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Enzyme Nomenclature
Information on EC 1.1.1.345 - D-2-hydroxyacid dehydrogenase (NAD+)
Word Map on EC 1.1.1.345
- 1.1.1.345
- lactobacillus
- casei
- l-lactate
-
d-2-hydroxyacids
- 4-methyl-2-oxopentanoate
- benzoylformate
-
enterococcus
-
acinetobacter
- hydroxypyruvate
- calcoaceticus
- l-mandelate
- faecalis
-
2-ketoacid
- d-3-phosphoglycerate
- l-malate
- plantarum
- d-glycerate
- 2-ketoisovalerate
- cholate
- 2-ketoisocaproate
-
nadp-dependent
- synthesis
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The expected taxonomic range for this enzyme is: Bacteria, Archaea, Eukaryota
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EC NUMBER 
COMMENTARY 
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
ORGANISM
UNIPROT
LITERATURE
an (R)-2-hydroxycarboxylate + NAD+ = a 2-oxocarboxylate + NADH + H+
-
-
-
-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
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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+).
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
malfunction
metabolism
malfunction
-
the inactivation of panE does not affect the total percentage of leucine degraded but totally prevented 4-methyl-2-oxopentanoate reduction to 2-hydroxyisocaproate and slightly decreased the production of isovalerate
malfunction
-
the inactivation of panE does not affect the total percentage of leucine degraded but totally prevents KIC reduction to 2-hydroxyisocaproate and slightly decreases the production of isovalerate
malfunction
-
the inactivation of panE does not affect the total percentage of leucine degraded but totally prevents KIC reduction to 2-hydroxyisocaproate and slightly decreases the production of isovalerate
-
malfunction
-
the inactivation of panE does not affect the total percentage of leucine degraded but totally prevented 4-methyl-2-oxopentanoate reduction to 2-hydroxyisocaproate and slightly decreased the production of isovalerate
-
metabolism
-
its probable physiological role is to regenerate the NAD+ necessary to catabolize branched-chain amino acids, leading to the production of ATP and aroma compounds responsible for the reduction of the 2-keto acids derived from leucine, isoleucine, and valine
metabolism
-
its probable physiological role is to regenerate the NAD+ necessary to catabolize branched-chain amino acids, leading to the production of ATP and aroma compounds responsible for the reduction of the 2-keto acids derived from leucine, isoleucine, and valine
-
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
(R)-2-hydroxy-4-methylpentanoate + NAD+
4-methyl-2-oxopentanoate + NADH + H+
-
i.e. (R)-2-hydroxyisocaproate
i.e. 2-oxoisocaproate
-
r
3-(4-hydroxyphenyl)-2-oxopropanoate + NADH + H+
2-hydroxy-3-(4-hydroxyphenyl)propanoate + NAD+
-
-
-
-
?
4-methyl-2-oxopentanoate + NAD+
4-methyl-2-hydroxypentanoate + NADH + H+
-
highest Vmax/Km value of all substrates tested
-
-
?
(R)-mandelate + NAD+
phenylglyoxylate + NADH + H+
-
Vmax/Km is 0.4% compared to 4-methyl-2-oxopentanoate
-
-
?
(R)-mandelate + NAD+
phenylglyoxylate + NADH + H+
-
Vmax/Km is 1% compared to 4-methyl-2-oxopentanoate
-
-
?
(R)-mandelate + NAD+
phenylglyoxylate + NADH + H+
-
Vmax/KM is less than 1% compared to 4-methyl-2-oxopentanoate
-
-
?
2-formylbutanethioate + NADH + H+
?
-
i.e. 2-ketomethylthiobutyrate
-
-
?
2-formylbutanethioate + NADH + H+
?
-
i.e. 2-ketomethylthiobutyrate
-
-
?
2-oxo-3-phenylpropanoate + NAD+
2-hydroxy-3-phenylpropanoate + NADH + H+
-
Vmax/KM is 1.9% compared to 4-methyl-2-oxopentanoate
-
-
?
