Protein Variants | Comment | Organism |
---|---|---|
G81A | higher specificity for small substrates, compared to that of wild-type enzyme, the affinity for (S)-mandelate is relatively unchanged. The rate of the first half-reaction is slower than the wild-type rate. 2fold increase in oxidative half-reaction. Affinity for oxygen increases 10-15fold | Pseudomonas putida |
G81D | extreme low activity, reduction in activity is due to the decrease in electrophilicity of the FMN | Pseudomonas putida |
G81S | the rate of the first half-reaction is slower than the wild-type rate. The rate of the first half-reaction is slower than the wild-type rate. Affinity for O2 increases 10-15fold | Pseudomonas putida |
G81V | extreme low activity, reduction in activity is due to the decrease in electrophilicity of the FMN | Pseudomonas putida |
additional information | MDH-GOX2, a chimeric mutant of (S)-mandelate dehydrogenase with residues 177-215 replaced by residues 176-195 of glycolate oxidase from spinach. G81A/MDH-GOX2, a chimeric mutant of (S)-mandelate dehydrogenase with residues 177-215 replaced by residues 176-195 of glycolate oxidase from spinach and a G81A mutation in MDH | Pseudomonas putida |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
sulfite | - |
Pseudomonas putida |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.04 | - |
O2 | pH 7.5, 20°C, mutant enzyme G81V | Pseudomonas putida | |
0.04 | - |
(S)-Mandelate | pH 7.5, 20°C, mutant enzyme, G81A/MDH-GOX2, a chimeric mutant of (S)-mandelate dehydrogenase with residues 177-215 replaced by residues 176-195 of glycolate oxidase from spinach and a G81A mutation in MDH | Pseudomonas putida | |
0.09 | - |
(S)-Mandelate | pH 7.5, 20°C, mutant enzyme MDH-GOX2, a chimeric mutant of (S)-mandelate dehydrogenase with residues 177-215 replaced by residues 176-195 of glycolate oxidase from spinach | Pseudomonas putida | |
0.13 | - |
(S)-Mandelate | pH 7.5, 20°C, wild-type enzyme | Pseudomonas putida | |
0.14 | - |
2-Hydroxyoctanoate | pH 7.5, 20°C, mutant enzyme G81A | Pseudomonas putida | |
0.15 | - |
(S)-Mandelate | pH 7.5, 20°C, mutant enzyme G81A | Pseudomonas putida | |
0.17 | - |
3-indolelactate | pH 7.5, 20°C, mutant enzyme G81A | Pseudomonas putida | |
0.17 | - |
(S)-Mandelate | pH 7.5, 20°C, mutant enzyme G81V | Pseudomonas putida | |
0.22 | - |
O2 | pH 7.5, 20°C, mutant enzyme G81D | Pseudomonas putida | |
0.24 | - |
Mandelate | pH 7.5, 20°C, mutant enzyme G81A | Pseudomonas putida | |
0.24 | - |
Mandelate | pH 7.5, 20°C, wild-type enzyme | Pseudomonas putida | |
0.27 | - |
4-chloromandelate | pH 7.5, 20°C, wild-type enzyme | Pseudomonas putida | |
0.37 | - |
3-Phenyllactate | pH 7.5, 20°C, mutant enzyme G81A | Pseudomonas putida | |
0.4 | - |
indoleglycolate | pH 7.5, 20°C, wild-type enzyme | Pseudomonas putida | |
0.43 | - |
4-chloromandelate | pH 7.5, 20°C, mutant enzyme G81A | Pseudomonas putida | |
0.49 | - |
O2 | pH 7.5, 20°C, mutant enzyme, G81A/MDH-GOX2, a chimeric mutant of (S)-mandelate dehydrogenase with residues 177-215 replaced by residues 176-195 of glycolate oxidase from spinach and a G81A mutation in MDH | Pseudomonas putida | |
0.63 | - |
indoleglycolate | pH 7.5, 20°C, mutant enzyme G81A | Pseudomonas putida | |
0.68 | - |
O2 | pH 7.5, 20°C, mutant enzyme MDH-GOX2, a chimeric mutant of (S)-mandelate dehydrogenase with 205 residues 177-215 replaced by residues 176-195 of glycolate oxidase from spinach | Pseudomonas putida | |
0.8 | - |
2-Hydroxyoctanoate | pH 7.5, 20°C, wild-type enzyme | Pseudomonas putida | |
0.9 | - |
3-indolelactate | pH 7.5, 20°C, wild-type enzyme | Pseudomonas putida | |
