Information on EC 1.1.1.345 - D-2-hydroxyacid dehydrogenase (NAD+)

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The expected taxonomic range for this enzyme is: Bacteria, Archaea, Eukaryota

EC NUMBER
COMMENTARY hide
1.1.1.345
-
RECOMMENDED NAME
GeneOntology No.
D-2-hydroxyacid dehydrogenase (NAD+)
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
an (R)-2-hydroxycarboxylate + NAD+ = a 2-oxocarboxylate + NADH + H+
show the reaction diagram
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
leucine degradation IV
-
-
SYSTEMATIC NAME
IUBMB Comments
(R)-2-hydroxycarboxylate:NAD+ oxidoreductase
The enzymes, characterized from bacteria (Peptoclostridium difficile, Enterococcus faecalis and from lactic acid bacteria) prefer substrates with a main chain of 5 carbons (such as 4-methyl-2-oxopentanoate) to those with a shorter chain. It also utilizes phenylpyruvate. The enzyme from the halophilic archaeon Haloferax mediterranei prefers substrates with a main chain of 3-4 carbons (pyruvate and 2-oxobutanoate). cf. EC 1.1.1.272, (D)-2-hydroxyacid dehydrogenase (NADP+).
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
metabolism
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(R)-2-hydroxy-4-methylpentanoate + NAD+
4-methyl-2-oxopentanoate + NADH + H+
show the reaction diagram
-
i.e. (R)-2-hydroxyisocaproate
i.e. 2-oxoisocaproate
-
r
(R)-mandelate + NAD+
phenylglyoxylate + NADH + H+
show the reaction diagram
2-formylbutanethioate + NADH + H+
?
show the reaction diagram
2-hydroxyhexanoate + NAD+
2-oxohexanoate + NADH + H+
show the reaction diagram
-
-
-
-
r
2-hydroxyoctanoate + NAD+
2-oxooctanoate + NADH + H+
show the reaction diagram
-
-
-
-
r
2-hydroxypentanoate + NAD+
2-oxopentanoate + NADH + H+
show the reaction diagram
-
-
-
-
r
2-oxo-3-phenylpropanoate + NAD+
2-hydroxy-3-phenylpropanoate + NADH + H+
show the reaction diagram
2-oxobutanoate + NAD+
2-hydroxybutanoate + NADH + H+
show the reaction diagram
2-oxobutyrate + NADH + H+
?
show the reaction diagram
2-oxobutyrate + NADH + H+
? + NAD+
show the reaction diagram
2-oxobutyrate + NADH + H+
D-2-hydroxybutyrate + NAD+
show the reaction diagram
2-oxocaproate + NADH + H+
?
show the reaction diagram
2-oxocaproate + NADH + H+
? + NAD+
show the reaction diagram
2-oxocarboxylate + NADH + H+
(R)-2-hydroxcarboxylate + NAD+
show the reaction diagram
2-oxohexanoate + NAD+
2-hydroxyhexanoate + NADH + H+
show the reaction diagram
2-oxohexanoate + NADH + H+
2-hydroxyhexanoate + NAD+
show the reaction diagram
2-oxoisocaproate + NADH + H+
?
show the reaction diagram
2-oxoisocaproate + NADH + H+
? + NAD+
show the reaction diagram
2-oxoisocaproate + NADH + H+
L-2-hydroxyisocaproate + NAD+
show the reaction diagram
2-oxoisovalerate + NADH + H+
?
show the reaction diagram
2-oxoisovalerate + NADH + H+
? + NAD+
show the reaction diagram
-
-
-
-
r
2-oxomethylthiobutyrate + NADH + H+
? + NAD+
show the reaction diagram
-
-
-
-
r
2-oxomethylvalerate + NADH + H+
? + NAD+
show the reaction diagram
-
-
-
-
r
2-oxooctanoate + NADH + H+
2-hydroxyoctanoate + NAD+
show the reaction diagram
-
-
-
-
r
2-oxopentanoate + NAD+
2-hydroxypentanoate + NADH + H+
show the reaction diagram
2-oxopentanoate + NADH + H+
2-hydroxypentanoate + NAD+
show the reaction diagram
2-oxovalerate + NADH + H+
?
show the reaction diagram
-
-
-
-
-
2-oxovalerate + NADH + H+
? + NAD+
show the reaction diagram
-
-
-
-
r
3-(4-hydroxyphenyl)-2-oxopropanoate + NADH + H+
2-hydroxy-3-(4-hydroxyphenyl)propanoate + NAD+
show the reaction diagram
-
-
-
-
?
