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Literature summary for 1.1.1.244 extracted from

  • Ochsner, A.M.; Mueller, J.E.; Mora, C.A.; Vorholt, J.A.
    In vitro activation of NAD-dependent alcohol dehydrogenases by Nudix hydrolases is more widespread than assumed (2014), FEBS Lett., 588, 2993-2999 .
    View publication on PubMed

Activating Compound

Activating Compound Comment Organism Structure
Act activator protein, UniProt ID I3EA59, the in vitro activity of Bacillus methanolicus Mdh is increased by the endogenous activator protein Act, a Nudix hydrolase, in a pH-dependent manner. Act increases the Vmax of the enzyme and decreases the Km for methanol, the increase was 23fold at pH 7.4 and 50fold at pH 9.0 Bacillus methanolicus
activator protein ACT UniProt ID I3EA59, the in vitro activity of Bacillus methanolicus Mdh is increased by the endogenous activator protein Act, a Nudix hydrolase, in a pH-dependent manner. Act increases the Vmax of the enzyme and decreases the Km for methanol, the increase is 20fold at pH 7.4 and 9fold at pH 9.0 Bacillus methanolicus
additional information the enzyme is activated by several Nudix hydrolases, relative activities of Adh enzymes, overview Bacillus methanolicus
NudBC a Nudix hydrolase from Bacillus coagulans, UniProt ID G2TKZ3 Bacillus methanolicus
NudDK a Nudix hydrolase from Desulfotomaculum kuznetsovii, UniProt ID F6CJI1 Bacillus methanolicus
NudLF a Nudix hydrolase from Lysinibacillus fusiformis, UniProt ID D7WXY3 Bacillus methanolicus
NudLS a Nudix hydrolase from Lysinibacillus sphaericus, UniProt ID B1HRL7 Bacillus methanolicus

Cloned(Commentary)

Cloned (Comment) Organism
gene mdh, sequence comparisons and phylogenetic tree, recombinant expression of His-tagged enzyme in Escherichia coli strain BL21(DE3) Bacillus methanolicus
gene mdh2, sequence comparisons and phylogenetic tree, recombinant expression of His-tagged enzyme in Escherichia coli strain BL21(DE3) Bacillus methanolicus

Protein Variants

Protein Variants Comment Organism
D38G site-directed mutagenesis, the mutant is active with NADP+ and NAD+ in contrast to the wild-type enzyme Bacillus methanolicus
D41G site-directed mutagenesis, the mutant is active with NADP+ and NAD+ in contrast to the wild-type enzyme, it shows increased activity compared to the wild-type enzyme Bacillus methanolicus
S101G site-directed mutagenesis, the mutant shows altered kinetics, reduced activity, and altered pH-dependency compared to the wild-type enzyme, overview Bacillus methanolicus
S98G site-directed mutagenesis, the mutant shows altered kinetics, reduced activity, and altered pH-dependency compared to the wild-type enzyme, overview Bacillus methanolicus

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
additional information Michaelis-Menten kinetics Bacillus methanolicus
9
-
methanol recombinant enzyme, pH 9.0, 30┬░C, in presence of activator Act Bacillus methanolicus
25
-
methanol recombinant enzyme, pH 7.4, 30┬░C, in presence of activator Act Bacillus methanolicus
51
-
ethanol recombinant enzyme, pH 7.4, 30┬░C, in presence of activator Act Bacillus methanolicus
96
-
methanol recombinant enzyme, pH 9.0, 30┬░C, in presence of activator Act Bacillus methanolicus
137
-
methanol recombinant enzyme mutant D38G, pH 7.4, 30┬░C, in presence of activator Act, with NADP+ Bacillus methanolicus
150
-
methanol recombinant enzyme, pH 9.0, 30┬░C, in absence of activator Act Bacillus methanolicus
161
-
ethanol recombinant enzyme, pH 9.0, 30┬░C, in presence of activator Act Bacillus methanolicus
224
-
ethanol recombinant enzyme, pH 9.0, 30┬░C, in absence of activator Act Bacillus methanolicus
225
-
ethanol recombinant enzyme, pH 7.4, 30┬░C, in absence of activator Act Bacillus methanolicus
255
-
methanol recombinant enzyme, pH 7.4, 30┬░C, in presence of activator Act Bacillus methanolicus
349
-
methanol recombinant enzyme, pH 7.4, 30┬░C, in absence of activator Act Bacillus methanolicus
379
-
methanol recombinant enzyme mutant D38G, pH 7.4, 30┬░C, in absence of activator Act, with NADP+ Bacillus methanolicus
416
-
methanol recombinant enzyme, pH 9.0, 30┬░C, in absence of activator Act Bacillus methanolicus
733
-
methanol recombinant enzyme, pH 7.4, 30┬░C, in absence of activator Act Bacillus methanolicus
847
-
methanol recombinant enzyme mutant S98G, pH 7.4, 30┬░C, in presence of activator Act Bacillus methanolicus
1151
-
methanol recombinant enzyme mutant S98G, pH 7.4, 30┬░C, in absence of activator Act Bacillus methanolicus