2-oxo-3-phenylpropanoate + NAD+
2-hydroxy-3-phenylpropanoate + NADH + H+
-
Vmax/Km is 2.7% compared to 4-methyl-2-oxopentanoate
-
-
?
2-oxobutanoate + NAD+
2-hydroxybutanoate + NADH + H+
-
Vmax/Km is 0.25% compared to 4-methyl-2-oxopentanoate
-
-
?
2-oxobutanoate + NAD+
2-hydroxybutanoate + NADH + H+
-
Vmax/KM is less than 1% compared to 4-methyl-2-oxopentanoate
-
-
?
2-oxobutyrate + NADH + H+
D-2-hydroxybutyrate + NAD+
-
55% activity compared to pyruvate
-
-
r
2-oxobutyrate + NADH + H+
D-2-hydroxybutyrate + NAD+
-
55% activity compared to pyruvate
-
-
r
2-oxocarboxylate + NADH + H+
(R)-2-hydroxcarboxylate + NAD+
-
-
-
-
r
2-oxohexanoate + NAD+
2-hydroxyhexanoate + NADH + H+
-
Vmax/KM is 3% compared to 4-methyl-2-oxopentanoate
-
-
?
2-oxohexanoate + NAD+
2-hydroxyhexanoate + NADH + H+
-
Vmax/Km is 4.1% compared to 4-methyl-2-oxopentanoate
-
-
?
2-oxohexanoate + NAD+
2-hydroxyhexanoate + NADH + H+
-
Vmax/Km is 5.1% compared to 4-methyl-2-oxopentanoate
-
-
?
2-oxohexanoate + NADH + H+
2-hydroxyhexanoate + NAD+
-
i.e. 2-oxovalerate
-
-
r
2-oxohexanoate + NADH + H+
2-hydroxyhexanoate + NAD+
i.e. 2-oxocaproate
-
-
?
2-oxohexanoate + NADH + H+
2-hydroxyhexanoate + NAD+
-
i.e. 2-oxocaproate
-
-
r
2-oxoisocaproate + NADH + H+
L-2-hydroxyisocaproate + NAD+
-
29% activity compared to pyruvate
-
-
r
2-oxoisocaproate + NADH + H+
L-2-hydroxyisocaproate + NAD+
-
29% activity compared to pyruvate
-
-
r
2-oxopentanoate + NAD+
2-hydroxypentanoate + NADH + H+
-
Vmax/KM is 10.6% compared to 4-methyl-2-oxopentanoate
-
-
?
2-oxopentanoate + NAD+
2-hydroxypentanoate + NADH + H+
-
Vmax/Km is 5.8% compared to 4-methyl-2-oxopentanoate
-
-
?
2-oxopentanoate + NAD+
2-hydroxypentanoate + NADH + H+
-
Vmax/Km is 7.6% compared to 4-methyl-2-oxopentanoate
-
-
?
2-oxopentanoate + NADH + H+
2-hydroxypentanoate + NAD+
-
i.e. 2-oxocaproate
-
-
r
2-oxopentanoate + NADH + H+
2-hydroxypentanoate + NAD+
i.e. 2-oxovalerate
-
-
?
2-oxopentanoate + NADH + H+
2-hydroxypentanoate + NAD+
-
i.e. 2-oxovalerate
-
-
r
3-methyl-2-oxobutanoate + NAD+
3-methyl-2-hydroxybutanoate + NADH + H+
-
Vmax/Km is 51% compared to 4-methyl-2-oxopentanoate
-
-
?
3-methyl-2-oxobutanoate + NAD+
3-methyl-2-hydroxybutanoate + NADH + H+
-
Vmax/KM is 56% compared to 4-methyl-2-oxopentanoate
-
-
?
3-methyl-2-oxobutanoate + NAD+
3-methyl-2-hydroxybutanoate + NADH + H+
-
Vmax/Km is 85.7% compared to 4-methyl-2-oxopentanoate
-
-
?
