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Literature summary extracted from

  • Holton, S.; Anandhakrishnan, M.; Geerlof, A.; Wilmanns, M.
    Structural characterization of a D-isomer specific 2-hydroxyacid dehydrogenase from Lactobacillus delbrueckii ssp. bulgaricus (2013), J. Struct. Biol., 181, 179-184 .
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.1.1.345 gene Ldb1010, recombinant expression of His-tagged D2-HDH in Escherichia coli BL21(DE3) pLysS. The enzyme containing the non-native C-terminal hexahistidine tag and a 4-residue linker (Thr Ala Ser Gly linker) is enzymatically active Lactobacillus delbrueckii subsp. bulgaricus

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.1.1.345 purified enzyme in apoform and complexed with coenzyme NAD+, hanging drop vapor diffusion method, mixing of 400 nl of 10 mg/ml protein in 40 mM HEPES, pH 7.4, 300 mM NaCl, and 0.02% v/v monothioglycerol, with 400 nl reservoir solution containing 25% PEG 3350, 200 mM MgCl2, 100 mM HEPES, pH 7.5, and equilibration against 0.1 ml of reservoir solution at 19°C, crystals are supplemented with 10 mM NAD+ for the enzyme complex crystals, X-ray diffraction structure determination at 3.45 A and 2.75 A resolution, respectively, molecular replacement and modeling using the monomer structure of D-2-hydroxyisocaproate dehydrogenase (D-HicDH) from Lactobacillus casei, PDB ID 1DXY Lactobacillus delbrueckii subsp. bulgaricus

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.1.1.345 additional information
-
 additional information Michaelis-Menten kinetics, recombinant enzyme Lactobacillus delbrueckii subsp. bulgaricus
1.1.1.345 0.0591
-
 phenylpyruvate recombinant His-tagged enzyme, pH and temperature not specified in the publication Lactobacillus delbrueckii subsp. bulgaricus

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.1.1.345 (R)-2-hydroxycarboxylate + NAD+ Lactobacillus delbrueckii subsp. bulgaricus
-
 a 2-oxocarboxylate + NADH + H+
-
 r
1.1.1.345 (R)-2-hydroxycarboxylate + NAD+ Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842
-
 a 2-oxocarboxylate + NADH + H+
-
 r

Organism

EC Number Organism UniProt Comment Textmining
1.1.1.345 Lactobacillus delbrueckii subsp. bulgaricus Q1GAA2
-
-
1.1.1.345 Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 Q1GAA2
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.1.1.345 recombinant His-tagged D2-HDH from Escherichia coli BL21(DE3) pLysS by nickel affinity chromatography and gel filtration Lactobacillus delbrueckii subsp. bulgaricus

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.1.1.345 (R)-2-hydroxycarboxylate + NAD+
-
 Lactobacillus delbrueckii subsp. bulgaricus a 2-oxocarboxylate + NADH + H+
-
 r
1.1.1.345 (R)-2-hydroxycarboxylate + NAD+
-
 Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 a 2-oxocarboxylate + NADH + H+
-
 r
1.1.1.345 phenylpyruvate + NADH + H+
-
 Lactobacillus delbrueckii subsp. bulgaricus phenyl-D-lactate + NAD+
-
 r
1.1.1.345 phenylpyruvate + NADH + H+
-
 Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 phenyl-D-lactate + NAD+
-
 r

Subunits

EC Number Subunits Comment Organism
1.1.1.345 More enzyme three-dimensional structure analysis, overview Lactobacillus delbrueckii subsp. bulgaricus

Synonyms

EC Number Synonyms Comment Organism
1.1.1.345 D-isomer specific 2-hydroxyacid dehydrogenase
-
 Lactobacillus delbrueckii subsp. bulgaricus
1.1.1.345 D2-HDH
-
 Lactobacillus delbrueckii subsp. bulgaricus
1.1.1.345 Ldb1010
-
 Lactobacillus delbrueckii subsp. bulgaricus

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.1.1.345 8.48
-
 phenylpyruvate recombinant His-tagged enzyme, pH and temperature not specified in the publication Lactobacillus delbrueckii subsp. bulgaricus

Cofactor

EC Number Cofactor Comment Organism Structure
1.1.1.345 NAD+ enzyme binding structure analysis, overview Lactobacillus delbrueckii subsp. bulgaricus
1.1.1.345 NADH
-
 Lactobacillus delbrueckii subsp. bulgaricus

General Information

EC Number General Information Comment Organism
1.1.1.345 evolution the enzyme belongs to the the NAD-dependent dehydrogenase family. Comparison with closely related members of the NAD-dependent dehydrogenase family reveals that whilst the D2-HDH core fold is structurally conserved, the substrate-binding site has a number of non-canonical features that may influence substrate selection and thus dictate the physiological function of the enzyme. The protein, 2-hydroxyisocaproate dehydrogenase (HO-HxoDH), is virtually identical to the D2-HDH, with only three amino-acid differences between the two proteins, all at sites not known to be biologically relevant Lactobacillus delbrueckii subsp. bulgaricus
1.1.1.345 additional information enzyme three-dimensional structure analysis, active site and cofactor binding site structures, overview Lactobacillus delbrueckii subsp. bulgaricus
1.1.1.345 physiological function the substrate-binding site has a number of non-canonical features that may influence substrate selection and thus dictate the physiological function of the enzyme Lactobacillus delbrueckii subsp. bulgaricus