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
methanol + NAD+ Bacillus methanolicus
-
formaldehyde + NADH + H+
-
r
methanol + NAD+ Bacillus methanolicus MGA3
-
formaldehyde + NADH + H+
-
r

Organism

Organism UniProt Comment Textmining
Bacillus methanolicus I3DTM5
-
-
Bacillus methanolicus I3E2P9
-
-
Bacillus methanolicus MGA3 I3DTM5
-
-
Bacillus methanolicus MGA3 I3E2P9
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography Bacillus methanolicus

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
ethanol + NAD+
-
Bacillus methanolicus acetaldehyde + NADH + H+
-
r
ethanol + NAD+
-
Bacillus methanolicus MGA3 acetaldehyde + NADH + H+
-
r
methanol + NAD+
-
Bacillus methanolicus formaldehyde + NADH + H+
-
r
methanol + NAD+
-
Bacillus methanolicus MGA3 formaldehyde + NADH + H+
-
r
methanol + NADP+ Mdh enzyme mutant D41G, no activity of the wild-type enzyme with NADP(H) Bacillus methanolicus formaldehyde + NADPH + H+
-
r
methanol + NADP+ Mdh enzyme mutant D98G, no activity of the wild-type enzyme with NADP(H) Bacillus methanolicus formaldehyde + NADPH + H+
-
r
methanol + NADP+ Mdh enzyme mutant D98G, no activity of the wild-type enzyme with NADP(H) Bacillus methanolicus MGA3 formaldehyde + NADPH + H+
-
r
methanol + NADP+ Mdh enzyme mutant D41G, no activity of the wild-type enzyme with NADP(H) Bacillus methanolicus MGA3 formaldehyde + NADPH + H+
-
r
additional information poor activity with 2-propanol and 1,2-popanediol Bacillus methanolicus ?
-
?
additional information poor activity with 2-propanol and 1,2-popanediol Bacillus methanolicus MGA3 ?
-
?
n-butanol + NAD+
-
Bacillus methanolicus butyraldehyde + NADH + H+
-
r
n-butanol + NAD+
-
Bacillus methanolicus MGA3 butyraldehyde + NADH + H+
-
r
n-propanol + NAD+
-
Bacillus methanolicus propionaldehyde + NADH + H+
-
r

Synonyms

Synonyms Comment Organism
MDH
-
Bacillus methanolicus
MDH2
-
Bacillus methanolicus
NAD-dependent methanol dehydrogenase
-
Bacillus methanolicus

Temperature Optimum [┬░C]

Temperature Optimum [┬░C] Temperature Optimum Maximum [┬░C] Comment Organism
30
-
assay at Bacillus methanolicus

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
9
-
-
Bacillus methanolicus

Cofactor

Cofactor Comment Organism Structure
NAD+
-
Bacillus methanolicus

General Information

General Information Comment Organism
evolution the enzyme belongs to the type III alcohol dehydrogenase (Adh) family Bacillus methanolicus

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.00004
-
methanol recombinant enzyme, pH 7.4, 30┬░C, in absence of activator Act Bacillus methanolicus
0.0001
-
methanol recombinant enzyme mutant D38G, pH 7.4, 30┬░C, in absence of activator Act, with NADP+ Bacillus methanolicus
0.0003
-
methanol recombinant enzyme mutant S98G, pH 7.4, 30┬░C, in absence of activator Act Bacillus methanolicus
0.0003
-
methanol recombinant enzyme, pH 7.4, 30┬░C, in absence of activator Act Bacillus methanolicus
0.0003
-
methanol recombinant enzyme, pH 9.0, 30┬░C, in absence of activator Act Bacillus methanolicus
0.0006
-
methanol recombinant enzyme mutant D38G, pH 7.4, 30┬░C, in presence of activator Act, with NADP+ Bacillus methanolicus
0.0006
-
ethanol recombinant enzyme, pH 7.4, 30┬░C, in absence of activator Act Bacillus methanolicus
0.0007
-
methanol recombinant enzyme mutant S98G, pH 7.4, 30┬░C, in presence of activator Act Bacillus methanolicus
0.0007
-
methanol recombinant enzyme, pH 9.0, 30┬░C, in absence of activator Act Bacillus methanolicus
0.0008
-
methanol recombinant enzyme, pH 7.4, 30┬░C, in presence of activator Act Bacillus methanolicus
0.0014
-
ethanol recombinant enzyme, pH 9.0, 30┬░C, in absence of activator Act Bacillus methanolicus
0.0028
-
methanol recombinant enzyme, pH 9.0, 30┬░C, in presence of activator Act Bacillus methanolicus
0.0068
-
methanol recombinant enzyme, pH 7.4, 30┬░C, in presence of activator Act Bacillus methanolicus
0.0074
-
ethanol recombinant enzyme, pH 7.4, 30┬░C, in presence of activator Act Bacillus methanolicus
0.0143
-
ethanol recombinant enzyme, pH 9.0, 30┬░C, in presence of activator Act Bacillus methanolicus
0.0353
-
methanol recombinant enzyme, pH 9.0, 30┬░C, in presence of activator Act Bacillus methanolicus