1.1 | - |
O2 | pH 7.5, 20°C, mutant enzyme G81A | Pseudomonas putida | |
1.4 | - |
2-hydroxyhexanoate | pH 7.5, 20°C, mutant enzyme G81A | Pseudomonas putida | |
1.4 | - |
O2 | pH 7.5, 20°C, mutant enzyme G81S | Pseudomonas putida | |
1.6 | - |
2-hydroxyisocaproate | pH 7.5, 20°C, mutant enzyme G81A | Pseudomonas putida | |
2 | - |
(S)-Mandelate | pH 7.5, 20°C, mutant enzyme G81D | Pseudomonas putida | |
2 | - |
3-Phenyllactate | pH 7.5, 20°C, wild-type enzyme | Pseudomonas putida | |
2.3 | - |
(S)-Mandelate | pH 7.5, 20°C, mutant enzyme G81S | Pseudomonas putida | |
3.2 | - |
O2 | pH 7.5, 20°C, wild-type enzyme | Pseudomonas putida | |
4.3 | - |
2-Hydroxy-3-butynoate | pH 7.5, 20°C, mutant enzyme G81A | Pseudomonas putida | |
4.3 | - |
2-hydroxyisocaproate | pH 7.5, 20°C, wild-type enzyme | Pseudomonas putida | |
4.4 | - |
2-Hydroxybutyrate | pH 7.5, 20°C, mutant enzyme G81A | Pseudomonas putida | |
4.9 | - |
2-hydroxyhexanoate | pH 7.5, 20°C, wild-type enzyme | Pseudomonas putida | |
6.4 | - |
2-hydroxyvalerate | pH 7.5, 20°C, mutant enzyme G81A | Pseudomonas putida | |
15.3 | - |
2-hydroxyvalerate | pH 7.5, 20°C, wild-type enzyme | Pseudomonas putida | |
22 | - |
2-Hydroxy-3-butynoate | pH 7.5, 20°C, wild-type enzyme | Pseudomonas putida | |
32 | - |
2-Hydroxybutyrate | pH 7.5, 20°C, wild-type enzyme | Pseudomonas putida |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Pseudomonas putida | - |
- |
- |
Purification (Comment) | Organism |
---|---|
wild-type and mutant enzymes | Pseudomonas putida |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
(S)-2-hydroxy-2-phenylacetate + O2 | in absence of any other electron acceptor, oxygen is used by the wild-type enzyme for reoxidation of the reduced flavin, at a rather slow rate. Ping-pong kinetics | Pseudomonas putida | 2-oxo-2-phenylacetate + H2O2 | - |
? | |
(S)-mandelate + acceptor | phenazine methosulfate and 2,6-dichloroindophenol as acceptor. In absence of any other electron acceptor, oxygen is used by the wild-type enzyme for reoxidation of the reduced flavin, at a rather slow rate | Pseudomonas putida | 2-oxo-2-phenylacetate + reduced acceptor | - |
? | |
2-hydroxy-3-butynoate + acceptor | - |
Pseudomonas putida | 2-oxo-3-butynoate + reduced acceptor | - |
? | |
2-hydroxybutyrate + acceptor | - |
Pseudomonas putida | 2-oxobutyrate + reduced acceptor | - |
? | |
2-hydroxyhexanoate + acceptor | - |
Pseudomonas putida | 2-oxohexanoate + reduced acceptor | - |
? | |
2-hydroxyisocaproate + acceptor | - |
Pseudomonas putida | 2-oxoisocaproate + reduced acceptor | - |
? | |
2-hydroxyoctanoate + acceptor | - |
Pseudomonas putida | 2-oxooctanoate + reduced acceptor | - |
? | |
2-hydroxyvalerate + acceptor | - |
Pseudomonas putida | 2-oxovalerate + reduced acceptor | - |
? | |
3-indolelactate + acceptor | - |
Pseudomonas putida | 3-(1H-indol-2-yl)-2-oxopropanoate + reduced acceptor | - |
? | |
3-phenyllactate + acceptor | - |
Pseudomonas putida | 2-oxo-3-phenylpropanoate + reduced acceptor | - |
? | |
4-chloromandelate + acceptor | - |
Pseudomonas putida | 2-oxo-2-(4-chlorophenyl)acetate + reduced acceptor | - |
? | |
indoleglycolate + acceptor | - |
Pseudomonas putida | indol-2-yl(oxo)acetate + reduced acceptor | - |
? | |
mandelate + acceptor | - |
Pseudomonas putida | 2-oxo-2-phenylacetate + reduced acceptor | - |
? |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.03 | - |
O2 | pH 7.5, 20°C, mutant enzyme MDH-GOX2, a chimeric mutant of (S)-mandelate dehydrogenase with 205 residues 177-215 replaced by residues 176-195 of glycolate oxidase from spinach | Pseudomonas putida | |
0.05 | - |
