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Literature summary for 1.1.1.40 extracted from
- Directed evolution of thermotolerant malic enzyme for improved malate production (2014), J. Biosci. Bioeng., 117, 147-152.
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| I310V | site-directed mutagenesis | Thermococcus kodakarensis |
| K228R | site-directed mutagenesis | Thermococcus kodakarensis |
| additional information | directed evolution of the thermotolerant NADP(H)-dependent malic enzyme from Thermococcus kodakarensis is conducted to alter the cofactor preference of the enzyme from NADP(H) to NAD(H). Integration of the thermotolerant NADPH-dependent malic enzyme (EC 1.1.1.40) from Thermococcus kodakarensis (TkME) to the chimeric EM pathway enables the construction of a cofactor-balanced and HCO3- fixing synthetic pathway, through which the direct conversion of glucose to malate can be achieved. The thermal degradation of the redox cofactors NADP+ and NADPH tends to be a major obstacle to the long-term operation of the in vitro metabolic system because, unlike living biological systems, it is not equipped with the complete enzyme apparatus for the de novo synthesis of these cofactors. No significant change is observed in the thermal stability of the wild type and mutant enzymes | Thermococcus kodakarensis |
| R221G | site-directed mutagenesis | Thermococcus kodakarensis |
| R221G/K228R | site-directed mutagenesis, substitution of Arg221 with Gly is responsible for the shift in reaction specificity | Thermococcus kodakarensis |
| R221G/K228R/I310V | site-directed mutagenesis, the reaction specificity of the triple mutant is significantly shifted to malate production and the mutant gives a reduced amount of the byproduct than the wild-type. When the triple mutant enzyme is used as a catalyst for pyruvate carboxylation with NADH, the enzyme gives 1.2times higher concentration of malate than the wild-type with NADPH. Single-point mutation analysis reveals that the substitution of Arg221 with Gly is responsible for the shift in reaction specificity | Thermococcus kodakarensis |
| V393V | 1179 GTC /GTT results in a synonymous mutation of V393V | Thermococcus kodakarensis |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.008 | - |
NADP+ | pH 8.0, 70°C, recombinant wild-type enzyme | Thermococcus kodakarensis |  |
| 0.019 | - |
NADP+ | pH 8.0, 70°C, recombinant mutant K228R | Thermococcus kodakarensis | |
| 0.021 | - |
NADP+ | pH 8.0, 70°C, recombinant mutant I310V | Thermococcus kodakarensis | |
| 0.025 | - |
NADP+ | pH 8.0, 70°C, recombinant mutant R221G | Thermococcus kodakarensis | |
| 0.244 | - |
NADP+ | pH 8.0, 70°C, recombinant mutant R221G/K228R | Thermococcus kodakarensis | |
| 0.384 | - |
NADP+ | pH 8.0, 70°C, recombinant mutant R221G/K22P8R/I310V | Thermococcus kodakarensis | |
| 0.96 | - |
NAD+ | pH 8.0, 70°C, recombinant mutant R221G/K228R | Thermococcus kodakarensis | |
| 1.28 | - |
NAD+ | pH 8.0, 70°C, recombinant mutant K228R | Thermococcus kodakarensis | |
| 1.54 | - |
NAD+ | pH 8.0, 70°C, recombinant mutant R221G/K228R/I310V | Thermococcus kodakarensis | |
| 2.06 | - |
NAD+ | pH 8.0, 70°C, recombinant wild-type enzyme | Thermococcus kodakarensis | |
| 2.29 | - |
NAD+ | pH 8.0, 70°C, recombinant mutant R221G | Thermococcus kodakarensis | |
| 6.55 | - |
NAD+ | pH 8.0, 70°C, recombinant mutant I310V | Thermococcus kodakarensis | |
Metals/Ions
| Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|
| Mn2+ | required | Thermococcus kodakarensis | |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| (S)-malate + NAD+ | Thermococcus kodakarensis | when the R221G/K228R/I310V mutant is used with NADH, the mutant gives 1.2 and 2.7 times higher malate concentration than the wild-type with NADPH and NADH, respectively. These results can be partly explained by the alteration of the cofactor preference of the mutant enzyme, since the half-life of NADH is approximately 1.3times longer than that of NADPH at 50°C. However, the Km of the triple mutant for NAD+ remains 190times higher than that of the wild-type for NADP+ | pyruvate + CO2 + NADH | - |
r | |
| (S)-malate + NADP+ | Thermococcus kodakarensis | - |
pyruvate + CO2 + NADPH | - |
r | |
| additional information | Thermococcus kodakarensis | malic enzymes can reversibly catalyze the NAD(P)H-dependent reductive carboxylation of pyruvate to malate | ? | - |
? | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Thermococcus kodakarensis | Q5JGC7 | - |
