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Literature summary for 1.1.1.B4 extracted from

  • Machielsen, R.; Looger, L.; Raedts, J.; Dijkhuizen, S.; Hummel, W.; Henneman, H.; Daussmann, T.; van der Oost, J.
    Cofactor engineering of Lactobacillus brevis alcohol dehydrogenase by computational design (2009), Eng. Life Sci., 9, 38-44.
No PubMed abstract available

Cloned(Commentary)

Cloned (Comment) Organism
expression in Escherichia coli Levilactobacillus brevis

Protein Variants

Protein Variants Comment Organism
A90S activity with NADPH as cofactor decreased to about 50% of wild-type, activity with NADH strongly decreased Levilactobacillus brevis
G37D/R38P activity with NADH is decreased relative to Arg38Pro alone, but is higher than that of the wild-type enzyme Levilactobacillus brevis
M140I no activity with NADPH as cofactor Levilactobacillus brevis
R38P no activity with NADPH as cofactor, fourfold increase in activity with NADH Levilactobacillus brevis
V112D no activity with NADPH as cofactor, strongly decreased activity with NADH Levilactobacillus brevis

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.017
-
NADPH wild-type, pH 6.5, 30°C Levilactobacillus brevis
0.028
-
NADPH mutant R38P, pH 5.5, 30°C Levilactobacillus brevis
0.12
-
NADH mutant R38P, pH 5.5, 30°C Levilactobacillus brevis
0.13
-
NADH wild-type, pH 6.5, 30°C Levilactobacillus brevis

Organism

Organism UniProt Comment Textmining
Levilactobacillus brevis
-
-
-

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
acetophenone + NADH + H+
-
Levilactobacillus brevis phenylethanol + NAD+
-
?
acetophenone + NADPH + H+
-
Levilactobacillus brevis phenylethanol + NADP+
-
?

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
30
-
wild-type, half-life 160 h, mutant R38P, half-life 220 h Levilactobacillus brevis
42
-
wild-type, half-life 1.25 h, mutant R38P, half-life 3 h Levilactobacillus brevis

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
5.5
-
wild-type, cofactor NADH. Mutant R38P, both cofactors NADH and NADPH Levilactobacillus brevis
6 6.5 wild-type, cofactor NADPH Levilactobacillus brevis

Cofactor

Cofactor Comment Organism Structure
NADPH clear preference for NADPH as cofactor. The phosphate group of NADPH interacts with both the side chains and main-chain amides of Arg38 and Thr15, stabilizing this cofactor significantly better than NADH Levilactobacillus brevis

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
75
-
NADH wild-type, pH 6.5, 30°C Levilactobacillus brevis
300
-
NADH mutant R38P, pH 5.5, 30°C Levilactobacillus brevis
5100
-
NADPH mutant R38P, pH 5.5, 30°C Levilactobacillus brevis
9300
-
NADPH wild-type, pH 6.5, 30°C Levilactobacillus brevis