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Literature summary 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

EC Number Activating Compound Comment Organism Structure
1.1.1.244 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)

EC Number Cloned (Comment) Organism
1.1.1.244 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

EC Number Protein Variants Comment Organism
1.1.1.244 A169C site-directed mutagenesis Cupriavidus necator
1.1.1.244 A169I site-directed mutagenesis Cupriavidus necator
1.1.1.244 A169L site-directed mutagenesis Cupriavidus necator
1.1.1.244 A169M site-directed mutagenesis Cupriavidus necator
1.1.1.244 A169P site-directed mutagenesis Cupriavidus necator
1.1.1.244 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
1.1.1.244 A26V site-directed mutagenesis, mutant CT2-2, the mutation A26V alone demolishes Mdh activity, inactive mutant Cupriavidus necator
1.1.1.244 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
1.1.1.244 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
1.1.1.244 A31V from library screening, mutant CT1-1 Cupriavidus necator
1.1.1.244 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

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

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.1.1.244 0.0072
-
 n-butanol pH 9.5, 30°C, recombinant wild-type enzyme Cupriavidus necator
1.1.1.244 0.0113
-
 n-butanol pH 9.5, 30°C, recombinant mutant A31V Cupriavidus necator
1.1.1.244 0.0216
-
 methanol pH 9.5, 30°C, recombinant mutant A26V/A31V/A169V Cupriavidus necator
1.1.1.244 0.0269
-
 methanol pH 9.5, 30°C, recombinant mutant A169V Cupriavidus necator
1.1.1.244 0.0307
-
 methanol pH 9.5, 30°C, recombinant mutant A26V/A169V Cupriavidus necator
1.1.1.244 0.0665
-
 n-butanol pH 9.5, 30°C, recombinant mutant A169V Cupriavidus necator
1.1.1.244 0.0759
-
 n-butanol pH 9.5, 30°C, recombinant mutant A26V/A169V Cupriavidus necator
1.1.1.244 0.093
-
 NAD+ pH 9.5, 30°C, recombinant wild-type enzyme Cupriavidus necator
1.1.1.244 0.12
-
 n-butanol pH 9.5, 30°C, recombinant mutant A26V/A31V/A169V Cupriavidus necator
1.1.1.244 0.129
-
 methanol pH 9.5, 30°C, recombinant mutant A31V Cupriavidus necator
1.1.1.244 0.132
-
 methanol pH 9.5, 30°C, recombinant wild-type enzyme Cupriavidus necator

Metals/Ions

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

Natural Substrates/ Products (Substrates)

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

Organism

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

Substrates and Products (Substrate)

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

Subunits

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

Synonyms

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

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.1.1.244 30
-
 forward reaction Cupriavidus necator

Temperature Stability [°C]

EC Number Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
1.1.1.244 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]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.1.1.244 0.13
-
 methanol pH 9.5, 30°C, recombinant mutant A169V Cupriavidus necator
1.1.1.244 0.2
-
 methanol pH 9.5, 30°C, recombinant mutant A26V/A31V/A169V Cupriavidus necator
1.1.1.244 0.21
-
 methanol pH 9.5, 30°C, recombinant mutant A26V/A169V Cupriavidus necator
1.1.1.244 0.22
-
 methanol pH 9.5, 30°C, recombinant wild-type enzyme Cupriavidus necator
1.1.1.244 0.24
-
 NAD+ pH 9.5, 30°C, recombinant wild-type enzyme Cupriavidus necator
1.1.1.244 0.25
-
 methanol pH 9.5, 30°C, recombinant mutant A31V Cupriavidus necator
1.1.1.244 3.3
-
 n-butanol pH 9.5, 30°C, recombinant mutant A169V Cupriavidus necator
1.1.1.244 5
-
 n-butanol pH 9.5, 30°C, recombinant mutant A26V/A169V Cupriavidus necator
1.1.1.244 5.7
-
 n-butanol pH 9.5, 30°C, recombinant mutant A26V/A31V/A169V Cupriavidus necator
1.1.1.244 6.5
-
 n-butanol pH 9.5, 30°C, recombinant wild-type enzyme Cupriavidus necator
1.1.1.244 7.2
-
 n-butanol pH 9.5, 30°C, recombinant mutant A31V Cupriavidus necator

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.1.1.244 9.5
-
 forward reaction Cupriavidus necator

pH Range

EC Number pH Minimum pH Maximum Comment Organism
1.1.1.244 6 10.5 activity range, forward reaction, inactive below pH 6.0 and above pH 10.5 Cupriavidus necator

Cofactor

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

General Information

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

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.1.1.244 0.0016
-
 methanol pH 9.5, 30°C, recombinant wild-type enzyme Cupriavidus necator
1.1.1.244 0.0019
-
 methanol pH 9.5, 30°C, recombinant mutant A31V Cupriavidus necator
1.1.1.244 0.0048
-
 methanol pH 9.5, 30°C, recombinant mutant A169V Cupriavidus necator
1.1.1.244 0.0068
-
 methanol pH 9.5, 30°C, recombinant mutant A26V/A169V Cupriavidus necator
1.1.1.244 0.0093
-
 methanol pH 9.5, 30°C, recombinant mutant A26V/A31V/A169V Cupriavidus necator
1.1.1.244 0.048
-
 n-butanol pH 9.5, 30°C, recombinant mutant A26V/A31V/A169V Cupriavidus necator
1.1.1.244 0.05
-
 n-butanol pH 9.5, 30°C, recombinant mutant A169V Cupriavidus necator
1.1.1.244 0.066
-
 n-butanol pH 9.5, 30°C, recombinant mutant A26V/A169V Cupriavidus necator
1.1.1.244 0.258
-
 NAD+ pH 9.5, 30°C, recombinant wild-type enzyme Cupriavidus necator
1.1.1.244 0.637
-
 n-butanol pH 9.5, 30°C, recombinant mutant A31V Cupriavidus necator
1.1.1.244 0.903
-
 n-butanol pH 9.5, 30°C, recombinant wild-type enzyme Cupriavidus necator