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Literature summary for 1.1.1.244 extracted from

  • Wu, T.Y.; Chen, C.T.; Liu, J.T.; Bogorad, I.W.; Damoiseaux, R.; Liao, J.C.
    Characterization and evolution of an activator-independent methanol dehydrogenase from Cupriavidus necator N-1 (2016), Appl. Microbiol. Biotechnol., 100, 4969-4983 .
    View publication on PubMed

Activating Compound

Activating Compound Comment Organism Structure
additional information Mdh2 from Cupriavidus necator does not require activation by and is insensitive to known activators such as Bacillus methanolicus ACT and Escherichia coli Nudix hydrolase NudF, or putative native Cupriavidus necator activators in the Nudix family under mesophilic condition. The enzyme exhibits higher or comparable activity and affinity toward methanol relative to the Bacillus methanolicus Mdh with or without activator ACT in a wide range of temperatures Cupriavidus necator

Cloned(Commentary)

Cloned (Comment) Organism
gene mdh2 or CNE_2c13570, DNA and amino acid sequence determination and analysis, sequence comparisons, recombinant expression of wild-type and mutant enzymes in Escherichia coli strain MG1655, recombinant expression of His-tagged enzyme Mdh2 in Escherichia coli strain XL-1 Cupriavidus necator

Protein Variants

Protein Variants Comment Organism
A169C site-directed mutagenesis Cupriavidus necator
A169I site-directed mutagenesis Cupriavidus necator
A169L site-directed mutagenesis Cupriavidus necator
A169M site-directed mutagenesis Cupriavidus necator
A169P site-directed mutagenesis Cupriavidus necator
A169V from library screening, mutant CT1-2, mutant variants CT1-2, CT2-1, and CT4-1 show 5 to 10fold reduced specific activity towards ethanol and 6 to 8fold reduced for propanol compared to wild-type Cupriavidus necator
A26V site-directed mutagenesis, mutant CT2-2, the mutation A26V alone demolishes Mdh activity, inactive mutant Cupriavidus necator
A26V/A169V site-directed mutagenesis, mutant CT2-1, synergistic effect of mutation A26V and A169V in enzyme function increasing the activity. Mutant variants CT1-2, CT2-1, and CT4-1 show 5 to 10fold reduced specific activity towards ethanol and 6 to 8fold reduced for propanol compared to wild-type Cupriavidus necator
A26V/A31V/A169V site-directed mutagenesis, mutant CT4-1. Engineering of a mutant enzyme chimeric variant CT4-1 of Mdh2 that shows a 6fold higher Kcat/Km for methanol and 10fold lower Kcat/Km for n-butanol. CT4-1 represents an NAD-dependent Mdh with much improved catalytic efficiency and specificity toward methanol compared with the existing NAD-dependent Mdhs with or without ACT activation. Development of automatic high throughput screening (HTS) for Mdh evolution, overview. Mutant variants CT1-2, CT2-1, and CT4-1 show 5 to 10fold reduced specific activity towards ethanol and 6 to 8fold reduced for propanol compared to wild-type. CT4-1 significantly improves its methanol over C2 to C4 alcohol activity ratio compared to wild-type Cupriavidus necator
A31V from library screening, mutant CT1-1 Cupriavidus necator
additional information engineering of a mutant enzyme variants results in enzymes with reduced activity with n-butanol and increased activity with methanol compared to wild-type Cupriavidus necator

Inhibitors

Inhibitors Comment Organism Structure
Ca2+ slight inhibition at 1 mM Cupriavidus necator
Cu2+ strong inhibition at 1 mM Cupriavidus necator
EDTA slight inhibition at 1 mM Cupriavidus necator
Na+ slight inhibition at 1 mM Cupriavidus necator
Zn2+ strong inhibition at 1 mM Cupriavidus necator

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.0072
-
n-butanol pH 9.5, 30°C, recombinant wild-type enzyme Cupriavidus necator
0.0113
-
n-butanol pH 9.5, 30°C, recombinant mutant A31V Cupriavidus necator
0.0216
-
methanol pH 9.5, 30°C, recombinant mutant A26V/A31V/A169V Cupriavidus necator
0.0269
-
methanol pH 9.5, 30°C, recombinant mutant A169V Cupriavidus necator
0.0307
-
methanol pH 9.5, 30°C, recombinant mutant A26V/A169V Cupriavidus necator
0.0665
-
n-butanol pH 9.5, 30°C, recombinant mutant A169V Cupriavidus necator
0.0759
-
n-butanol pH 9.5, 30°C, recombinant mutant A26V/A169V Cupriavidus necator
0.093
-
NAD+ pH 9.5, 30°C, recombinant wild-type enzyme Cupriavidus necator
0.12
-
n-butanol pH 9.5, 30°C, recombinant mutant A26V/A31V/A169V Cupriavidus necator
0.129
-
methanol pH 9.5, 30°C, recombinant mutant A31V Cupriavidus necator
0.132
-
methanol pH 9.5, 30°C, recombinant wild-type enzyme Cupriavidus necator

Metals/Ions

Metals/Ions Comment Organism Structure
Co2+ slightly activating at 1 mM Cupriavidus necator
additional information no or poor effects by 1 mM of Fe2+, Mn2+, K+, and Mg2+ Cupriavidus necator
Ni2+ activates at 1 mM Cupriavidus necator

