
0.0061 | - |
4-hydroxybenzoyl-CoA | mutant T121A, pH 7.5, 25°C | Arthrobacter sp. | |
0.0128 | - |
4-hydroxybenzoyl-CoA | mutant D31N, pH 7.5, 25°C | Arthrobacter sp. | |
0.013 | - |
4-hydroxybenzoyl-CoA | mutant E73D/T77S, pH 7.5, 25°C | Arthrobacter sp. | |
0.014 | - |
4-hydroxybenzoyl-CoA | mutant R102A, pH 7.5, 25°C | Arthrobacter sp. | |
0.0142 | - |
4-hydroxybenzoyl-CoA | mutant E73Q, pH 7.5, 25°C | Arthrobacter sp. | |
0.015 | - |
4-hydroxybenzoyl-CoA | mutant R150A, pH 7.5, 25°C | Arthrobacter sp. | |
0.018 | - |
4-hydroxybenzoyl-CoA | mutant E73D, pH 7.5, 25°C | Arthrobacter sp. | |
0.096 | - |
4-hydroxybenzoyl-CoA | mutant E78A, pH 7.5, 25°C | Arthrobacter sp. | |
0.18 | - |
4-hydroxybenzoyl-CoA | mutant Q58D, pH 7.5, 25°C | Arthrobacter sp. | |
0.237 | - |
4-hydroxybenzoyl-CoA | mutant H64Q, pH 7.5, 25°C | Arthrobacter sp. | |
0.51 | - |
4-hydroxybenzoyl-CoA | mutant H64A, pH 7.5, 25°C | Arthrobacter sp. |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Arthrobacter sp. | Q04416 | - |
- |
Arthrobacter sp. SU / DSM 20407 | Q04416 | - |
- |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
4-Hydroxybenzoyl-CoA + H2O | - |
Arthrobacter sp. | 4-Hydroxybenzoate + CoA | - |
? | |
4-Hydroxybenzoyl-CoA + H2O | - |
Arthrobacter sp. SU / DSM 20407 | 4-Hydroxybenzoate + CoA | - |
? | |
additional information | residue Glu73 functions in nucleophilic catalysis, Gly65 and Gln58 contribute to transition-state stabilization via hydrogen bond formation with the thioester moiety and Thr77 orients the water nucleophile for attack at the 4-hydroxybenzoyl carbon of the enzymeanhydride intermediate. Mutation E73D switches the function of the carboxylate residue from nucleophilic catalysis to base catalysis and thus, the reaction from a two-step process involving a covalent enzyme intermediate to a single-step hydrolysis reaction. The substrate nucleotide unit is bound to the enzyme surface | Arthrobacter sp. | ? | - |
? | |
additional information | residue Glu73 functions in nucleophilic catalysis, Gly65 and Gln58 contribute to transition-state stabilization via hydrogen bond formation with the thioester moiety and Thr77 orients the water nucleophile for attack at the 4-hydroxybenzoyl carbon of the enzymeanhydride intermediate. Mutation E73D switches the function of the carboxylate residue from nucleophilic catalysis to base catalysis and thus, the reaction from a two-step process involving a covalent enzyme intermediate to a single-step hydrolysis reaction. The substrate nucleotide unit is bound to the enzyme surface | Arthrobacter sp. SU / DSM 20407 | ? | - |
? |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.0001 | - |
4-hydroxybenzoyl-CoA | mutant E73A, pH 7.5, 25°C | Arthrobacter sp. | |
0.0017 | - |
4-hydroxybenzoyl-CoA | mutant E73Q, pH 7.5, 25°C | Arthrobacter sp. | |
0.0103 | - |
4-hydroxybenzoyl-CoA | mutant T77D, pH 7.5, 25°C | Arthrobacter sp. | |
0.0417 | - |
4-hydroxybenzoyl-CoA | mutant D31A, pH 7.5, 25°C | Arthrobacter sp. | |
0.123 | - |
4-hydroxybenzoyl-CoA | mutant E73D, pH 7.5, 25°C | Arthrobacter sp. | |
0.127 | - |
4-hydroxybenzoyl-CoA | mutant Q58A, pH 7.5, 25°C | Arthrobacter sp. | |
0.129 | - |