3-hydroxypyruvate + NADH + H+
?
show the reaction diagram
-
-
-
-
-
3-methyl-2-oxobutanoate + NAD+
3-methyl-2-hydroxybutanoate + NADH + H+
show the reaction diagram
3-methyl-2-oxobutanoate + NADH + H+
2-hydroxy-3-methylbutanoate + NAD+
show the reaction diagram
3-methyl-2-oxopentanoate + NADH + H+
2-hydroxy-3-methylpentanoate + NAD+
show the reaction diagram
4-methyl-2-oxopentanoate + NAD+
4-methyl-2-hydroxypentanoate + NADH + H+
show the reaction diagram
-
highest Vmax/Km value of all substrates tested
-
-
?
4-methyl-2-oxopentanoate + NADH + H+
(R)-2-hydroxy-4-methylpentanoate + NAD+
show the reaction diagram
benzoylformate + NADH + H+
?
show the reaction diagram
benzoylformate + NADH + H+
? + NAD+
show the reaction diagram
-
-
-
-
r
D-2-hydroxybutyrate + NAD+
2-oxobutyrate + NADH + H+
show the reaction diagram
D-lactate + NAD+
pyruvate + NADH + H+
show the reaction diagram
-
-
-
-
r
D-mandelate + NADH + H+
? + NAD+
show the reaction diagram
-
-
-
-
r
DL-2-hydroxyisocaproate + NADH + H+
? + NAD+
show the reaction diagram
-
-
-
-
r
L-2-hydroxycaproate + NAD+
2-oxocaproate + NADH + H+
show the reaction diagram
-
-
-
-
r
phenylglyoxylate + NAD+
hydroxy(phenyl)acetic acid + NADH + H+
show the reaction diagram
phenylpyruvate + NADH + H+
?
show the reaction diagram
-
-
-
-
-
phenylpyruvate + NADH + H+
? + NAD+
show the reaction diagram
phenylpyruvate + NADH + H+
phenyllactate + NAD+
show the reaction diagram
pyruvate + NADH + H+
D-lactate + NAD+
show the reaction diagram
pyruvate + NADH + H+
lactate + NAD+
show the reaction diagram
additional information
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
NaCl
-
maximum activity in the presence of 4 M NaCl
additional information
-
no influence on the enzymatic activity can be detected with Mg2+ and Ca2+ and only very weak effects with Cd2+, Co2+ , and Mn2+
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1,10-phenanthroline
-
10 mM, decrease of 13% in the enzymatic activity
2-Oxohexanoate
substrate inhibition
2-Oxopentanoate
substrate inhibition
4-methyl-2-oxopentanoate
substrate inhibition
Ca2+
-
72% residual activity at 1 mM
CaCl2
-
1 mM, 28% inhibition
Cu2+
-
complete inhibition at 0.1 mM
CuSO4
-
0.1 mM, complete inhibition
DEPC
-
65% residual activity at 1 mM, 12% residual activity at 2 mM
diethyl dicarbonate
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2 mM, 88% inhibition
HgCl2
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0.1 mM, complete inhibition
iodoacetamide
Mg2+
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83% residual activity at 1 mM
MgCl2
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1 mM, 17% inhibition
phenylpyruvate
substrate inhibition
additional information
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the addition of citrate and EDTA (up to 10 mM) has no effect on the enzymatic activity. The addition of of iodoacetamide, KCN, 2-mercaptoethanol, dithiothreitol, and reduced glutathione in concentrations up to 20 mM has no effect
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
dithiothreitol
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2.3
(R)-2-hydroxy-4-methylpentanoate
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pH 7.0, temperature not specified in the publication
-
0.76 - 0.9
(R)-mandelate
1.26
2-formylbutanethioate
-
pH 7.0, 37C
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2.7
2-hydroxyhexanoate
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pH 7.0, temperature not specified in the publication
1.6
2-Hydroxyoctanoate
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pH 7.0, temperature not specified in the publication
3.3
2-hydroxypentanoate
-
pH 7.0, temperature not specified in the publication
3.4 - 5.7
2-oxo-3-phenylpropanoate
4 - 9.5
2-oxobutanoate
0.36 - 28
2-oxobutyrate
2.4 - 40
2-oxocaproate
0.02 - 1.5
2-Oxohexanoate
0.3 - 71
2-oxoisocaproate
0.29 - 46
2-oxoisovalerate
0.21
2-oxomethylvalerate
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at pH 7.0 and 37C
0.12
2-oxooctanoate
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pH 7.0, temperature not specified in the publication
0.057 - 0.4
2-Oxopentanoate
0.53 - 62
2-oxovalerate
