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Literature summary extracted from

  • Morimoto, Y.; Honda, K.; Ye, X.; Okano, K.; Ohtake, H.
    Directed evolution of thermotolerant malic enzyme for improved malate production (2014), J. Biosci. Bioeng., 117, 147-152.
    View publication on PubMed

Protein Variants

EC Number Protein Variants Comment Organism
1.1.1.40 I310V site-directed mutagenesis Thermococcus kodakarensis
1.1.1.40 K228R site-directed mutagenesis Thermococcus kodakarensis
1.1.1.40 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
1.1.1.40 R221G site-directed mutagenesis Thermococcus kodakarensis
1.1.1.40 R221G/K228R site-directed mutagenesis, substitution of Arg221 with Gly is responsible for the shift in reaction specificity Thermococcus kodakarensis
1.1.1.40 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
1.1.1.40 V393V 1179 GTC /GTT results in a synonymous mutation of V393V Thermococcus kodakarensis

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.1.1.40 0.008
-
NADP+ pH 8.0, 70°C, recombinant wild-type enzyme Thermococcus kodakarensis
1.1.1.40 0.019
-
NADP+ pH 8.0, 70°C, recombinant mutant K228R Thermococcus kodakarensis
1.1.1.40 0.021
-
NADP+ pH 8.0, 70°C, recombinant mutant I310V Thermococcus kodakarensis
1.1.1.40 0.025
-
NADP+ pH 8.0, 70°C, recombinant mutant R221G Thermococcus kodakarensis
1.1.1.40 0.244
-
NADP+ pH 8.0, 70°C, recombinant mutant R221G/K228R Thermococcus kodakarensis
1.1.1.40 0.384
-
NADP+ pH 8.0, 70°C, recombinant mutant R221G/K22P8R/I310V Thermococcus kodakarensis
1.1.1.40 0.96
-
NAD+ pH 8.0, 70°C, recombinant mutant R221G/K228R Thermococcus kodakarensis
1.1.1.40 1.28
-
NAD+ pH 8.0, 70°C, recombinant mutant K228R Thermococcus kodakarensis
1.1.1.40 1.54
-
NAD+ pH 8.0, 70°C, recombinant mutant R221G/K228R/I310V Thermococcus kodakarensis
1.1.1.40 2.06
-
NAD+ pH 8.0, 70°C, recombinant wild-type enzyme Thermococcus kodakarensis
1.1.1.40 2.29
-
NAD+ pH 8.0, 70°C, recombinant mutant R221G Thermococcus kodakarensis
1.1.1.40 6.55
-
NAD+ pH 8.0, 70°C, recombinant mutant I310V Thermococcus kodakarensis

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
1.1.1.40 Mn2+ required Thermococcus kodakarensis

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.1.1.40 (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
1.1.1.40 (S)-malate + NADP+ Thermococcus kodakarensis
-
pyruvate + CO2 + NADPH
-
r
1.1.1.40 additional information Thermococcus kodakarensis malic enzymes can reversibly catalyze the NAD(P)H-dependent reductive carboxylation of pyruvate to malate ?
-
?

Organism

EC Number Organism UniProt Comment Textmining
1.1.1.40 Thermococcus kodakarensis Q5JGC7
-
-

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.1.1.40 (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
1.1.1.40 (S)-malate + NADP+
-
Thermococcus kodakarensis pyruvate + CO2 + NADPH
-
r
1.1.1.40 additional information malic enzymes can reversibly catalyze the NAD(P)H-dependent reductive carboxylation of pyruvate to malate Thermococcus kodakarensis ?
-
?
1.1.1.40 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

EC Number Synonyms Comment Organism
1.1.1.40 NADP(H)-dependent malic enzyme
-
Thermococcus kodakarensis

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.1.1.40 70
-
assay at Thermococcus kodakarensis

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.1.1.40 2.97
-
NADP+ pH 8.0, 70°C, recombinant mutant K228R Thermococcus kodakarensis
1.1.1.40 3.37
-
NAD+ pH 8.0, 70°C, recombinant mutant K228R Thermococcus kodakarensis
1.1.1.40 4.69
-
NAD+ pH 8.0, 70°C, recombinant wild-type enzyme Thermococcus kodakarensis
1.1.1.40 6.48
-
NADP+ pH 8.0, 70°C, recombinant mutant R221G/K228R Thermococcus kodakarensis
1.1.1.40 7.44
-
NAD+ pH 8.0, 70°C, recombinant mutant R221G/K228R Thermococcus kodakarensis
1.1.1.40 12.7
-
NAD+ pH 8.0, 70°C, recombinant mutant R221G Thermococcus kodakarensis
1.1.1.40 21.3
-
NAD+ pH 8.0, 70°C, recombinant mutant R221G/K228R/I310V Thermococcus kodakarensis
1.1.1.40 31.1
-
NADP+ pH 8.0, 70°C, recombinant wild-type enzyme Thermococcus kodakarensis
1.1.1.40 37.7
-
NAD+ pH 8.0, 70°C, recombinant mutant I310V Thermococcus kodakarensis
1.1.1.40 40.1
-
NADP+ pH 8.0, 70°C, recombinant mutant R221G/K22P8R/I310V Thermococcus kodakarensis
1.1.1.40 42.6
-
NADP+ pH 8.0, 70°C, recombinant mutant R221G Thermococcus kodakarensis
1.1.1.40 43.4
-
NADP+ pH 8.0, 70°C, recombinant mutant I310V Thermococcus kodakarensis

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.1.1.40 8
-
assay at Thermococcus kodakarensis

Cofactor

EC Number Cofactor Comment Organism Structure
1.1.1.40 additional information the enzyme mutant R221G utilizes NAD+/NADH with high efficiency in contrast to the wild-type enzyme Thermococcus kodakarensis
1.1.1.40 NADP+
-
Thermococcus kodakarensis
1.1.1.40 NADPH
-
Thermococcus kodakarensis

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.1.1.40 2.27
-
NAD+ pH 8.0, 70°C, recombinant wild-type enzyme Thermococcus kodakarensis
1.1.1.40 2.64
-
NAD+ pH 8.0, 70°C, recombinant mutant K228R Thermococcus kodakarensis
1.1.1.40 5.57
-
NAD+ pH 8.0, 70°C, recombinant mutant R221G Thermococcus kodakarensis
1.1.1.40 5.76
-
NAD+ pH 8.0, 70°C, recombinant mutant I310V Thermococcus kodakarensis
1.1.1.40 7.72
-
NAD+ pH 8.0, 70°C, recombinant mutant R221G/K228R Thermococcus kodakarensis
1.1.1.40 13.8
-
NAD+ pH 8.0, 70°C, recombinant mutant R221G/K228R/I310V Thermococcus kodakarensis
1.1.1.40 27.5
-
NADP+ pH 8.0, 70°C, recombinant mutant R221G/K228R Thermococcus kodakarensis
1.1.1.40 104
-
NADP+ pH 8.0, 70°C, recombinant mutant R221G/K22P8R/I310V Thermococcus kodakarensis
1.1.1.40 156
-
NADP+ pH 8.0, 70°C, recombinant mutant K228R Thermococcus kodakarensis
1.1.1.40 1700
-
NADP+ pH 8.0, 70°C, recombinant mutant R221G Thermococcus kodakarensis
1.1.1.40 2070
-
NADP+ pH 8.0, 70°C, recombinant mutant I310V Thermococcus kodakarensis
1.1.1.40 3760
-
NADP+ pH 8.0, 70°C, recombinant wild-type enzyme Thermococcus kodakarensis