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Literature summary for 1.4.1.9 extracted from

  • Xu, J.M.; Cheng, F.; Fu, F.T.; Hu, H.F.; Zheng, Y.G.
    Semi-rational engineering of leucine dehydrogenase for L-2-aminobutyric acid production (2016), Appl. Biochem. Biotechnol., 182, 898-909 .
    View publication on PubMed

Application

Application Comment Organism
synthesis enzyme cascade from threonine to synthesis of L-2-aminobutanoate. In this cascade, the threonine deaminase is used for threonine to 2-oxobutanoate, then LeuDH mutant and formate dehydrogenase are used for synthesis of L-2-aminobutanoate. Under optimized conditions, 1 M threonine is catalyzed by whole cells of Escherichia coli harboring the enzymes in 12 h in sodium phosphate buffer to the optically pure L-2-aminobutanoate with a yield of 99% and ee above 99% Bacillus cereus

Crystallization (Commentary)

Crystallization (Comment) Organism
homology modeling of structure and semirational engineering to increase the catalytic efficiency Bacillus cereus
modeling of structure. Active site residues Lys81, Asp116, and Lys69 are catalytically important. Lys81 linkes the -OH group of the substrate 2-oxobutanoate by a 2.3-A long H-bond, and Asp116 is involved in proton transfer during catalysis Bacillus cereus

Protein Variants

Protein Variants Comment Organism
M347G 251% of wild-type activity Bacillus cereus
M347G 341% of wild-type activity Bacillus cereus
M347G/Q358N 400% of wild-type activity Bacillus cereus
M347N 228% of wild-type activity Bacillus cereus
N262N 121% of wild-type activity Bacillus cereus
N70F 128% of wild-type activity Bacillus cereus
Q358N 310% of wild-type activity Bacillus cereus
Q358N 459% of wild-type activity Bacillus cereus
Q358T 248% of wild-type activity Bacillus cereus
Q358T 257% of wild-type activity Bacillus cereus

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
13.5
-
2-oxobutanoate mutant Q358N, pH 7.5, 37°C Bacillus cereus
13.53
-
2-oxobutanoate mutant Q358N, pH 7.5, 37°C Bacillus cereus
16.8
-
2-oxobutanoate mutant M347G/Q358N, pH 7.5, 37°C Bacillus cereus
21.3
-
2-oxobutanoate mutant M374G, pH 7.5, 37°C Bacillus cereus
21.33
-
2-oxobutanoate mutant M347G, pH 7.5, 37°C Bacillus cereus
22.7
-
2-oxobutanoate wild-type, pH 7.5, 37°C Bacillus cereus
22.74
-
2-oxobutanoate wild-type, pH 7.5, 37°C Bacillus cereus

Organism

Organism UniProt Comment Textmining
Bacillus cereus
-
-
-

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
2.6
-
wild-type, pH 7.5, 37°C Bacillus cereus
8.88
-
mutant M374G, pH 7.5, 37°C Bacillus cereus
10.4
-
mutant M347G/Q358N, pH 7.5, 37°C Bacillus cereus
11.94
-
mutant Q358N, pH 7.5, 37°C Bacillus cereus

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2-oxobutanoate + NH3 + NADH + H+
-
Bacillus cereus L-2-aminobutanoate + H2O + NAD+
-
?
2-oxobutanoate + NH3 + NADH + H+
-
Bacillus cereus L-2-aminobutanoate + H2O + NAD+
-
r
L-leucine + H2O + NAD+
-
Bacillus cereus 4-methyl-2-oxopentanoate + NH3 + NADH + H+
-
r

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.055
-
2-oxobutanoate wild-type, pH 7.5, 37°C Bacillus cereus
0.056
-
2-oxobutanoate wild-type, pH 7.5, 37°C Bacillus cereus
0.15
-
2-oxobutanoate mutant Q358N, pH 7.5, 37°C Bacillus cereus
0.46
-
2-oxobutanoate mutant M347G, pH 7.5, 37°C Bacillus cereus
0.461
-
2-oxobutanoate mutant M374G, pH 7.5, 37°C Bacillus cereus
0.826
-
2-oxobutanoate mutant M347G/Q358N, pH 7.5, 37°C Bacillus cereus
0.83
-
2-oxobutanoate mutant M347G/Q358N, pH 7.5, 37°C Bacillus cereus
1.5
-
2-oxobutanoate mutant Q358N, pH 7.5, 37°C Bacillus cereus

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.0025
-
2-oxobutanoate wild-type, pH 7.5, 37°C Bacillus cereus
0.0217
-
2-oxobutanoate mutant M374G, pH 7.5, 37°C Bacillus cereus
0.022
-
2-oxobutanoate mutant M347G, pH 7.5, 37°C Bacillus cereus
0.0492
-
2-oxobutanoate mutant M347G/Q358N, pH 7.5, 37°C Bacillus cereus
0.05
-
2-oxobutanoate mutant M347G/Q358N, pH 7.5, 37°C Bacillus cereus
0.11
-
2-oxobutanoate mutant Q358N, pH 7.5, 37°C Bacillus cereus
0.1112
-
2-oxobutanoate mutant Q358N, pH 7.5, 37°C Bacillus cereus