0.6
3-(4-hydroxyphenyl)-2-oxopropanoate
-
pH 7.0, temperature not specified in the publication
2.9
3-hydroxypyruvate
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in 100 mM Tris-HC1 buffer, pH 8.0, at 30C
0.15 - 0.29
3-methyl-2-oxobutanoate
0.21
3-methyl-2-oxopentanoate
-
pH 7.0, 37C
0.022 - 0.4
4-methyl-2-oxopentanoate
1.26
4-methylthio-2-oxobutanoate
-
at pH 7.0 and 37C
1.5
benzoylformate
0.14
D-lactate
-
in 0.1 M acetate buffer, pH 5.0, 4 M NaCl, at 52C
3
D-mandelate
0.5
NAD+
-
pH 7.0, 25C
0.01 - 0.14
NADH
0.25 - 0.26
phenylglyoxylate
0.031 - 20
phenylpyruvate
0.56 - 1.2
pyruvate
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
43
(R)-2-hydroxy-4-methylpentanoate
Lactobacillus casei
-
pH 7.0, temperature not specified in the publication
-
26
2-hydroxyhexanoate
Lactobacillus casei
-
pH 7.0, temperature not specified in the publication
15
2-Hydroxyoctanoate
Lactobacillus casei
-
pH 7.0, temperature not specified in the publication
24
2-hydroxypentanoate
Lactobacillus casei
-
pH 7.0, temperature not specified in the publication
139
2-Oxohexanoate
Lactobacillus casei
-
pH 7.0, temperature not specified in the publication
1200
2-oxooctanoate
Lactobacillus casei
-
pH 7.0, temperature not specified in the publication
197
2-Oxopentanoate
Lactobacillus casei
-
pH 7.0, temperature not specified in the publication
788
3-(4-hydroxyphenyl)-2-oxopropanoate
Lactobacillus casei
-
pH 7.0, temperature not specified in the publication
84
4-methyl-2-oxopentanoate
Lactobacillus casei
-
pH 7.0, temperature not specified in the publication
968
phenylpyruvate
Lactobacillus casei
-
pH 7.0, temperature not specified in the publication
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
18
(R)-2-hydroxy-4-methylpentanoate
Lactobacillus casei
-
pH 7.0, temperature not specified in the publication
202156
9.7
2-hydroxyhexanoate
Lactobacillus casei
-
pH 7.0, temperature not specified in the publication
9020
25
2-Hydroxyoctanoate
Lactobacillus casei
-
pH 7.0, temperature not specified in the publication
3780
7.2
2-hydroxypentanoate
Lactobacillus casei
-
pH 7.0, temperature not specified in the publication
15498
1100
2-Oxohexanoate
Lactobacillus casei
-
pH 7.0, temperature not specified in the publication
1100
139
2-oxooctanoate
Lactobacillus casei
-
pH 7.0, temperature not specified in the publication
5900
1600
2-Oxopentanoate
Lactobacillus casei
-
pH 7.0, temperature not specified in the publication
968
1300
3-(4-hydroxyphenyl)-2-oxopropanoate
Lactobacillus casei
-
pH 7.0, temperature not specified in the publication
2815
210
4-methyl-2-oxopentanoate
Lactobacillus casei
-
pH 7.0, temperature not specified in the publication
722
4800
phenylpyruvate
Lactobacillus casei
-
pH 7.0, temperature not specified in the publication
198
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.427
-
with 2-oxoisocaproate as substrate, in 0.1 M glycine-NaOH buffer, pH 9.0, 4 M NaCl, at 52C
0.799
-
with 2-oxobutyrate as substrate, in 0.1 M glycine-NaOH buffer, pH 9.0, 4 M NaCl, at 52C
1.462
-
with pyruvate as substrate, in 0.1 M glycine-NaOH buffer, pH 9.0, 4 M NaCl, at 52C
2.2
-
crude extract, in 100 mM Tris-HC1 buffer, pH 8.0, at 30C
2.6
-
pH 7.5, 30C, D-mandelate dehydrogenase D-ManDH2
3.7
-
pH 7.5, 30C, D-mandelate dehydrogenase D-ManDH1
170
-
after 77fold purification, in 100 mM Tris-HC1 buffer, pH 8.0, at 30C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.5
-
maximal catalytic efficiency, D-mandelate dehydrogenase D-ManDH1; maximal catalytic efficiency, D-mandelate dehydrogenase D-ManDH2
5
-
optimum pH for reaction with pyruvate or 2-oxobutyrate
5.5 - 7
7.5 - 8.5
-
optimum pH values for reaction with 2-oxoisocaproate
8 - 9
-
oxidation of (R)-2-hydroxy-4-methylpentanoate
9
-
reverse reaction
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 8
-
activity is reduced by only about 10% at pH 5.0 and 8.0
5.5 - 7
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reduction of 4-methyl-2-oxopentanoate
9
-
oxidation of (R)-2-hydroxy-4-methylpentanoate
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30 - 40
-
D-mandelate dehydrogenase D-ManDH1
35 - 45
-
D-mandelate dehydrogenase D-ManDH2
