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 (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 | 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 | 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 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 | 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 | 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 | UniProt | Comment | Textmining |
---|---|---|---|
Cupriavidus necator | F8GNE5 | formerly Ralstonia eutropha | - |
Cupriavidus necator N-1 | F8GNE5 | formerly Ralstonia eutropha | - |
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 | Comment | Organism |
---|---|---|
? | x * 40700, recombinant His-tagged enzyme Mdh2, SDS-PAGE | Cupriavidus necator |
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 Maximum [°C] | Comment | Organism |
---|---|---|---|
30 | - |
forward reaction | Cupriavidus necator |
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 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 Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
9.5 | - |
forward reaction | Cupriavidus necator |
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 | Comment | Organism | Structure |
---|---|---|---|
additional information | no methanol oxidation activity is detected when NADP+ is used | Cupriavidus necator | |
NAD+ | specific for | Cupriavidus necator |
General Information | Comment | Organism |
---|---|---|
evolution | Mdh2 is a group III Adh | Cupriavidus necator |
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 |