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
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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
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-
-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
leucine degradation IV
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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
evolution
malfunction
metabolism
physiological function
additional information
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
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i.e. (R)-2-hydroxyisocaproate
i.e. 2-oxoisocaproate
-
r
(R)-2-hydroxycarboxylate + NAD+
a 2-oxocarboxylate + NADH + H+
show the reaction diagram
(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
-
-
-
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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
-
-
-
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r
2-oxomethylvalerate + NADH + H+
? + NAD+
show the reaction diagram
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-
-
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r
2-oxooctanoate + NADH + H+
2-hydroxyoctanoate + NAD+
show the reaction diagram
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-
-
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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
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-
-
-
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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
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-
-
-
?
3-hydroxypyruvate + NADH + H+
?
show the reaction diagram
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-
-
-
-
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
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highest Vmax/Km value of all substrates tested
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-
?
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
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-
-
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r
D-2-hydroxybutyrate + NAD+
2-oxobutyrate + NADH + H+
show the reaction diagram
D-lactate + NAD+
pyruvate + NADH + H+
show the reaction diagram
-
-
-
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r
D-mandelate + NADH + H+
? + NAD+
show the reaction diagram
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-
-
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r
DL-2-hydroxyisocaproate + NADH + H+
? + NAD+
show the reaction diagram
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-
-
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r
L-2-hydroxycaproate + NAD+
2-oxocaproate + NADH + H+
show the reaction diagram
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-
-
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r
phenylglyoxylate + NAD+
hydroxy(phenyl)acetic acid + NADH + H+
show the reaction diagram
phenylpyruvate + NADH + H+
?
show the reaction diagram
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-
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phenylpyruvate + NADH + H+
? + NAD+
show the reaction diagram
phenylpyruvate + NADH + H+
phenyl-D-lactate + 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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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
(R)-2-hydroxycarboxylate + NAD+
a 2-oxocarboxylate + NADH + H+
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
NaCl
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maximum activity in the presence of 4 M NaCl
additional information
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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
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10 mM, decrease of 13% in the enzymatic activity
2-Oxohexanoate
substrate inhibition
2-Oxopentanoate
substrate inhibition
4-methyl-2-oxopentanoate
substrate inhibition
Ca2+
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72% residual activity at 1 mM
CaCl2
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1 mM, 28% inhibition
Cu2+
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complete inhibition at 0.1 mM
CuSO4
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0.1 mM, complete inhibition
DEPC
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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
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pH 7.0, 37°C
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
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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 37°C
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
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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 30°C
0.15 - 0.29
3-methyl-2-oxobutanoate
0.21
3-methyl-2-oxopentanoate
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pH 7.0, 37°C
0.022 - 0.4
4-methyl-2-oxopentanoate
1.26
4-methylthio-2-oxobutanoate
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at pH 7.0 and 37°C
1.5
benzoylformate
0.14
D-lactate
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in 0.1 M acetate buffer, pH 5.0, 4 M NaCl, at 52°C
3
D-mandelate
-
at pH 7.0 and 37°C
3
DL-2-hydroxyisocaproate
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at pH 7.0 and 37°C
0.5
NAD+
-
pH 7.0, 25°C
0.01 - 0.14
NADH
0.25 - 0.26
phenylglyoxylate
0.031 - 20
phenylpyruvate
0.56 - 1.2
pyruvate
additional information
additional information
Michaelis-Menten kinetics, recombinant enzyme
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
43
(R)-2-hydroxy-4-methylpentanoate
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pH 7.0, temperature not specified in the publication
26
2-hydroxyhexanoate
-
pH 7.0, temperature not specified in the publication
15
2-Hydroxyoctanoate
-
pH 7.0, temperature not specified in the publication
24
2-hydroxypentanoate
-
pH 7.0, temperature not specified in the publication
139
2-Oxohexanoate
-
pH 7.0, temperature not specified in the publication
1200
2-oxooctanoate
-
pH 7.0, temperature not specified in the publication
197
2-Oxopentanoate
-
pH 7.0, temperature not specified in the publication
788
3-(4-hydroxyphenyl)-2-oxopropanoate
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pH 7.0, temperature not specified in the publication
84
4-methyl-2-oxopentanoate
-
pH 7.0, temperature not specified in the publication
8.48 - 968
phenylpyruvate
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
18
(R)-2-hydroxy-4-methylpentanoate
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pH 7.0, temperature not specified in the publication
9.7
2-hydroxyhexanoate
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pH 7.0, temperature not specified in the publication
25
2-Hydroxyoctanoate
-
pH 7.0, temperature not specified in the publication
7.2
2-hydroxypentanoate