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37 - 55
-
at 37C, the activity reaches about 65% of peak activity at 55C
65 - 75
-
above 60C there is a pronounced activity decrease, while there is nearly no activity at 75C
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
-
most abundant expression
Manually annotated by BRENDA team
-
-
Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
74000
-
gel filtration
101400
-
gel filtration
110000
-
gel filtration, D-mandelate dehydrogenase D-ManDH1
130000
-
gel filtration, D-mandelate dehydrogenase D-ManDH2
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homodimer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystals are grown from a 1:1 mixture of a protein solution (10 mg/ml in 10 mM TrisHCl (pH 7.5)) and a reservoir solution (0.085 M HEPES-Na (pH 7.5), 0.17 M ammonium acetate and 22.5% PEG8000) using the hanging-drop vapor diffusion method at 25C. The overall structure shows that the enzyme has a similar fold to 2-ketopantoate reductase, which catalyzes the conversion of 2-ketopantoate to D-pantoate using NADP+ as a coenzyme. They share conserved catalytic residues, indicating that D-mandelate dehydrogenase ManDH2 has the same reaction mechanism as 2-ketopantoate reductase. However, D-mandelate dehydrogenase ManDH2 exhibits significant structural variations in the coenzyme and substrate binding sites compared to 2-ketopantoate reductase. These structural observations can explain their different coenzyme and substrate specificities
hanging drop vapor diffusion method, using 2.0-3.5 M ammonium sulfate or 14-26% PEG 3350 as precipitant
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hexagonal crystals of recombinant D-HicDH in the presence of NAD+ and 2-oxoisocaproate (4-methyl-2-oxopentanoate) are grown with ammonium sulfate as precipitating agent. The structure of the crystals is solved by molecular replacement and refined to a final R-factor of 19.6% for all measured X-ray reflections in the resolution range 1.9 A resolution
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vapor diffusion using ammonium sulfate as precipitant. The crystals belong to hexagonal space group type P6(3)22 with a = b = 134.1 A, c = 124.1 A and diffract X-rays to 3.0 A resolution. Packing considerations show that there are either one or two D-HicDH monomers in the asymmetric unit
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
3.3 - 9.7
-
6 weeks, more than 80% of the activity is recovered
731190
5 - 8
-
activity is reduced by about 10% at pH 5.0 and 8.0
712292
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
45
-
60 min, 95% of the activity can be recovered
50
-
at temperatures above 50C the stability of the enzyme decreases drastically
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4C, concentrated enzyme solution at pH 7.0 (10.6 mg/ml), 3 months, without significant loss of activity
-
4C, diluted enzyme solution at pH 7.0 (0.004 mg/ml), 1 h, 40% loss of activity
-
4C, pH 7.0, concentrated enzyme solution (10.6 mg/ml) is stable for at least 2-3 months
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
DEAE-cellulose DE32 column chromatography, AMP-Sepharose column chromatography, and Mono Q column chromatography
-
Sepharose column chromatography, DEAE-cellulose column chromatography, Q-Sepharose column chromatography, Sephacryl S-300 gel filtration, and Sephadex G25 gel filtration
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli
-
expressed in Escherichia coli BL21(DE3) cells
-
expressed in Escherichia coli MV1184 cells
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expressed in Escherichia coli Rosetta cells
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expression in Escherichia coli
overexpression in Escherichia coli
-
overexpression in Escherichia coli as His-tagged fusion protein
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
K187A
mutant has completely lost its activity
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
synthesis
-
the enzyme can be utilized for preparation of enantiomerically pure (R)-2-hydroxy-4-methylpentanoate in 88% yield, using formate dehydrogenase recycling of the NADH coenzyme