Protein Variants | Comment | Organism |
---|---|---|
R165E | mutant is less stable than wild-type enzyme, mutant enzyme is only partially reduced by (S)-mandelate. 81.8fold decrease in kcat for (S)-mandelate, 199fold increase in KM-value for (S)-mandelate compared to wild-type enzyme | Pseudomonas putida |
R165G | mutant is less stable than wild-type enzyme, mutant enzyme is only partially reduced by (S)-mandelate. 27.3fold decrease in kcat for (S)-mandelate, 48.3fold increase in KM-value for (S)-mandelate compared to wild-type enzyme | Pseudomonas putida |
R165G/R277G | no activity. Double mutant is less stable than single Arg165 mutant in term of long-term storage | Pseudomonas putida |
R165G/R277K | double mutant is less stable than single Arg165 mutant in term of long-term storage. 9000fold decrease in kcat for (S)-mandelate, 261.7fold increase in KM-value for (S)-mandelate compared to wild-type enzyme | Pseudomonas putida |
R165K | mutant is less stable than wild-type enzyme, mutant enzyme is fully reduced on addition of (S)-mandelate. 3fold decrease in kcat for (S)-mandelate, 12.5fold increase in KM-value for (S)-mandelate compared to wild-type enzyme | Pseudomonas putida |
R165K/R277G | no activity. Double mutant is less stable than single Arg165 mutant in term of long-term storage | Pseudomonas putida |
R165K/R277K | double mutant is less stable than single Arg165 mutant in term of long-term storage. 327fold decrease in kcat for (S)-mandelate, 126.7fold increase in KM-value for (S)-mandelate compared to wild-type enzyme | Pseudomonas putida |
R165M | mutant is less stable than wild-type enzyme, mutant enzyme is fully reduced on addition of (S)-mandelate. 19.6fold decrease in kcat for (S)-mandelate, 36.7fold increase in KM-value for (S)-mandelate compared to wild-type enzyme | Pseudomonas putida |
R277G | 1565fold decrease in kcat for (S)-mandelate, 141fold increase in KM-value for (S)-mandelate compared to wild-type enzyme | Pseudomonas putida |
R277K | 5.5 fold decrease in kcat for (S)-mandelate, 46.7fold increase in KM-value for (S)-mandelate compared to wild-type enzyme | Pseudomonas putida |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
(R)-mandelate | - |
Pseudomonas putida | |
2-phenylethanoate | - |
Pseudomonas putida | |
phenylethanediol | poor competitive inhibitor of wild-type enzyme | Pseudomonas putida |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.12 | - |
(S)-Mandelate | pH 7.5, 20°C, wild-type enzyme | Pseudomonas putida | |
1.5 | - |
(S)-Mandelate | pH 7.5, 20°C, mutant enzyme R165K | Pseudomonas putida | |
4.4 | - |
(S)-Mandelate | pH 7.5, 20°C, mutant enzyme R165M | Pseudomonas putida | |
5.6 | - |
(S)-Mandelate | pH 7.5, 20°C, mutant enzyme R277K | Pseudomonas putida | |
5.8 | - |
(S)-Mandelate | pH 7.5, 20°C, mutant enzyme R165G | Pseudomonas putida | |
12 | - |
(S)-Mandelate | pH 7.5, 20°C, mutant enzyme R165E | Pseudomonas putida | |
15.2 | - |
(S)-Mandelate | pH 7.5, 20°C, mutant enzyme R165K/R277K | Pseudomonas putida | |
17 | - |
(S)-Mandelate | pH 7.5, 20°C, mutant enzyme R277G | Pseudomonas putida | |
31.4 | - |
(S)-Mandelate | pH 7.5, 20°C, mutant enzyme R165G/R277K | Pseudomonas putida |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Pseudomonas putida | - |
- |
- |
Purification (Comment) | Organism |
---|---|
- |
Pseudomonas putida |
Storage Stability | Organism |
---|---|
-70°C, wild-type enzyme retains activity for more than 1 year. The single Arg165 mutant lose activity after 2-3 months, double mutants R165K/R277K and R165G/R277K are inactivated after 2-3 weeks in storage | Pseudomonas putida |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
(S)-mandelate + acceptor | reduction of 2,6-dichloroindophenol in presence of N-methylphenazonium methosulfate | Pseudomonas putida | 2-oxo-2-phenylacetate + reduced acceptor | - |
? |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.04 | - |
(S)-Mandelate | pH 7.5, 20°C, mutant enzyme R165G/R277K | Pseudomonas putida | |
0.23 | - |
(S)-Mandelate | pH 7.5, 20°C, mutant enzyme R277G | Pseudomonas putida | |
1.1 | - |
(S)-Mandelate | pH 7.5, 20°C, mutant enzyme R165K/R277K | Pseudomonas putida | |
4.4 | - |
(S)-Mandelate | pH 7.5, 20°C, mutant enzyme R165E | Pseudomonas putida | |
13.2 | - |
(S)-Mandelate | pH 7.5, 20°C, mutant enzyme R165G | Pseudomonas putida | |
18.4 | - |
(S)-Mandelate | pH 7.5, 20°C, mutant enzyme R165M | Pseudomonas putida | |
66 | - |
(S)-Mandelate | pH 7.5, 20°C, mutant enzyme R277K | Pseudomonas putida | |
120 | - |
(S)-Mandelate | pH 7.5, 20°C, mutant enzyme R165K | Pseudomonas putida | |
360 | - |
(S)-Mandelate | pH 7.5, 20°C, wild-type enzyme | Pseudomonas putida |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
FMN | - |
Pseudomonas putida |
Ki Value [mM] | Ki Value maximum [mM] | Inhibitor | Comment | Organism | Structure |
---|---|---|---|---|---|
2.9 | - |
(R)-mandelate | pH 7.5, 20°C, wild-type enzyme | Pseudomonas putida | |
3.1 | - |
(R)-mandelate | pH 7.5, 20°C, mutant enzyme R165K | Pseudomonas putida | |
7.2 | - |
(R)-mandelate | pH 7.5, 20°C, mutant enzyme R277K | Pseudomonas putida | |
11 | - |
2-phenylethanoate | pH 7.5, 20°C, mutant enzyme R165K | Pseudomonas putida | |
11 | - |
2-phenylethanoate | pH 7.5, 20°C, mutant enzyme R165K/R277K | Pseudomonas putida | |
12 | - |
2-phenylethanoate | pH 7.5, 20°C, mutant enzyme R277K | Pseudomonas putida | |
12 | - |
2-phenylethanoate | pH 7.5, 20°C, wild-type enzyme | Pseudomonas putida | |
19.5 | - |
(R)-mandelate | pH 7.5, 20°C, mutant enzyme R277G | Pseudomonas putida | |
20.3 | - |
2-phenylethanoate | pH 7.5, 20°C, mutant enzyme R165G/R277K | Pseudomonas putida | |
22.5 | - |
2-phenylethanoate | pH 7.5, 20°C, mutant enzyme R277G | Pseudomonas putida | |
67 | - |
2-phenylethanoate | pH 7.5, 20°C, mutant enzyme R165G | Pseudomonas putida | |
73 | - |
(R)-mandelate | pH 7.5, 20°C, mutant enzyme R165G | Pseudomonas putida | |
700 | - |
(R)-mandelate | pH 7.5, 20°C, mutant enzyme R165G/R277K | Pseudomonas putida |