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Literature summary for 1.2.1.59 extracted from
- A de novo NADPH generation pathway for improving lysine production of Corynebacterium glutamicum by rational design of the coenzyme specificity of glyceraldehyde 3-phosphate dehydrogenase (2014), Metab. Eng., 25, 30-37 .
Application
| Application | Comment | Organism |
|---|---|---|
| synthesis | engineering the coenzyme specificity of (GAPDH) as a promising NADPH source is of interest for the metabolic engineering of NADPH-dependent bioproduction systems, e.g. for lysine production | Corynebacterium glutamicum |
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| gene gapA, recombinant overexpression of mutant enzymes in Escherichia coli strain BL21(DE3) | Corynebacterium glutamicum |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| D35G | site-directed mutagenesis, the mutant enzyme accepts both NAD+ and NADP+ , the catalytic efficiency with NADP+ is 3fold lower than with NAD+ | Corynebacterium glutamicum |
| D35G/L36R/P192S | site-directed mutagenesis, the mutant enzyme accepts both NAD+ and NADP+ with similar catalytic efficiency | Corynebacterium glutamicum |
| D35G/L36T/T37K | site-directed mutagenesis, introducing a third mutation T37K into the mutant D35G/L36T completely reverses the coenzyme specificity of the enzyme | Corynebacterium glutamicum |
| D35G/L36T/T37K/P192S | site-directed mutagenesis, the mutant shows high catalytic efficiency with NADP+ while the catalytic efficiency with NAD+ also increases. The replacement of Pro192 to Ser benefits the binding affinity of both NAD+ and NADP+ | Corynebacterium glutamicum |
| L36T | site-directed mutagenesis, the mutant enzyme accepts both NAD+ and NADP+ , the catalytic efficiency with NADP+ is lower than with NAD+ | Corynebacterium glutamicum |
| additional information | the coenzyme specificity of GAPDH, EC 1.2.1.12, of Corynebacterium glutamicum is systematically manipulated by rational protein design and the effect of the manipulation for cellular metabolism and lysine production is evaluated. By a combinatorial modification of four key residues within the coenzyme binding sites, different GAPDH mutants with varied coenzyme specificity are constructed. While increasing the catalytic efficiency of GAPDH towards NADP+ enhances lysine production in all of the tested mutants, the most significant improvement of lysine production (about 60%) is achieved with the mutant showing similar preference towards both NAD+ and NADP+, EC 1.2.1.59 | Corynebacterium glutamicum |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.058 | - |
NAD+ | pH 8.0, 25°C, recombinant mutant D35G/L36T/P192S | Corynebacterium glutamicum |  |
| 0.07 | - |
NADP+ | pH 8.0, 25°C, recombinant mutant D35G/L36T/T37K/P192S | Corynebacterium glutamicum | |
| 0.1 | - |
NADP+ | pH 8.0, 25°C, recombinant mutant D35G/L36T/P192S | Corynebacterium glutamicum | |
| 0.1 | - |
NADP+ | pH 8.0, 25°C, recombinant mutant D35G/L36T/T37K | Corynebacterium glutamicum | |
| 0.1 | - |
NAD+ | pH 8.0, 25°C, recombinant mutant D35G/L36T/T37K/P192S | Corynebacterium glutamicum | |
| 0.14 | - |
NAD+ | pH 8.0, 25°C, recombinant mutant L36T | Corynebacterium glutamicum | |
| 0.47 | - |
NAD+ | pH 8.0, 25°C, recombinant mutant D35G/L36T/T37K | Corynebacterium glutamicum | |
| 0.54 | - |
NAD+ | pH 8.0, 25°C, recombinant mutant D35G | Corynebacterium glutamicum | |
| 0.83 | - |
NADP+ | pH 8.0, 25°C, recombinant mutant D35G | Corynebacterium glutamicum | |
| 1.46 | - |
NADP+ | pH 8.0, 25°C, recombinant mutant L36T | Corynebacterium glutamicum | |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | 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 ATCC 13032 | - |
3-phospho-D-glyceroyl phosphate + NADH + H+ | - |
? | |
| D-glyceraldehyde 3-phosphate + phosphate + NAD+ | Corynebacterium glutamicum ATCC13032 | - |
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 ATCC 13032 | - |
3-phospho-D-glyceroyl phosphate + NADPH + H+ | - |
? | |
| D-glyceraldehyde 3-phosphate + phosphate + NADP+ | Corynebacterium glutamicum ATCC13032 | - |