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
methanol + NAD+ Cupriavidus necator
-
formaldehyde + NADH + H+
-
r
methanol + NAD+ Cupriavidus necator N-1
-
formaldehyde + NADH + H+
-
r

Organism

Organism UniProt Comment Textmining
Cupriavidus necator F8GNE5 formerly Ralstonia eutropha
-
Cupriavidus necator N-1 F8GNE5 formerly Ralstonia eutropha
-

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
ethanol + NAD+
-
Cupriavidus necator acetaldehyde + NADH + H+
-
r
ethanol + NAD+
-
Cupriavidus necator N-1 acetaldehyde + NADH + H+
-
r
methanol + NAD+
-
Cupriavidus necator formaldehyde + NADH + H+
-
r
methanol + NAD+
-
Cupriavidus necator N-1 formaldehyde + NADH + H+
-
r
n-butanol + NAD+
-
Cupriavidus necator butyraldehyde + NADH + H+
-
r
n-butanol + NAD+
-
Cupriavidus necator N-1 butyraldehyde + NADH + H+
-
r
propanol + NAD+
-
Cupriavidus necator propionaldehyde + NADH + H+
-
r
propanol + NAD+
-
Cupriavidus necator N-1 propionaldehyde + NADH + H+
-
r

Subunits

Subunits Comment Organism
? x * 40700, recombinant His-tagged enzyme Mdh2, SDS-PAGE Cupriavidus necator

Synonyms

Synonyms Comment Organism
activator-independent methanol dehydrogenase
-
Cupriavidus necator
CNE_2c13570
-
Cupriavidus necator
group III NAD-dependent alcohol dehydrogenase
-
Cupriavidus necator
MDH2
-
Cupriavidus necator
NAD-dependent MDH
-
Cupriavidus necator

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
30
-
forward reaction Cupriavidus necator

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
30 60 purified recombinant His-tagged enzyme, 10 min, pH 9.5, stable up to 30°C, inactivation above, 13 % of activity remains after preincubation at 55°C and the activity is abolished at 60°C Cupriavidus necator

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.13
-
methanol pH 9.5, 30°C, recombinant mutant A169V Cupriavidus necator
0.2
-
methanol pH 9.5, 30°C, recombinant mutant A26V/A31V/A169V Cupriavidus necator
0.21
-
methanol pH 9.5, 30°C, recombinant mutant A26V/A169V Cupriavidus necator
0.22
-
methanol pH 9.5, 30°C, recombinant wild-type enzyme Cupriavidus necator
0.24
-
NAD+ pH 9.5, 30°C, recombinant wild-type enzyme Cupriavidus necator
0.25
-
methanol pH 9.5, 30°C, recombinant mutant A31V Cupriavidus necator
3.3
-
n-butanol pH 9.5, 30°C, recombinant mutant A169V Cupriavidus necator
5
-
n-butanol pH 9.5, 30°C, recombinant mutant A26V/A169V Cupriavidus necator
5.7
-
n-butanol pH 9.5, 30°C, recombinant mutant A26V/A31V/A169V Cupriavidus necator
6.5
-
n-butanol pH 9.5, 30°C, recombinant wild-type enzyme Cupriavidus necator
7.2
-
n-butanol pH 9.5, 30°C, recombinant mutant A31V Cupriavidus necator

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
9.5
-
forward reaction Cupriavidus necator

pH Range

pH Minimum pH Maximum Comment Organism
6 10.5 activity range, forward reaction, inactive below pH 6.0 and above pH 10.5 Cupriavidus necator

Cofactor

Cofactor Comment Organism Structure
additional information no methanol oxidation activity is detected when NADP+ is used Cupriavidus necator
NAD+ specific for Cupriavidus necator

General Information

General Information Comment Organism
evolution Mdh2 is a group III Adh Cupriavidus necator

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.0016
-
methanol pH 9.5, 30°C, recombinant wild-type enzyme Cupriavidus necator
0.0019
-
methanol pH 9.5, 30°C, recombinant mutant A31V Cupriavidus necator
0.0048
-
methanol pH 9.5, 30°C, recombinant mutant A169V Cupriavidus necator
0.0068
-
methanol pH 9.5, 30°C, recombinant mutant A26V/A169V Cupriavidus necator
0.0093
-
methanol pH 9.5, 30°C, recombinant mutant A26V/A31V/A169V Cupriavidus necator
0.048
-
n-butanol pH 9.5, 30°C, recombinant mutant A26V/A31V/A169V Cupriavidus necator
0.05
-
n-butanol pH 9.5, 30°C, recombinant mutant A169V Cupriavidus necator
0.066
-
n-butanol pH 9.5, 30°C, recombinant mutant A26V/A169V Cupriavidus necator
0.258
-
NAD+ pH 9.5, 30°C, recombinant wild-type enzyme Cupriavidus necator
0.637
-
n-butanol pH 9.5, 30°C, recombinant mutant A31V Cupriavidus necator
0.903
-
n-butanol pH 9.5, 30°C, recombinant wild-type enzyme Cupriavidus necator