Cloned (Comment) | Organism |
---|---|
gene ldhD, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) | Lactobacillus delbrueckii subsp. bulgaricus |
Protein Variants | Comment | Organism |
---|---|---|
F299Y | site-directed mutagenesis | Lactobacillus delbrueckii subsp. bulgaricus |
additional information | highly stereoselective biosynthesis of (R)-alpha-hydroxy carboxylic acids through rationally re-designed mutation of D-lactate dehydrogenase, asymmetric reduction of a homologous series of alpha-keto carboxylic acids such as phenylpyruvic acid, 2-oxobutyric acid, 2-oxovaleric acid, beta-hydroxypyruvate, overview. Compared with wild-type D-nLDH, the Y52L mutant D-nLDH shows elevated activities toward unnatural substrates especially with large substitutes at C-3. By the biocatalysis combined with a formate dehydrogenase for in situ generation of NADH, the corresponding (R)-alpha-hydroxy carboxylic acids can be produced at high yields and highly optical purity. Production of chiral (R)-phenyllactic acid. 50 mM PPA is completely reduced to (R)-phenyllactate in 90 min with a high yield of 99.0% and a highly optical purity (99.9% e.e.) by the engineered coupled production system. Activties of the F299Y mutant are similar to the wild-type enzyme | Lactobacillus delbrueckii subsp. bulgaricus |
Y52L | site-directed mutagenesis | Lactobacillus delbrueckii subsp. bulgaricus |
Y52L/F299Y | site-directed mutagenesis | Lactobacillus delbrueckii subsp. bulgaricus |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.27 | - |
phenylpyruvate | pH 7.4, 37°C, recombinant His-taged mutant Y52L | Lactobacillus delbrueckii subsp. bulgaricus | |
0.32 | - |
phenylpyruvate | pH 7.4, 37°C, recombinant His-taged mutant F299Y | Lactobacillus delbrueckii subsp. bulgaricus | |
1.4 | - |
phenylpyruvate | pH 7.4, 37°C, recombinant His-taged mutant Y52L/F299Y | Lactobacillus delbrueckii subsp. bulgaricus | |
11.4 | - |
phenylpyruvate | pH 7.4, 37°C, recombinant His-taged wild-type enzyme | Lactobacillus delbrueckii subsp. bulgaricus |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
(R)-lactate + NAD+ | Lactobacillus delbrueckii subsp. bulgaricus | - |
pyruvate + NADH + H+ | - |
r | |
(R)-lactate + NAD+ | Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 | - |
pyruvate + NADH + H+ | - |
r | |
pyruvate + NADH + H+ | Lactobacillus delbrueckii subsp. bulgaricus | - |
(R)-lactate + NAD+ | - |
r | |
pyruvate + NADH + H+ | Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 | - |
(R)-lactate + NAD+ | - |
r |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Lactobacillus delbrueckii subsp. bulgaricus | - |
- |
- |
Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 | - |
- |
- |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) to homogeneity by nickel affinity chromatgraphy | Lactobacillus delbrueckii subsp. bulgaricus |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
(R)-lactate + NAD+ | - |
Lactobacillus delbrueckii subsp. bulgaricus | pyruvate + NADH + H+ | - |
r | |
(R)-lactate + NAD+ | - |
Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 | pyruvate + NADH + H+ | - |
r | |
2-oxobutyrate + NADH + H+ | - |
Lactobacillus delbrueckii subsp. bulgaricus | 2-hydroxybutyrate + NAD+ | - |
? | |
2-oxobutyrate + NADH + H+ | - |
Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 | 2-hydroxybutyrate + NAD+ | - |
? | |
2-oxovalerate + NADH + H+ | - |
Lactobacillus delbrueckii subsp. bulgaricus | 2-hydroxyvalerate + NAD+ | - |
? | |
2-oxovalerate + NADH + H+ | - |
Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 | 2-hydroxyvalerate + NAD+ | - |
? | |
additional information | residues Tyr52 and Phe299 are mainly responsible for hindering larger substrates because of their short distance from the side chain of 2-oxocarboxylic acids and their steric orientation in the wild-type enzyme. Substrate specificity and enantioselectivity of D-nLDH and DnLDH mutants, overview | Lactobacillus delbrueckii subsp. bulgaricus | ? | - |
? | |
additional information | residues Tyr52 and Phe299 are mainly responsible for hindering larger substrates because of their short distance from the side chain of 2-oxocarboxylic acids and their steric orientation in the wild-type enzyme. Substrate specificity and enantioselectivity of D-nLDH and DnLDH mutants, overview | Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 | ? | - |
? | |
phenylpyruvate + NADH + H+ | - |
Lactobacillus delbrueckii subsp. bulgaricus | D-phenyllactate + NAD+ | - |
? | |
pyruvate + NADH + H+ | - |
Lactobacillus delbrueckii subsp. bulgaricus | (R)-lactate + NAD+ | - |
r | |
pyruvate + NADH + H+ | - |
Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 | (R)-lactate + NAD+ | - |
r |
Synonyms | Comment | Organism |
---|---|---|
D-nLDH | - |
Lactobacillus delbrueckii subsp. bulgaricus |
NAD-dependent D-lactate dehydrogenase | - |
Lactobacillus delbrueckii subsp. bulgaricus |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
37 | - |
assay at, 2-oxoacid reduction activity | Lactobacillus delbrueckii subsp. bulgaricus |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
1.8 | - |
phenylpyruvate | pH 7.4, 37°C, recombinant His-taged mutant F299Y | Lactobacillus delbrueckii subsp. bulgaricus | |
11.3 | - |
phenylpyruvate | pH 7.4, 37°C, recombinant His-taged wild-type enzyme | Lactobacillus delbrueckii subsp. bulgaricus | |
1447 | - |
phenylpyruvate | pH 7.4, 37°C, recombinant His-taged mutant Y52L/F299Y | Lactobacillus delbrueckii subsp. bulgaricus | |
2013 | - |
phenylpyruvate | pH 7.4, 37°C, recombinant His-taged mutant Y52L | Lactobacillus delbrueckii subsp. bulgaricus |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | - |
assay at, 2-oxoacid reduction activity | Lactobacillus delbrueckii subsp. bulgaricus |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
NAD+ | - |
Lactobacillus delbrueckii subsp. bulgaricus | |
NADH | - |
Lactobacillus delbrueckii subsp. bulgaricus |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
1 | - |
phenylpyruvate | pH 7.4, 37°C, recombinant His-taged wild-type enzyme | Lactobacillus delbrueckii subsp. bulgaricus | |
5.7 | - |
phenylpyruvate | pH 7.4, 37°C, recombinant His-taged mutant F299Y | Lactobacillus delbrueckii subsp. bulgaricus | |
1000 | - |
phenylpyruvate | pH 7.4, 37°C, recombinant His-taged mutant Y52L/F299Y | Lactobacillus delbrueckii subsp. bulgaricus | |
7500 | - |
phenylpyruvate | pH 7.4, 37°C, recombinant His-taged mutant Y52L | Lactobacillus delbrueckii subsp. bulgaricus |