3-phospho-D-glyceroyl phosphate + NADPH + H+ | - |
? | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Corynebacterium glutamicum | Q01651 | containing the point mutation Q298G in the lysC gene and mutation N917G in the ppc gene | - |
| Corynebacterium glutamicum ATCC13032 | Q01651 | containing the point mutation Q298G in the lysC gene and mutation N917G in the ppc gene | - |
Substrates and Products (Substrate)
| 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 ATCC 13032 | 3-phospho-D-glyceroyl phosphate + NADH + H+ | - |
? | |
| D-glyceraldehyde 3-phosphate + phosphate + NAD+ | - |
Corynebacterium glutamicum ATCC13032 | 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 ATCC 13032 | 3-phospho-D-glyceroyl phosphate + NADPH + H+ | - |
? | |
| D-glyceraldehyde 3-phosphate + phosphate + NADP+ | - |
Corynebacterium glutamicum ATCC13032 | 3-phospho-D-glyceroyl phosphate + NADPH + H+ | - |
? | |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| GAPDH | - |
Corynebacterium glutamicum |
| glyceraldehyde 3-phosphate dehydrogenase | - |
Corynebacterium glutamicum |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 25 | - |
assay at | Corynebacterium glutamicum |
Turnover Number [1/s]
| Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 1.45 | - |
NAD+ | pH 8.0, 25°C, recombinant mutant L36T | Corynebacterium glutamicum | |
| 1.58 | - |
NADP+ | pH 8.0, 25°C, recombinant mutant L36T | Corynebacterium glutamicum | |
| 2.73 | - |
NAD+ | pH 8.0, 25°C, recombinant mutant D35G/L36T/T37K/P192S | Corynebacterium glutamicum | |
| 2.87 | - |
NAD+ | pH 8.0, 25°C, recombinant mutant D35G/L36T/P192S | Corynebacterium glutamicum | |
| 2.97 | - |
NADP+ | pH 8.0, 25°C, recombinant mutant D35G | Corynebacterium glutamicum | |
| 4.2 | - |
NAD+ | pH 8.0, 25°C, recombinant mutant D35G/L36T/T37K | Corynebacterium glutamicum | |
| 6.37 | - |
NAD+ | pH 8.0, 25°C, recombinant mutant D35G | Corynebacterium glutamicum | |
| 6.48 | - |
NADP+ | pH 8.0, 25°C, recombinant mutant D35G/L36T/P192S | Corynebacterium glutamicum | |
| 7.13 | - |
NADP+ | pH 8.0, 25°C, recombinant mutant D35G/L36T/T37K/P192S | Corynebacterium glutamicum | |
| 8 | - |
NADP+ | pH 8.0, 25°C, recombinant mutant D35G/L36T/T37K | Corynebacterium glutamicum | |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 8 | - |
assay at | Corynebacterium glutamicum |
Cofactor
| Cofactor | Comment | Organism | Structure |
|---|---|---|---|
| NAD+ | - |
Corynebacterium glutamicum | |
| NADP+ | - |
Corynebacterium glutamicum | |
General Information
| General Information | Comment | Organism |
|---|---|---|
| physiological function | glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is an enzyme that catalyzes an inevitable step in the central metabolism of most industrially important sugars such as glucose, fructose and sucrose. During the glycolysis of 1 mol glucose and 2 mol of NAD(P)H are generated at this enzymatic reaction with the oxidation of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate | Corynebacterium glutamicum |
kcat/KM [mM/s]
| kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 1.08 | - |
NADP+ | pH 8.0, 25°C, recombinant mutant L36T | Corynebacterium glutamicum | |
| 3.58 | - |
NADP+ | pH 8.0, 25°C, recombinant mutant D35G | Corynebacterium glutamicum | |
| 8.94 | - |
NAD+ | pH 8.0, 25°C, recombinant mutant D35G/L36T/T37K | Corynebacterium glutamicum | |
| 10.36 | - |
NAD+ | pH 8.0, 25°C, recombinant mutant L36T | Corynebacterium glutamicum | |
| 11.8 | - |
NAD+ | pH 8.0, 25°C, recombinant mutant D35G | Corynebacterium glutamicum | |
| 27.3 | - |
NAD+ | pH 8.0, 25°C, recombinant mutant D35G/L36T/T37K/P192S | Corynebacterium glutamicum | |
| 49.48 | - |
NAD+ | pH 8.0, 25°C, recombinant mutant D35G/L36T/P192S | Corynebacterium glutamicum | |
| 64.8 | - |
NADP+ | pH 8.0, 25°C, recombinant mutant D35G/L36T/P192S | Corynebacterium glutamicum | |
| 80 | - |
NADP+ | pH 8.0, 25°C, recombinant mutant D35G/L36T/T37K | Corynebacterium glutamicum | |
| 101.86 | - |
NADP+ | pH 8.0, 25°C, recombinant mutant D35G/L36T/T37K/P192S | Corynebacterium glutamicum | |
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