- |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| (S)-malate + NAD+ | when the R221G/K228R/I310V mutant is used with NADH, the mutant gives 1.2 and 2.7 times higher malate concentration than the wild-type with NADPH and NADH, respectively. These results can be partly explained by the alteration of the cofactor preference of the mutant enzyme, since the half-life of NADH is approximately 1.3times longer than that of NADPH at 50°C. However, the Km of the triple mutant for NAD+ remains 190times higher than that of the wild-type for NADP+ | Thermococcus kodakarensis | pyruvate + CO2 + NADH | - |
r | |
| (S)-malate + NADP+ | - |
Thermococcus kodakarensis | pyruvate + CO2 + NADPH | - |
r | |
| additional information | malic enzymes can reversibly catalyze the NAD(P)H-dependent reductive carboxylation of pyruvate to malate | Thermococcus kodakarensis | ? | - |
? | |
| additional information | when the R221G/K228R/I310V mutant is used with NADH, the mutant gives 1.2 and 2.7 times higher malate concentration than the wild-type with NADPH and NADH, respectively. These results can be partly explained by the alteration of the cofactor preference of the mutant enzyme, since the half-life of NADH is approximately 1.3times longer than that of NADPH at 50°C. However, the Km of the triple mutant for NAD+ remains 190times higher than that of the wild-type for NADP+ | Thermococcus kodakarensis | ? | - |
? | |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| NADP(H)-dependent malic enzyme | - |
Thermococcus kodakarensis |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 70 | - |
assay at | Thermococcus kodakarensis |
Turnover Number [1/s]
| Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 2.97 | - |
NADP+ | pH 8.0, 70°C, recombinant mutant K228R | Thermococcus kodakarensis | |
| 3.37 | - |
NAD+ | pH 8.0, 70°C, recombinant mutant K228R | Thermococcus kodakarensis | |
| 4.69 | - |
NAD+ | pH 8.0, 70°C, recombinant wild-type enzyme | Thermococcus kodakarensis | |
| 6.48 | - |
NADP+ | pH 8.0, 70°C, recombinant mutant R221G/K228R | Thermococcus kodakarensis | |
| 7.44 | - |
NAD+ | pH 8.0, 70°C, recombinant mutant R221G/K228R | Thermococcus kodakarensis | |
| 12.7 | - |
NAD+ | pH 8.0, 70°C, recombinant mutant R221G | Thermococcus kodakarensis | |
| 21.3 | - |
NAD+ | pH 8.0, 70°C, recombinant mutant R221G/K228R/I310V | Thermococcus kodakarensis | |
| 31.1 | - |
NADP+ | pH 8.0, 70°C, recombinant wild-type enzyme | Thermococcus kodakarensis | |
| 37.7 | - |
NAD+ | pH 8.0, 70°C, recombinant mutant I310V | Thermococcus kodakarensis | |
| 40.1 | - |
NADP+ | pH 8.0, 70°C, recombinant mutant R221G/K22P8R/I310V | Thermococcus kodakarensis | |
| 42.6 | - |
NADP+ | pH 8.0, 70°C, recombinant mutant R221G | Thermococcus kodakarensis | |
| 43.4 | - |
NADP+ | pH 8.0, 70°C, recombinant mutant I310V | Thermococcus kodakarensis | |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 8 | - |
assay at | Thermococcus kodakarensis |
Cofactor
| Cofactor | Comment | Organism | Structure |
|---|---|---|---|
| additional information | the enzyme mutant R221G utilizes NAD+/NADH with high efficiency in contrast to the wild-type enzyme | Thermococcus kodakarensis | |
| NADP+ | - |
Thermococcus kodakarensis | |
| NADPH | - |
Thermococcus kodakarensis | |
kcat/KM [mM/s]
| kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 2.27 | - |
NAD+ | pH 8.0, 70°C, recombinant wild-type enzyme | Thermococcus kodakarensis | |
| 2.64 | - |
NAD+ | pH 8.0, 70°C, recombinant mutant K228R | Thermococcus kodakarensis | |
| 5.57 | - |
NAD+ | pH 8.0, 70°C, recombinant mutant R221G | Thermococcus kodakarensis | |
| 5.76 | - |
NAD+ | pH 8.0, 70°C, recombinant mutant I310V | Thermococcus kodakarensis | |
| 7.72 | - |
NAD+ | pH 8.0, 70°C, recombinant mutant R221G/K228R | Thermococcus kodakarensis | |
| 13.8 | - |
NAD+ | pH 8.0, 70°C, recombinant mutant R221G/K228R/I310V | Thermococcus kodakarensis | |
| 27.5 | - |
NADP+ | pH 8.0, 70°C, recombinant mutant R221G/K228R | Thermococcus kodakarensis | |
| 104 | - |
NADP+ | pH 8.0, 70°C, recombinant mutant R221G/K22P8R/I310V | Thermococcus kodakarensis | |
| 156 | - |
NADP+ | pH 8.0, 70°C, recombinant mutant K228R | Thermococcus kodakarensis | |
| 1700 | - |
NADP+ | pH 8.0, 70°C, recombinant mutant R221G | Thermococcus kodakarensis | |
| 2070 | - |
NADP+ | pH 8.0, 70°C, recombinant mutant I310V | Thermococcus kodakarensis | |
| 3760 | - |
NADP+ | pH 8.0, 70°C, recombinant wild-type enzyme | Thermococcus kodakarensis | |
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