O2 | pH 7.5, 20°C, mutant enzyme G81D | Pseudomonas putida | |
0.05 | - |
(S)-Mandelate | pH 7.5, 20°C, mutant enzyme G81V | Pseudomonas putida | |
0.07 | - |
O2 | pH 7.5, 20°C, mutant enzyme G81V | Pseudomonas putida | |
0.1 | - |
2-Hydroxybutyrate | pH 7.5, 20°C, wild-type enzyme | Pseudomonas putida | |
0.11 | - |
(S)-Mandelate | pH 7.5, 20°C, mutant enzyme G81D | Pseudomonas putida | |
0.15 | - |
3-Phenyllactate | pH 7.5, 20°C, mutant enzyme G81A | Pseudomonas putida | |
0.19 | - |
2-hydroxyhexanoate | pH 7.5, 20°C, mutant enzyme G81A | Pseudomonas putida | |
0.22 | - |
O2 | pH 7.5, 20°C, mutant enzyme, G81A/MDH-GOX2, a chimeric mutant of (S)-mandelate dehydrogenase with residues 177-215 replaced by residues 176-195 of glycolate oxidase from spinach and a G81A mutation in MDH | Pseudomonas putida | |
0.26 | - |
2-Hydroxyoctanoate | pH 7.5, 20°C, mutant enzyme G81A | Pseudomonas putida | |
0.27 | - |
3-indolelactate | pH 7.5, 20°C, mutant enzyme G81A | Pseudomonas putida | |
0.27 | - |
2-hydroxyvalerate | pH 7.5, 20°C, mutant enzyme G81A | Pseudomonas putida | |
0.34 | - |
2-hydroxyhexanoate | pH 7.5, 20°C, wild-type enzyme | Pseudomonas putida | |
0.36 | - |
2-hydroxyvalerate | pH 7.5, 20°C, wild-type enzyme | Pseudomonas putida | |
0.37 | - |
2-Hydroxybutyrate | pH 7.5, 20°C, mutant enzyme G81A | Pseudomonas putida | |
0.48 | - |
2-hydroxyisocaproate | pH 7.5, 20°C, mutant enzyme G81A | Pseudomonas putida | |
0.5 | - |
2-Hydroxyoctanoate | pH 7.5, 20°C, wild-type enzyme | Pseudomonas putida | |
0.52 | - |
3-Phenyllactate | pH 7.5, 20°C, wild-type enzyme | Pseudomonas putida | |
0.61 | - |
O2 | pH 7.5, 20°C, mutant enzyme G81S | Pseudomonas putida | |
0.8 | - |
2-hydroxyisocaproate | pH 7.5, 20°C, wild-type enzyme | Pseudomonas putida | |
1 | - |
3-indolelactate | pH 7.5, 20°C, wild-type enzyme | Pseudomonas putida | |
1.2 | - |
indoleglycolate | pH 7.5, 20°C, mutant enzyme G81A | Pseudomonas putida | |
1.2 | - |
O2 | pH 7.5, 20°C, wild-type enzyme | Pseudomonas putida | |
2.3 | - |
(S)-Mandelate | pH 7.5, 20°C, mutant enzyme, G81A/MDH-GOX2, a chimeric mutant of (S)-mandelate dehydrogenase with residues 177-215 replaced by residues 176-195 of glycolate oxidase from spinach and a G81A mutation in MDH | Pseudomonas putida | |
2.8 | - |
(S)-Mandelate | pH 7.5, 20°C, mutant enzyme G81S | Pseudomonas putida | |
3.4 | - |
O2 | pH 7.5, 20°C, mutant enzyme G81A | Pseudomonas putida | |
3.9 | - |
2-Hydroxy-3-butynoate | pH 7.5, 20°C, wild-type enzyme | Pseudomonas putida | |
9.3 | - |
4-chloromandelate | pH 7.5, 20°C, mutant enzyme G81A | Pseudomonas putida | |
14.8 | - |
2-Hydroxy-3-butynoate | pH 7.5, 20°C, mutant enzyme G81A | Pseudomonas putida | |
15.2 | - |
Mandelate | pH 7.5, 20°C, mutant enzyme G81A | Pseudomonas putida | |
19.2 | - |
(S)-Mandelate | pH 7.5, 20°C, mutant enzyme G81A | Pseudomonas putida | |
122 | - |
indoleglycolate | pH 7.5, 20°C, wild-type enzyme | Pseudomonas putida | |
205 | - |
(S)-Mandelate | pH 7.5, 20°C, mutant enzyme MDH-GOX2, a chimeric mutant of (S)-mandelate dehydrogenase with 205 residues 177-215 replaced by residues 176-195 of glycolate oxidase from spinach | Pseudomonas putida | |
334 | - |
4-chloromandelate | pH 7.5, 20°C, wild-type enzyme | Pseudomonas putida | |
350 | - |
Mandelate | pH 7.5, 20°C, wild-type enzyme | Pseudomonas putida | |
360 | - |
(S)-Mandelate | pH 7.5, 20°C, wild-type enzyme | Pseudomonas putida |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
FMN | dependent on. In the reductive half-reaction (S)-mandelate is oxidized to 2-oxo-2-phenylacetate, resulting in the reduction of FMN | Pseudomonas putida |