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Literature summary for 1.4.1.1 extracted from

  • Lerchner, A.; Jarasch, A.; Skerra, A.
    Engineering of alanine dehydrogenase from Bacillus subtilis for novel cofactor specificity (2016), Biotechnol. Appl. Biochem., 63, 616-624 .
    View publication on PubMed

Application

Application Comment Organism
synthesis alterating of cofactor specificity from NADH to NADPH via protein engineering for application as a regenerating enzyme in coupled reactions with NADPH-dependent alcohol dehydrogenases. Mutant D196A/L197R can be applied in a coupled oxidation/transamination reaction of the dicyclic dialcohol isosorbide to its diamines, catalyzed by Ralstonia sp. alcohol dehydrogenase and Paracoccus denitrificans omega-aminotransferase, allowing recycling of NADP+ and L-Ala. Recycling factors of 33 for NADP+ and 13 for L-Ala are observed Bacillus subtilis

Cloned(Commentary)

Cloned (Comment) Organism
expression in Escherichia coli Bacillus subtilis

Protein Variants

Protein Variants Comment Organism
D196A alterating cofactor specificity from NADH to NADPH, 10fold decrease in activity with NADH, 4fold increase in activity with NADPH Bacillus subtilis
D196A/L197R alterating cofactor specificity from NADH to NADPH, almost the same activity with NADPH as the wild-type enzyme for NADH Bacillus subtilis
D196A/L197R/N198S/R201A alterating cofactor specificity from NADH to NADPH, loss of activity with NADH, 5fold increase in activity with NADPH Bacillus subtilis
D196A/L197R/R201A alterating cofactor specificity from NADH to NADPH, loss of activity with NADH, 2fold increase in activity with NADPH Bacillus subtilis

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.014
-
NADH wild-type, pH 8.3, 30°C Bacillus subtilis
0.032
-
NADPH mutant D196A/L197R, pH 8.3, 30°C Bacillus subtilis
0.119
-
NADPH mutant D196A/L197R/N198S/R201A, pH 8.3, 30°C Bacillus subtilis
0.219
-
NADPH mutant D196A/L197R/R201A, pH 8.3, 30°C Bacillus subtilis
0.39
-
NADPH mutant D196A, pH 8.3, 30°C Bacillus subtilis
0.429
-
NADH mutant D196A, pH 8.3, 30°C Bacillus subtilis
0.448
-
NADPH wild-type, pH 8.3, 30°C Bacillus subtilis
0.887
-
NADH mutant D196A/L197R, pH 8.3, 30°C Bacillus subtilis

Organism

Organism UniProt Comment Textmining
Bacillus subtilis
-
-
-

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
pyruvate + NH3 + NADH + H+
-
Bacillus subtilis L-Ala + H2O + NAD+
-
?
pyruvate + NH3 + NADPH + H+ NADPH is a poor cofactor for wild-type Bacillus subtilis L-Ala + H2O + NADP+
-
?

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
11.1
-
NADPH wild-type, pH 8.3, 30°C Bacillus subtilis
12.65
-
NADPH mutant D196A/L197R/R201A, pH 8.3, 30°C Bacillus subtilis
13.96
-
NADH wild-type, pH 8.3, 30°C Bacillus subtilis
16.57
-
NADPH mutant D196A/L197R/N198S/R201A, pH 8.3, 30°C Bacillus subtilis
28.83
-
NADPH mutant D196A/L197R, pH 8.3, 30°C Bacillus subtilis
36.07
-
NADH mutant D196A, pH 8.3, 30°C Bacillus subtilis
40.58
-
NADPH mutant D196A, pH 8.3, 30°C Bacillus subtilis
44.48
-
NADH mutant D196A/L197R, pH 8.3, 30°C Bacillus subtilis

Cofactor

Cofactor Comment Organism Structure
NADH
-
Bacillus subtilis
NADPH poor cofactor for wild-type Bacillus subtilis

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
25
-
NADPH wild-type, pH 8.3, 30°C Bacillus subtilis
50
-
NADH mutant D196A/L197R, pH 8.3, 30°C Bacillus subtilis
58
-
NADPH mutant D196A/L197R/R201A, pH 8.3, 30°C Bacillus subtilis
83
-
NADH mutant D196A, pH 8.3, 30°C Bacillus subtilis
103
-
NADPH mutant D196A, pH 8.3, 30°C Bacillus subtilis
140
-
NADPH mutant D196A/L197R/N198S/R201A, pH 8.3, 30°C Bacillus subtilis
901
-
NADPH mutant D196A/L197R, pH 8.3, 30°C Bacillus subtilis
998
-
NADH wild-type, pH 8.3, 30°C Bacillus subtilis