4-hydroxybenzoyl-CoA | mutant E73D/T77S, pH 7.5, 25°C | Arthrobacter sp. | |
0.13 | - |
4-hydroxybenzoyl-CoA | mutant Q58D, pH 7.5, 25°C | Arthrobacter sp. | |
0.15 | - |
4-hydroxybenzoyl-CoA | mutant H64A, pH 7.5, 25°C | Arthrobacter sp. | |
0.17 | - |
4-hydroxybenzoyl-CoA | mutant T77V, pH 7.5, 25°C | Arthrobacter sp. | |
0.28 | - |
4-hydroxybenzoyl-CoA | mutant H64Q, pH 7.5, 25°C | Arthrobacter sp. | |
0.364 | - |
4-hydroxybenzoyl-CoA | mutant Q58E, pH 7.5, 25°C | Arthrobacter sp. | |
1.08 | - |
4-hydroxybenzoyl-CoA | mutant E78A, pH 7.5, 25°C | Arthrobacter sp. | |
1.15 | - |
4-hydroxybenzoyl-CoA | mutant D31N, pH 7.5, 25°C | Arthrobacter sp. | |
1.54 | - |
4-hydroxybenzoyl-CoA | mutant E73D/T77A, pH 7.5, 25°C | Arthrobacter sp. | |
2.09 | - |
4-hydroxybenzoyl-CoA | mutant T77A, pH 7.5, 25°C | Arthrobacter sp. | |
2.4 | - |
4-hydroxybenzoyl-CoA | wild-type, solvent D2O, pH 7.5, 25°C | Arthrobacter sp. | |
3.16 | - |
4-hydroxybenzoyl-CoA | mutant T77S, pH 7.5, 25°C | Arthrobacter sp. | |
6.7 | - |
4-hydroxybenzoyl-CoA | wild-type, pH 7.5, 25°C | Arthrobacter sp. | |
9.5 | - |
4-hydroxybenzoyl-CoA | mutant R150A, pH 7.5, 25°C | Arthrobacter sp. | |
9.7 | - |
4-hydroxybenzoyl-CoA | mutant T121A, pH 7.5, 25°C | Arthrobacter sp. | |
10.1 | - |
4-hydroxybenzoyl-CoA | mutant S120A, pH 7.5, 25°C | Arthrobacter sp. | |
99 | - |
4-hydroxybenzoyl-CoA | mutant R102A, pH 7.5, 25°C | Arthrobacter sp. |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.12 | - |
4-hydroxybenzoyl-CoA | mutant E73Q, pH 7.5, 25°C | Arthrobacter sp. | |
0.29 | - |
4-hydroxybenzoyl-CoA | mutant H64A, pH 7.5, 25°C | Arthrobacter sp. | |
1.1 | - |
4-hydroxybenzoyl-CoA | mutant E78A, pH 7.5, 25°C | Arthrobacter sp. | |
1.2 | - |
4-hydroxybenzoyl-CoA | mutant H64Q, pH 7.5, 25°C | Arthrobacter sp. | |
6.8 | - |
4-hydroxybenzoyl-CoA | mutant E73D, pH 7.5, 25°C | Arthrobacter sp. | |
10 | - |
4-hydroxybenzoyl-CoA | mutant D31A, pH 7.5, 25°C | Arthrobacter sp. | |
10 | - |
4-hydroxybenzoyl-CoA | mutant E73D/T77S, pH 7.5, 25°C | Arthrobacter sp. | |
27 | - |
4-hydroxybenzoyl-CoA | mutant T77D, pH 7.5, 25°C | Arthrobacter sp. | |
31 | - |
4-hydroxybenzoyl-CoA | mutant T77V, pH 7.5, 25°C | Arthrobacter sp. | |
72 | - |
4-hydroxybenzoyl-CoA | mutant Q58D, pH 7.5, 25°C | Arthrobacter sp. | |
79 | - |
4-hydroxybenzoyl-CoA | mutant Q58A, pH 7.5, 25°C | Arthrobacter sp. | |
90 | - |
4-hydroxybenzoyl-CoA | mutant D31N, pH 7.5, 25°C | Arthrobacter sp. | |
91 | - |
4-hydroxybenzoyl-CoA | mutant Q58E, pH 7.5, 25°C | Arthrobacter sp. | |
340 | - |
4-hydroxybenzoyl-CoA | mutant E73D/T77A, pH 7.5, 25°C | Arthrobacter sp. | |
630 | - |
4-hydroxybenzoyl-CoA | mutant R150A, pH 7.5, 25°C | Arthrobacter sp. | |
710 | - |
4-hydroxybenzoyl-CoA | mutant R102A, pH 7.5, 25°C | Arthrobacter sp. | |
1600 | - |
4-hydroxybenzoyl-CoA | mutant T121A, pH 7.5, 25°C | Arthrobacter sp. | |
1700 | - |
4-hydroxybenzoyl-CoA | mutant T77A, pH 7.5, 25°C | Arthrobacter sp. | |
4100 | - |
4-hydroxybenzoyl-CoA | mutant T77S, pH 7.5, 25°C | Arthrobacter sp. | |
5400 | - |
4-hydroxybenzoyl-CoA | wild-type, pH 7.5, 25°C | Arthrobacter sp. | |
6300 | - |
4-hydroxybenzoyl-CoA | mutant S120A, pH 7.5, 25°C | Arthrobacter sp. |