3-methyl-2-oxobutanoate + NADH + H+
2-hydroxy-3-methylbutanoate + NAD+
-
i.e.2-oxoisovalerate
-
-
?
3-methyl-2-oxobutanoate + NADH + H+
2-hydroxy-3-methylbutanoate + NAD+
-
i.e.2-oxoisovalerate
-
-
?
3-methyl-2-oxopentanoate + NADH + H+
2-hydroxy-3-methylpentanoate + NAD+
-
i.e. 2-oxomethylvalerate
-
-
?
3-methyl-2-oxopentanoate + NADH + H+
2-hydroxy-3-methylpentanoate + NAD+
-
i.e. 2-oxomethylvalerate
-
-
?
4-methyl-2-oxopentanoate + NADH + H+
(R)-2-hydroxy-4-methylpentanoate + NAD+
-
i.e. 2-oxoisocaproate
i.e. (R)-2-hydroxyisocaproate
-
r
4-methyl-2-oxopentanoate + NADH + H+
(R)-2-hydroxy-4-methylpentanoate + NAD+
i.e. 2-oxoisocaproate
i.e. (R)-2-hydroxyisocaproate
-
?
4-methyl-2-oxopentanoate + NADH + H+
(R)-2-hydroxy-4-methylpentanoate + NAD+
-
i.e. 2-oxoisocaproate
i.e. (R)-2-hydroxyisocaproate
-
r
4-methyl-2-oxopentanoate + NADH + H+
(R)-2-hydroxy-4-methylpentanoate + NAD+
-
i.e. 2-oxoisocaproate. The apparent Vmax/Km ratio for the reverse reaction is about 0.5% of that for the forward reaction
i.e. (R)-2-hydroxyisocaproate
-
r
4-methyl-2-oxopentanoate + NADH + H+
(R)-2-hydroxy-4-methylpentanoate + NAD+
-
i.e. 2-oxoisocaproate. The apparent Vmax/Km ratio for the reverse reaction is about 0.5% of that for the forward reaction
i.e. (R)-2-hydroxyisocaproate
-
r
D-2-hydroxybutyrate + NAD+
2-oxobutyrate + NADH + H+
-
-
-
-
r
phenylglyoxylate + NAD+
hydroxy(phenyl)acetic acid + NADH + H+
-
Vmax/Km is 16.7% compared to 4-methyl-2-oxopentanoate
-
-
?
phenylglyoxylate + NAD+
hydroxy(phenyl)acetic acid + NADH + H+
-
Vmax/Km is 44% compared to 4-methyl-2-oxopentanoate
-
-
?
phenylglyoxylate + NAD+
hydroxy(phenyl)acetic acid + NADH + H+
-
Vmax/KM is 54% compared to 4-methyl-2-oxopentanoate
-
-
?
pyruvate + NADH + H+
D-lactate + NAD+
-
100% activity
-
-
r
additional information
?
-
-
both enzymes (D-mandelate dehydrogenase D-ManDH1 and D-mandelate dehydrogenase D-ManDH2) exhibit no or very little activity toward small 2-ketoacid substrates, such as pyruvate, hydroxypyruvate, and 2-ketobutyrate, and much higher activity toward substrates with larger aliphatic or aromatic side chains. The two enzymes exhibit higher activity (smaller Km and larger Vmax) for 2-ketoacid substrates branched at the C3 or C4 position than for unbranched substrates; i.e., 2-ketoisovalerate and 2-ketoisocaproate are more favorable than 2-ketovalerate and 2-ketocaproate, respectively. Among aromatic substrates, the two enzymes preferr benzoylformate to phenylpyruvate by 9- and 17-fold, respectively
-
-
-
additional information
?
