Application | Comment | Organism |
---|---|---|
synthesis | alterating of cofactor specificity from NADH to NADPH via protein engineering for application as a regenerating enzyme in coupled reactions with NADPH-dependent alcohol dehydrogenases. Mutant D196A/L197R can be applied in a coupled oxidation/transamination reaction of the dicyclic dialcohol isosorbide to its diamines, catalyzed by Ralstonia sp. alcohol dehydrogenase and Paracoccus denitrificans omega-aminotransferase, allowing recycling of NADP+ and L-Ala. Recycling factors of 33 for NADP+ and 13 for L-Ala are observed | Bacillus subtilis |
Cloned (Comment) | Organism |
---|---|
expression in Escherichia coli | Bacillus subtilis |
Protein Variants | Comment | Organism |
---|---|---|
D196A | alterating cofactor specificity from NADH to NADPH, 10fold decrease in activity with NADH, 4fold increase in activity with NADPH | Bacillus subtilis |
D196A/L197R | alterating cofactor specificity from NADH to NADPH, almost the same activity with NADPH as the wild-type enzyme for NADH | Bacillus subtilis |
D196A/L197R/N198S/R201A | alterating cofactor specificity from NADH to NADPH, loss of activity with NADH, 5fold increase in activity with NADPH | Bacillus subtilis |
D196A/L197R/R201A | alterating cofactor specificity from NADH to NADPH, loss of activity with NADH, 2fold increase in activity with NADPH | Bacillus subtilis |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.014 | - |
NADH | wild-type, pH 8.3, 30°C | Bacillus subtilis | |
0.032 | - |
NADPH | mutant D196A/L197R, pH 8.3, 30°C | Bacillus subtilis | |
0.119 | - |
NADPH | mutant D196A/L197R/N198S/R201A, pH 8.3, 30°C | Bacillus subtilis | |
0.219 | - |
NADPH | mutant D196A/L197R/R201A, pH 8.3, 30°C | Bacillus subtilis | |
0.39 | - |
NADPH | mutant D196A, pH 8.3, 30°C | Bacillus subtilis | |
0.429 | - |
NADH | mutant D196A, pH 8.3, 30°C | Bacillus subtilis | |
0.448 | - |
NADPH | wild-type, pH 8.3, 30°C | Bacillus subtilis | |
0.887 | - |
NADH | mutant D196A/L197R, pH 8.3, 30°C | Bacillus subtilis |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Bacillus subtilis | - |
- |
- |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
pyruvate + NH3 + NADH + H+ | - |
Bacillus subtilis | L-Ala + H2O + NAD+ | - |
? | |
pyruvate + NH3 + NADPH + H+ | NADPH is a poor cofactor for wild-type | Bacillus subtilis | L-Ala + H2O + NADP+ | - |
? |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
11.1 | - |
NADPH | wild-type, pH 8.3, 30°C | Bacillus subtilis | |
12.65 | - |
NADPH | mutant D196A/L197R/R201A, pH 8.3, 30°C | Bacillus subtilis | |
13.96 | - |
NADH | wild-type, pH 8.3, 30°C | Bacillus subtilis | |
16.57 | - |
NADPH | mutant D196A/L197R/N198S/R201A, pH 8.3, 30°C | Bacillus subtilis | |
28.83 | - |
NADPH | mutant D196A/L197R, pH 8.3, 30°C | Bacillus subtilis | |
36.07 | - |
NADH | mutant D196A, pH 8.3, 30°C | Bacillus subtilis | |
40.58 | - |
NADPH | mutant D196A, pH 8.3, 30°C | Bacillus subtilis | |
44.48 | - |
NADH | mutant D196A/L197R, pH 8.3, 30°C | Bacillus subtilis |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
NADH | - |
Bacillus subtilis | |
NADPH | poor cofactor for wild-type | Bacillus subtilis |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
25 | - |
NADPH | wild-type, pH 8.3, 30°C | Bacillus subtilis | |
50 | - |
NADH | mutant D196A/L197R, pH 8.3, 30°C | Bacillus subtilis | |
58 | - |
NADPH | mutant D196A/L197R/R201A, pH 8.3, 30°C | Bacillus subtilis | |
83 | - |
NADH | mutant D196A, pH 8.3, 30°C | Bacillus subtilis | |
103 | - |
NADPH | mutant D196A, pH 8.3, 30°C | Bacillus subtilis | |
140 | - |
NADPH | mutant D196A/L197R/N198S/R201A, pH 8.3, 30°C | Bacillus subtilis | |
901 | - |
NADPH | mutant D196A/L197R, pH 8.3, 30°C | Bacillus subtilis | |
998 | - |
NADH | wild-type, pH 8.3, 30°C | Bacillus subtilis |