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1.4.1.9: leucine dehydrogenase

This is an abbreviated version!
For detailed information about leucine dehydrogenase, go to the full flat file.

Word Map on EC 1.4.1.9

Reaction

L-leucine
+
H2O
 +
NAD+
=
4-methyl-2-oxopentanoate
 +
NH3
 +
NADH
 +
H+

Synonyms

BCD, dehydrogenase, leucine, L-leucine dehydrogenase, L-leucine:NAD+ oxidoreductase, deaminating, LeuDH

ECTree

     1 Oxidoreductases
         1.4 Acting on the CH-NH2 group of donors
             1.4.1 With NAD+ or NADP+ as acceptor
                1.4.1.9 leucine dehydrogenase

Engineering

Engineering on EC 1.4.1.9 - leucine dehydrogenase

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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D126E
2fold increase in kcat/Km value for trimethylpyruvate
E116V
E24A/D126E
affinity for trimethylpyruvate and NADH is increased by 7.7- and 2.8fold, respectively
E24V/E116V
5fold increase in kcat/Km value for trimethylpyruvate
M347G
M347G/Q358N
-
400% of wild-type activity
M347N
-
228% of wild-type activity
N262N
-
121% of wild-type activity
N70F
-
128% of wild-type activity
Q358N
Q358T
T45M
an additional hydrogen bond in beta5 fold is formed, which results in a more rigid beta5 fold leading to better stability
T45M/E116V
mutant displays higher productivities, melting temperature and half-lives of thermal inactivation are increased compared to wild type
E116V
-
123% of wild-type specific activity. Variant is associated with the presence of more hydrophobic tunnels that allow easy substrate diffusion
-
T45M
-
an additional hydrogen bond in beta5 fold is formed, which results in a more rigid beta5 fold leading to better stability
-
T45M/E116V
-
mutant displays higher productivities, melting temperature and half-lives of thermal inactivation are increased compared to wild type
-
A113G
-
mutant enzyme with altered substrate specificity. 17.9fold decrease in turnover number for L-Leu, 1.2fold decrease in turnover-number for L-Ile, 13.8fold increase in turnover number of L-norleucine, 1.7fold decrease in turnover-number for L-norvaline, 3fold decrease in turnover number for alpha-keto-isocaproate, 1.2fold decrease in turnover number for alpha-ketocaproate, 1.3fold increase in turnover number for alpha-ketocaproate, 3.6fold decrease in Km-value for L-Leu, 3.3fold increase in Km-value for L-Ile, 1.1fold decrease in Km-value for L-norleucine, 3.5fold increase in Km-value for L-norvaline, 1.9fold increase in Km-value for alpha-keto-isocaproate, 2.5fold increase in Km-value for alpha-keto-beta-methylvalerate, 2.4fold decrease in Km-value for alpha-ketocaproate, 1.2fold increase in Km-value for NAD+, 1.2fold increase in Km-value for NADH as compared to wild-type enzyme. L-Ethionine and L-Phe are not substrates of the wild-type enzyme but are deaminated by mutant enzyme. Phenylpyruvate is not a substrate of the wild-type enzyme, but is aminated by mutant enzyme
A113G/V291L
-
mutant enzyme with altered substrate specificity. 67.6fold decrease in turnover number for L-Leu, 20fold decrease in turnover-number for L-Ile, 2.2fold decrease in turnover number of L-norleucine, 44.8fold decrease in turnover-number for L-norvaline, 9.7fold decrease in turnover number for alpha-keto-isocaproate, 7.6fold decrease in turnover number for alpha-ketocaproate, 4.6fold decrease in turnover number for alpha-ketocaproate, 6.9fold increase in Km-value for L-Leu, 13.8fold increase in Km-value for L-Ile, 5.5fold increase in Km-value for L-norleucine, 9fold increase in Km-value for L-norvaline, 34fold increase in Km-value for alpha-keto-isocaproate, 18.2fold increase in Km-value for alpha-keto-beta-methylvalerate, 6fold increase in Km-value for alpha-ketocaproate, 4.4fold increase in Km-value for NAD+, 2fold decrease in Km-value for NADH as compared to wild-type enzyme. L-Ethionine and L-Phe are not substrates of the wild-type enzyme but are deaminated by mutant enzyme. Phenylpyruvate is not a substrate of the wild-type enzyme, but is aminated by mutant enzyme
A94E
