Application | Comment | Organism |
---|---|---|
synthesis | generation of a de novo NADPH generation pathway by altering the coenzyme specificity of native NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPDH) to NADP, to produce additional NADPH in the glycolytic pathway. Increasing the catalytic efficiency of GAPDH towards NADP enhances lysine production in all of the tested mutants, the most significant improvement of lysine production (60%) is achieved with the mutant showing similar preference towards both NAD and NADP. There is no significant change of flux towards the pentose phosphate pathway and the increased lysine yield is mainly attributed to the NADPH generated by the mutated GAPDH | Corynebacterium glutamicum |
Cloned (Comment) | Organism |
---|---|
expression in Escherichia coli | Corynebacterium glutamicum |
Protein Variants | Comment | Organism |
---|---|---|
D35G | mutation enables GAPDH to accept both NAD and NADP | Corynebacterium glutamicum |
D35G/L36R/P192S | mutant accepts both Nad AND nadp WITH SIMILAR EFFICINCY | Corynebacterium glutamicum |
D35G/L36T/T37K | catalytic efficiency with NADP is about 10fold hihger than with NAD | Corynebacterium glutamicum |
D35G/L36T/T37K/P192S | Mutant shows the highest catalytic efficiency with NADP while the catalytic efficiency with NAD also increases | Corynebacterium glutamicum |
L36T | mutation enables GAPDH to accept both NAD and NADP | Corynebacterium glutamicum |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.048 | - |
NAD+ | wild-type, pH 8.5, 25°C | Corynebacterium glutamicum | |
0.058 | - |
NAD+ | mutant D35G/L36T/P192S, pH 8.5, 25°C | Corynebacterium glutamicum | |
0.07 | - |
NADP+ | mutant D35G/L36T/T37K/P192S, pH 8.5, 25°C | Corynebacterium glutamicum | |
0.1 | - |
NADP+ | mutant D35G/L36T/P192S, pH 8.5, 25°C | Corynebacterium glutamicum | |
0.1 | - |
NADP+ | mutant D35G/L36T/T37K, pH 8.5, 25°C | Corynebacterium glutamicum | |
0.1 | - |
NAD+ | mutant D35G/L36T/T37K/P192S, pH 8.5, 25°C | Corynebacterium glutamicum | |
0.14 | - |
NAD+ | mutant L36T, pH 8.5, 25°C | Corynebacterium glutamicum | |
0.47 | - |
NAD+ | mutant D35G/L36T/T37K, pH 8.5, 25°C | Corynebacterium glutamicum | |
0.54 | - |
NAD+ | mutant D35G, pH 8.5, 25°C | Corynebacterium glutamicum | |
0.83 | - |
NADP+ | mutant D35G, pH 8.5, 25°C | Corynebacterium glutamicum | |
1.46 | - |
NADP+ | mutant L36T, pH 8.5, 25°C | Corynebacterium glutamicum |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Corynebacterium glutamicum | Q01651 | - |
- |
Corynebacterium glutamicum DSM 20300 | Q01651 | - |
- |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
D-glyceraldehyde 3-phosphate + phosphate + NAD+ | - |
Corynebacterium glutamicum | 3-phospho-D-glyceroyl phosphate + NADH + H+ | - |
? | |
D-glyceraldehyde 3-phosphate + phosphate + NAD+ | - |
Corynebacterium glutamicum DSM 20300 | 3-phospho-D-glyceroyl phosphate + NADH + H+ | - |
? | |
D-glyceraldehyde 3-phosphate + phosphate + NADP+ | - |
Corynebacterium glutamicum | 3-phospho-D-glyceroyl phosphate + NADPH + H+ | - |
? | |
D-glyceraldehyde 3-phosphate + phosphate + NADP+ | - |
Corynebacterium glutamicum DSM 20300 | 3-phospho-D-glyceroyl phosphate + NADPH + H+ | - |
? |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
1.45 | - |
NAD+ | mutant L36T, pH 8.5, 25°C | Corynebacterium glutamicum | |
1.58 | - |
NADP+ | mutant L36T, pH 8.5, 25°C | Corynebacterium glutamicum | |
2.73 | - |
NAD+ | mutant D35G/L36T/T37K/P192S, pH 8.5, 25°C | Corynebacterium glutamicum | |
2.87 | - |
NAD+ | mutant D35G/L36T/P192S, pH 8.5, 25°C | Corynebacterium glutamicum | |
2.97 | - |
NADP+ | mutant D35G, pH 8.5, 25°C | Corynebacterium glutamicum | |
4.2 | - |
NAD+ | mutant D35G/L36T/T37K, pH 8.5, 25°C | Corynebacterium glutamicum | |
6.37 | - |
NAD+ | mutant D35G, pH 8.5, 25°C | Corynebacterium glutamicum | |
6.45 | - |
NAD+ | wild-type, pH 8.5, 25°C | Corynebacterium glutamicum | |
6.48 | - |
NADP+ | mutant D35G/L36T/P192S, pH 8.5, 25°C | Corynebacterium glutamicum | |
7.13 | - |
NADP+ | mutant D35G/L36T/T37K/P192S, pH 8.5, 25°C | Corynebacterium glutamicum | |
8 | - |
NADP+ | mutant D35G/L36T/T37K, pH 8.5, 25°C | Corynebacterium glutamicum |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
NAD+ | - |
Corynebacterium glutamicum | |
NADP+ | no cofactor for wild-type | Corynebacterium glutamicum |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
1.1 | - |
NADP+ | mutant L36T, pH 8.5, 25°C | Corynebacterium glutamicum | |
3.6 | - |
NADP+ | mutant D35G, pH 8.5, 25°C | Corynebacterium glutamicum | |
8.9 | - |
NAD+ | mutant D35G/L36T/T37K, pH 8.5, 25°C | Corynebacterium glutamicum | |
10.3 | - |
NAD+ | mutant L36T, pH 8.5, 25°C | Corynebacterium glutamicum | |
11.8 | - |
NAD+ | mutant D35G, pH 8.5, 25°C | Corynebacterium glutamicum | |
27.3 | - |
NAD+ | mutant D35G/L36T/T37K/P192S, pH 8.5, 25°C | Corynebacterium glutamicum | |
49.2 | - |
NAD+ | mutant D35G/L36T/P192S, pH 8.5, 25°C | Corynebacterium glutamicum | |
64.75 | - |
NADP+ | mutant D35G/L36T/P192S, pH 8.5, 25°C | Corynebacterium glutamicum | |
80 | - |
NADP+ | mutant D35G/L36T/T37K, pH 8.5, 25°C | Corynebacterium glutamicum | |
97.8 | - |
NADP+ | mutant D35G/L36T/T37K/P192S, pH 8.5, 25°C | Corynebacterium glutamicum | |
134.4 | - |
NAD+ | wild-type, pH 8.5, 25°C | Corynebacterium glutamicum |