-
-
both enzymes (D-mandelate dehydrogenase D-ManDH1 and D-mandelate dehydrogenase D-ManDH2) exhibit no or very little activity toward small 2-oxoacid substrates, such as pyruvate, hydroxypyruvate, and 2-oxobutyrate, and much higher activity toward substrates with larger aliphatic or aromatic side chains. The two enzymes exhibit higher activity (smaller Km and larger Vmax) for 2-ketoacid substrates branched at the C3 or C4 position than for unbranched substrates; i.e., 2-oxoisovalerate and 2-oxoisocaproate are more favorable than 2-oxovalerate and 2-oxocaproate, respectively. Among aromatic substrates, the two enzymes prefer benzoylformate to phenylpyruvate by 9- and 17-fold, respectively
-
-
-
additional information
?
-
-
the recombinant enzyme exhibits high catalytic activity toward various 2-oxoacid substrates with bulky hydrophobic side chains, particularly C3-branched substrates such as benzoylformate and 2-oxoisovalerate
-
-
-
additional information
?
-
-
no significant activity with L-lactate, L-2-hydroxybutyrate, L-hydroxyisocaproate, phenylpyruvate, 2-oxomethyl-n-valerate, and 2-oxoisovalerate
-
-
-
additional information
?
-
-
no significant activity with L-lactate, L-2-hydroxybutyrate, L-hydroxyisocaproate, phenylpyruvate, 2-oxomethyl-n-valerate, and 2-oxoisovalerate
-
-
-
additional information
?
-
-
very low specificity regarding size and chemical constitution of the accepted D-2-hydroxycarboxylates
-
-
-
additional information
?
-
-
the enzyme accepts D-2-hydroxyacids but not L-2-hydroxyacids and shows no NADP-dependent 2-ketopantoate reductase activity. No reactions are observed with 10 mM L-2-hydroxyisocaproate and 1 mM NAD+, 10 mM pyruvate and 0.3 mM NADH, 10 mM 2-oxooisocaproate and 0.3 mM NADPH, and 10 mM 2-oxopantoate and 0.3 mM NADH or NADPH
-
-
-
additional information
?
-
-
the enzyme accepts D-2-hydroxyacids but not L-2-hydroxyacids and shows no NADP-dependent 2-ketopantoate reductase activity. No reactions are observed with 10 mM L-2-hydroxyisocaproate and 1 mM NAD+, 10 mM pyruvate and 0.3 mM NADH, 10 mM 2-oxooisocaproate and 0.3 mM NADPH, and 10 mM 2-oxopantoate and 0.3 mM NADH or NADPH
-
-
-
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
NAD+
-
with NADP+ no reaction was observed in concentrations up to 4.5 mM
NADH
-
NADH can not be replaced by NADPH in the benzoylformate reduction by D-mandelate dehydrogenase D-ManDH1 or D-ManDH2
NADH
-
rhe enzyme requires NADH as a coenzyme for the hydrogenation reaction, NADPH does not support the reaction in concentrations up to 0.1 mM
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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+
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INHIBITORS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
1,10-phenanthroline
-
10 mM, decrease of 13% in the enzymatic activity
additional information
-
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
LITERATURE
IMAGE
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
2.3
(R)-2-hydroxy-4-methylpentanoate
-
pH 7.0, temperature not specified in the publication
-
2.7
2-hydroxyhexanoate
-
pH 7.0, temperature not specified in the publication
1.6
2-Hydroxyoctanoate
-
pH 7.0, temperature not specified in the publication
3.3
2-hydroxypentanoate
-
pH 7.0, temperature not specified in the publication
0.12
2-oxooctanoate
-
pH 7.0, temperature not specified in the publication
0.6
3-(4-hydroxyphenyl)-2-oxopropanoate
-
pH 7.0, temperature not specified in the publication
3.4
2-oxo-3-phenylpropanoate
-
pH 7.5, 30°C, D-mandelate dehydrogenase D-ManDH2
0.36
2-oxobutyrate
-
in 0.1 M glycine-NaOH buffer, pH 9.0, 4 M NaCl, at 52°C
0.13
2-Oxohexanoate
-
pH 7.0, temperature not specified in the publication
2.3
2-oxoisocaproate
-
in 0.1 M glycine-NaOH buffer, pH 9.0, 4 M NaCl, at 52°C
0.12
2-Oxopentanoate
-
pH 7.0, temperature not specified in the publication
0.15
3-methyl-2-oxobutanoate
-
