Cloned (Comment) | Organism |
---|---|
expression in Escherichia coli | Pseudomonas fluorescens |
Protein Variants | Comment | Organism |
---|---|---|
N191D | the internal equilibrium of enzyme-NADH-fructose and enzyme-NAD+-mannitol is altered 10000- to 100000fold from being balanced in the wild-type enzyme to favoring enzyme-NAD+-mannitol in the single site mutants, N191D and N300D. N191D and N300D appear to lose fructose binding affinity due to deprotonation of the respective Asp above apparent pK values of 5.3 0.1 and 6.3 0.2, respectively | Pseudomonas fluorescens |
N191D/N300D | mutant behaves as a slow fructose reductase at pH 5.2, lacking measurable activity for mannitol oxidation in the pH range 6.8-10 | Pseudomonas fluorescens |
N300D | the internal equilibrium of enzyme-NADH-fructose and enzyme-NAD+-mannitol is altered 10000- to 100000fold from being balanced in the wild-type enzyme to favoring enzyme-NAD+-mannitol in the single site mutants, N191D and N300D. N191D and N300D appear to lose fructose binding affinity due to deprotonation of the respective Asp above apparent pK values of 5.3 0.1 and 6.3 0.2, respectively | Pseudomonas fluorescens |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
NAD+ | - |
Pseudomonas fluorescens | |
NADH | - |
Pseudomonas fluorescens |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.001 | - |
NAD+ | mutant N300D, pH 6.8, temperature not specified in the publication | Pseudomonas fluorescens | |
0.008 | - |
NADH | mutant N191D, pH 7.1, temperature not specified in the publication | Pseudomonas fluorescens | |
0.009 | - |
NADH | mutant N300D, pH 10.0, temperature not specified in the publication | Pseudomonas fluorescens | |
0.011 | - |
NADH | mutant N191D/N300D, pH 10.0, temperature not specified in the publication | Pseudomonas fluorescens | |
0.016 | - |
NADH | mutant N191D, pH 10.0, temperature not specified in the publication | Pseudomonas fluorescens | |
0.02 | - |
NADH | mutant N300D, pH 6.8, temperature not specified in the publication | Pseudomonas fluorescens | |
0.054 | - |
NAD+ | mutant N191D, pH 10.0, temperature not specified in the publication | Pseudomonas fluorescens | |
0.067 | - |
NADH | wild-type, pH 7.1, temperature not specified in the publication | Pseudomonas fluorescens | |
0.074 | - |
NAD+ | mutant N300D, pH 10.0, temperature not specified in the publication | Pseudomonas fluorescens | |
0.231 | - |
NAD+ | mutant N191D, pH 7.1, temperature not specified in the publication | Pseudomonas fluorescens | |
0.24 | - |
D-fructose | wild-type, pH 7.1, temperature not specified in the publication | Pseudomonas fluorescens | |
0.3 | - |
D-mannitol | mutant N300D, pH 10.0, temperature not specified in the publication | Pseudomonas fluorescens | |
0.32 | - |
D-mannitol | mutant N191D, pH 10.0, temperature not specified in the publication | Pseudomonas fluorescens | |
0.54 | - |
D-fructose | mutant N191D, pH 10.0, temperature not specified in the publication | Pseudomonas fluorescens | |
0.775 | - |
NAD+ | wild-type, pH 7.1, temperature not specified in the publication | Pseudomonas fluorescens | |
3.9 | - |
D-fructose | mutant N300D, pH 10.0, temperature not specified in the publication | Pseudomonas fluorescens | |
6 | - |
D-fructose | mutant N191D, pH 7.1, temperature not specified in the publication | Pseudomonas fluorescens | |
9 | - |
D-mannitol | mutant N191D, pH 7.1, temperature not specified in the publication | Pseudomonas fluorescens | |
20 | - |
D-fructose | mutant N191D/N300D, pH 10.0, temperature not specified in the publication | Pseudomonas fluorescens | |
21 | - |
D-mannitol | wild-type, pH 7.1, temperature not specified in the publication | Pseudomonas fluorescens | |
22 | - |
D-fructose | mutant N300D, pH 6.8, temperature not specified in the publication | Pseudomonas fluorescens | |
93 | - |
D-mannitol | mutant N300D, pH 6.8, temperature not specified in the publication | Pseudomonas fluorescens |
Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
55000 | - |
x * 55000, SDS-PAGE | Pseudomonas fluorescens |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Pseudomonas fluorescens | O08355 | - |
- |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
D-fructose + NADH + H+ | - |
Pseudomonas fluorescens | D-mannitol + NAD+ | - |
? | |
D-mannitol + NAD+ | - |
Pseudomonas fluorescens | D-fructose + NADH + H+ | - |
? |
Subunits | Comment | Organism |
---|---|---|
? | x * 55000, SDS-PAGE | Pseudomonas fluorescens |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.00055 | - |
D-mannitol | mutant N191D, pH 7.1, temperature not specified in the publication | Pseudomonas fluorescens | |
0.0011 | - |
D-mannitol | mutant N191D, pH 10.0, temperature not specified in the publication | Pseudomonas fluorescens | |
