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Literature summary for 1.2.1.12 extracted from

  • Bommareddy, R.R.; Chen, Z.; Rappert, S.; Zeng, A.P.
    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.
    View publication on PubMed

Application

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(Commentary)

Cloned (Comment) Organism
expression in Escherichia coli Corynebacterium glutamicum

Protein Variants

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 [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

Organism UniProt Comment Textmining
Corynebacterium glutamicum Q01651
-
-
Corynebacterium glutamicum DSM 20300 Q01651
-
-

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 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 [1/s]

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

Cofactor Comment Organism Structure
NAD+
-
Corynebacterium glutamicum
NADP+ no cofactor for wild-type Corynebacterium glutamicum

kcat/KM [mM/s]

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