proportion of residual activity of A94E is 19% of that of wild type after incubation at 70 °C for 10 min
G77A
-
turnover numver in oxidative deamination of L-Leu is 36% of that of the wild-type enzyme. In reductive amination the turnover number is comparable to that of the wild-type enzyme. The Km-value for 2-oxoisohexanoate is 6.3fold higher and the Km-value for NH4+ is 2.8fold higher than that of the wild-type enzyme. Mutant enzyme shows lowered unfolding temperature compared with the wild-type enzyme. Faster degradation than wild-type enzyme after incubation at 37°C for 15 h with trypsin or subtilisin at a protease-to-substrate ratio of 1:1
G78A
-
turnover number in oxidative deamination of L-Leu is 5.4% of that of the wild-type enzyme. In reductive amination the turnover number is comparable to that of the wild-type enzyme. The Km-value for 2-oxoisohexanoate is 8.8fold higher and the Km-value for NH4+ is 10fold higher than that of the wild-type enzyme. Mutant enzyme shows lowered unfolding temperature compared with the wild-type enzyme. Faster degradation than wild-type enzyme after incubation at 37°C for 15 h with trypsin or subtilisin at a protease-to-substrate ratio of 1:1
G79A
-
turnover number in oxidative deamination of L-Leu is 40% of that of the wild-type enzyme. In reductive amination the turnover number is comparable to that of the wild-type enzyme. The Km-value for 2-oxoisohexanoate is 6.4fold higher and the Km-value for NH4+ is 3.9fold higher than that of the wild-type enzyme. Mutant enzyme shows lowered unfolding temperature compared with the wild-type enzyme
K68A
-
nearly complete loss of activity in the oxidative deamination, marked increase in Km-values for both amino acid substrates and oxo acid substrates. An ionizable group in the wild-type enzyme with a pKa value of 10.1-10.7, which must be protonated for binding of substrate and competitive inhibitor with an alpha-carboxyl group, is unobservable in mutant enzyme
K68R
-
nearly complete loss of activity in the oxidative deamination. An ionizable group in the wild-type enzyme with a pKa value of 10.1-10.7, which must be protonated for binding of substrate and competitive inhibitor with an alpha-carboxyl group, is unobservable in mutant enzyme
K80A
-
markedly reduced activity in oxidative deamination, nearly 90% of the wild-type activity in reductive amination. Km-value for 2-oxoisohexanoate is 11fold higher than that of the wild-type enzyme, Km-value for L-Leu is lower than that of the wild-type enzyme
K80Q
-
markedly reduced activity in oxidative deamination. Km-value for 2-oxoisohexanoate is 28fold higher than that of the wild-type enzyme, Km-value for L-Leu is about 3times larger than that of the wild-type enzyme
K80R
-
markedly reduced activity in oxidative deamination, 0.6% of the wild-type activity in reductive amination, Km-value for L-Leu is lower than that of the wild-type enzyme
Y127N
proportion of residual activity of Y127N is 27% of that of wild type after incubation at 70 °C for 10 min
synthesis
-
production of L-tert-leucine in Escherichia coli coexpressing leucine dehydrogenase and a formate dehydrogenase. Under the optimized conditions, the double-plasmid strain transforms 1 mol/l trimethylpyruvate completely into L-tert-leucine with greater than 99.9% ee within 8 h
A43V/D124E
mutant shows improved efficiency of L-tert-leucine synthesis, 5fold increase in catalyic efficiency compared to wild-type
LeuDEL4
-
4 C-terminal amino acids deleted, mutant enzyme is a dimer
D153 N/H191 N
Roseibium aggregatum
-
25fold improved affinity for NADH and with 50fold enhanced catalytic efficiency
D153N
Roseibium aggregatum
-
affinity for NADH is improved, 11.3fold decrease in Km value for NADH
H191N
Roseibium aggregatum
-
affinity for NADH is improved, 6.0fold decrease in Km value for NADH
P130V
Roseibium aggregatum
-
affinity for NADH is improved, 8.6fold decrease in Km value for NADH
D203A
-
dual specificity for NAD+ and NADP+
D203A/I204R
-
high affinity for NADP+
D203A/I204R/D210R
-
high affinity for NADP+
additional information