pH 7.5, 30°C, D-mandelate dehydrogenase D-ManDH2
0.11
4-methyl-2-oxopentanoate
-
pH 7.5, 30°C, D-mandelate dehydrogenase D-ManDH2
0.4
4-methyl-2-oxopentanoate
-
pH 7.0, temperature not specified in the publication
0.05
NADH
-
with 2-oxobutyrate as cosubstrate, in 0.1 M glycine-NaOH buffer, pH 9.0, 4 M NaCl, at 52°C
0.086
NADH
-
with 2-oxoisocaproate as cosubstrate, in 0.1 M glycine-NaOH buffer, pH 9.0, 4 M NaCl, at 52°C
0.096
NADH
-
with pyruvate as cosubstrate, in 0.1 M glycine-NaOH buffer, pH 9.0, 4 M NaCl, at 52°C
0.26
phenylglyoxylate
-
pH 7.5, 30°C, D-mandelate dehydrogenase D-ManDH2
0.2
phenylpyruvate
-
pH 7.0, temperature not specified in the publication
0.56
pyruvate
-
in 0.1 M glycine-NaOH buffer, pH 9.0, 4 M NaCl, at 52°C
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
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
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
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
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
0.427
-
with 2-oxoisocaproate as substrate, in 0.1 M glycine-NaOH buffer, pH 9.0, 4 M NaCl, at 52°C
0.799
-
with 2-oxobutyrate as substrate, in 0.1 M glycine-NaOH buffer, pH 9.0, 4 M NaCl, at 52°C
1.462
-
with pyruvate as substrate, in 0.1 M glycine-NaOH buffer, pH 9.0, 4 M NaCl, at 52°C
170
-
after 77fold purification, in 100 mM Tris-HC1 buffer, pH 8.0, at 30°C
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
4.5
-
maximal catalytic efficiency, D-mandelate dehydrogenase D-ManDH1; maximal catalytic efficiency, D-mandelate dehydrogenase D-ManDH2
5.5 - 7
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pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
25
55
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TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
37 - 55
-
at 37°C, the activity reaches about 65% of peak activity at 55°C
65 - 75
-
above 60°C there is a pronounced activity decrease, while there is nearly no activity at 75°C
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SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
70000
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SUBUNITS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
dimer
homodimer
?
-
x * 34331, calculated from nucleotide sequence; x * 40000, SDS-PAGE
?
-
x * 32000, SDS-PAGE, D-mandelate dehydrogenase D-ManDH1; x * 40000, SDS-PAGE, D-mandelate dehydrogenase D-ManDH2
homodimer
-
2 * 37000, SDS-PAGE; 2 * 37183, calculated from amino acid sequence
homodimer
-
2 * 37000, SDS-PAGE; 2 * 37183, calculated from amino acid sequence
-
homodimer
-
2 * 34361, calculated from amino acid sequence; 2 * 36000, His-tagged fusion protein, SDS-PAGE
homodimer
-
2 * 34361, calculated from amino acid sequence; 2 * 36000, His-tagged fusion protein, SDS-PAGE
-
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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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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
LITERATURE
5 - 8
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activity is reduced by about 10% at pH 5.0 and 8.0
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
50
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at temperatures above 50°C the stability of the enzyme decreases drastically
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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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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
expression in Escherichia coli
overexpression in Escherichia coli as His-tagged fusion protein
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
synthesis
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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
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LINKS TO OTHER DATABASES (specific for EC-Number 1.1.1.345)
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