Literature summary for 1.1.1.345 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 .

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Cloned(Commentary)

Cloned (Comment)	Organism
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)

Crystallization (Comment)	Organism
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

Crystallization (Comment)

Organism

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]

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

Natural Substrates/ Products (Substrates)

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

Organism

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

Purification (Commentary)

Purification (Comment)	Organism
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)

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

Subunits

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

Synonyms

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

Turnover Number [1/s]

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

Cofactor

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

General Information

General Information	Comment	Organism
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
additional information	enzyme three-dimensional structure analysis, active site and cofactor binding site structures, overview	Lactobacillus delbrueckii subsp. bulgaricus
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