0.0014 | - |
D-mannitol | mutant N300D, pH 6.8, temperature not specified in the publication | Pseudomonas fluorescens | |
0.0015 | - |
D-mannitol | mutant N300D, pH 10.0, temperature not specified in the publication | Pseudomonas fluorescens | |
0.15 | - |
D-fructose | mutant N191D/N300D, pH 10.0, temperature not specified in the publication | Pseudomonas fluorescens | |
0.8 | - |
D-fructose | mutant N300D, pH 10.0, temperature not specified in the publication | Pseudomonas fluorescens | |
0.9 | - |
D-fructose | mutant N300D, pH 6.8, temperature not specified in the publication | Pseudomonas fluorescens | |
2.7 | - |
D-fructose | mutant N191D, pH 7.1, temperature not specified in the publication | Pseudomonas fluorescens | |
8.2 | - |
D-fructose | mutant N191D, pH 10.0, temperature not specified in the publication | Pseudomonas fluorescens | |
15.9 | - |
D-mannitol | wild-type, pH 7.1, temperature not specified in the publication | Pseudomonas fluorescens | |
61 | - |
D-fructose | wild-type, pH 7.1, temperature not specified in the publication | Pseudomonas fluorescens |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
NAD+ | - |
Pseudomonas fluorescens | |
NADH | - |
Pseudomonas fluorescens |
Ki Value [mM] | Ki Value maximum [mM] | Inhibitor | Comment | Organism | Structure |
---|---|---|---|---|---|
0.003 | - |
NAD+ | mutant N191D, pH 7.1, temperature not specified in the publication | Pseudomonas fluorescens | |
0.061 | - |
NADH | mutant N300D, pH 6.8, temperature not specified in the publication | Pseudomonas fluorescens | |
0.064 | - |
NADH | mutant N191D, pH 7.1, temperature not specified in the publication | Pseudomonas fluorescens | |
0.07 | - |
NAD+ | mutant N300D, pH 6.8, temperature not specified in the publication | Pseudomonas fluorescens | |
0.08 | - |
NAD+ | wild-type, pH 7.1, temperature not specified in the publication | Pseudomonas fluorescens | |
0.08 | - |
NADH | wild-type, pH 7.1, temperature not specified in the publication | Pseudomonas fluorescens |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.000015 | - |
D-mannitol | mutant N300D, pH 6.8, temperature not specified in the publication | Pseudomonas fluorescens | |
0.000061 | - |
D-mannitol | mutant N191D, pH 7.1, temperature not specified in the publication | Pseudomonas fluorescens | |
0.0024 | - |
NAD+ | mutant N191D, pH 7.1, temperature not specified in the publication | Pseudomonas fluorescens | |
0.0034 | - |
D-mannitol | mutant N191D, pH 10.0, temperature not specified in the publication | Pseudomonas fluorescens | |
0.0049 | - |
D-mannitol | mutant N300D, pH 10.0, temperature not specified in the publication | Pseudomonas fluorescens | |
0.0074 | - |
D-fructose | mutant N191D/N300D, pH 10.0, temperature not specified in the publication | Pseudomonas fluorescens | |
0.02 | - |
NAD+ | mutant N191D, pH 10.0, temperature not specified in the publication | Pseudomonas fluorescens | |
0.02 | - |
NAD+ | mutant N300D, pH 10.0, temperature not specified in the publication | Pseudomonas fluorescens | |
0.041 | - |
D-fructose | mutant N300D, pH 6.8, temperature not specified in the publication | Pseudomonas fluorescens | |
0.205 | - |
D-fructose | mutant N300D, pH 10.0, temperature not specified in the publication | Pseudomonas fluorescens | |
0.45 | - |
D-fructose | mutant N191D, pH 7.1, temperature not specified in the publication | Pseudomonas fluorescens | |
0.757 | - |
D-mannitol | wild-type, pH 7.1, temperature not specified in the publication | Pseudomonas fluorescens | |
1.4 | - |
NAD+ | mutant N300D, pH 6.8, temperature not specified in the publication | Pseudomonas fluorescens | |
15 | - |
D-fructose | mutant N191D, pH 10.0, temperature not specified in the publication | Pseudomonas fluorescens | |
15 | - |
NADH | mutant N191D/N300D, pH 10.0, temperature not specified in the publication | Pseudomonas fluorescens | |
20 | - |
NAD+ | wild-type, pH 7.1, temperature not specified in the publication | Pseudomonas fluorescens | |
45 | - |
NADH | mutant N300D, pH 6.8, temperature not specified in the publication | Pseudomonas fluorescens | |
89 | - |
NADH | mutant N300D, pH 10.0, temperature not specified in the publication | Pseudomonas fluorescens | |
250 | - |
D-fructose | wild-type, pH 7.1, temperature not specified in the publication | Pseudomonas fluorescens | |
340 | - |
NADH | mutant N191D, pH 7.1, temperature not specified in the publication | Pseudomonas fluorescens | |
510 | - |
NADH | mutant N191D, pH 10.0, temperature not specified in the publication | Pseudomonas fluorescens | |
910 | - |
NADH | wild-type, pH 7.1, temperature not specified in the publication | Pseudomonas fluorescens |