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pH 7.0, temperature not specified in the publication
1100
2-Oxohexanoate
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pH 7.0, temperature not specified in the publication
139
2-oxooctanoate
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pH 7.0, temperature not specified in the publication
1600
2-Oxopentanoate
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pH 7.0, temperature not specified in the publication
1300
3-(4-hydroxyphenyl)-2-oxopropanoate
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pH 7.0, temperature not specified in the publication
210
4-methyl-2-oxopentanoate
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pH 7.0, temperature not specified in the publication
4800
phenylpyruvate
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pH 7.0, temperature not specified in the publication
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.427
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with 2-oxoisocaproate as substrate, in 0.1 M glycine-NaOH buffer, pH 9.0, 4 M NaCl, at 52°C
0.799
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with 2-oxobutyrate as substrate, in 0.1 M glycine-NaOH buffer, pH 9.0, 4 M NaCl, at 52°C
1.462
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with pyruvate as substrate, in 0.1 M glycine-NaOH buffer, pH 9.0, 4 M NaCl, at 52°C
2.2
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crude extract, in 100 mM Tris-HC1 buffer, pH 8.0, at 30°C
2.6
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pH 7.5, 30°C, D-mandelate dehydrogenase D-ManDH2
3.7
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pH 7.5, 30°C, D-mandelate dehydrogenase D-ManDH1
170
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after 77fold purification, in 100 mM Tris-HC1 buffer, pH 8.0, at 30°C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.5
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maximal catalytic efficiency, D-mandelate dehydrogenase D-ManDH1; maximal catalytic efficiency, D-mandelate dehydrogenase D-ManDH2
5
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optimum pH for reaction with pyruvate or 2-oxobutyrate
5.5 - 7
7.5 - 8.5
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optimum pH values for reaction with 2-oxoisocaproate
8 - 9
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oxidation of (R)-2-hydroxy-4-methylpentanoate
9
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reverse reaction
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 8
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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
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oxidation of (R)-2-hydroxy-4-methylpentanoate
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30 - 40
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D-mandelate dehydrogenase D-ManDH1
35 - 45
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D-mandelate dehydrogenase D-ManDH2
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37 - 55
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at 37°C, the activity reaches about 65% of peak activity at 55°C
65 - 75
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above 60°C there is a pronounced activity decrease, while there is nearly no activity at 75°C
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
-
most abundant expression
Manually annotated by BRENDA team
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Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
32000
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x * 32000, SDS-PAGE, D-mandelate dehydrogenase D-ManDH1
33000
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x * 33000, SDS-PAGE
34331
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x * 34331, calculated from nucleotide sequence
34361
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2 * 34361, calculated from amino acid sequence
36948
x * 36948, calculated from sequence
37183
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2 * 37183, calculated from amino acid sequence
38000
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2 * 38000, SDS-PAGE
59500
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2 * 59500, CTAB-PAGE
62900
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2 * 62900, SDS-PAGE
74000
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gel filtration
101400
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gel filtration
110000
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gel filtration, D-mandelate dehydrogenase D-ManDH1
130000
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gel filtration, D-mandelate dehydrogenase D-ManDH2
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homodimer
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystals are grown from a 1:1 mixture of a protein solution (10 mg/ml in 10 mM Tris–HCl (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 25°C. 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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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
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
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6 weeks, more than 80% of the activity is recovered
731190
5 - 8
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activity is reduced by about 10% at pH 5.0 and 8.0
712292
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
45
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60 min, 95% of the activity can be recovered
50
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at temperatures above 50°C the stability of the enzyme decreases drastically
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4°C, concentrated enzyme solution at pH 7.0 (10.6 mg/ml), 3 months, without significant loss of activity
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4°C, diluted enzyme solution at pH 7.0 (0.004 mg/ml), 1 h, 40% loss of activity
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4°C, 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
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recombinant His-tagged D2-HDH from Escherichia coli BL21(DE3) pLysS by nickel affinity chromatography and gel filtration
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
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expressed in Escherichia coli BL21(DE3) cells
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expressed in Escherichia coli MV1184 cells
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expressed in Escherichia coli Rosetta cells
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expression in Escherichia coli
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
overexpression in Escherichia coli
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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