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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 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 wild-type and 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
0.048
-
NAD+ pH 8.0, 25°C, recombinant wild-type enzyme Corynebacterium glutamicum
0.058
-
NAD+ pH 8.0, 25°C, recombinant mutant D35G/L36T/P192S 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

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+
-
?

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 ATCC 13032 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+
-
?

Synonyms

Synonyms Comment Organism
GapA
-
Corynebacterium glutamicum
GAPDH
-
Corynebacterium glutamicum
glyceraldehyde 3-phosphate dehydrogenase
-
Corynebacterium glutamicum
NAD-dependent 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
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
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
6.45
-
NAD+ pH 8.0, 25°C, recombinant wild-type enzyme Corynebacterium glutamicum

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8
-
assay at Corynebacterium glutamicum

Cofactor

Cofactor Comment Organism Structure
additional information the wild-type enzyme shows no activity with NADP+. The carboxylate group of Asp35 forms a network of hydrogen bonds with the 2' and 3-hydroxylgroups of the adenosine ribose ring of NAD+. This seems to be a critical factor to discriminate against the 2'-phosphate group of NADP+, because of electrostatic repulsion between the carboxylate group and phosphate group. Mutation of residues D35, L36, T37, and P192 alters the enzyme's cofactor specificity. Molecular docking and modelling Corynebacterium glutamicum
NAD+ absolutely specific for 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 NADH 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
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
134.375
-
NAD+ pH 8.0, 25°C, recombinant wild-type enzyme Corynebacterium glutamicum