Information on EC 1.1.1.337 - L-2-hydroxycarboxylate dehydrogenase (NAD+)

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

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EC NUMBER
COMMENTARY hide
1.1.1.337
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RECOMMENDED NAME
GeneOntology No.
L-2-hydroxycarboxylate dehydrogenase (NAD+)
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
a (2S)-2-hydroxycarboxylate + NAD+ = a 2-oxocarboxylate + NADH + H+
show the reaction diagram
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
(R)-cysteate degradation
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coenzyme M biosynthesis I
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coenzyme M biosynthesis
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Methane metabolism
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SYSTEMATIC NAME
IUBMB Comments
(2S)-2-hydroxycarboxylate:NAD+ oxidoreductase
The enzyme from the archaeon Methanocaldococcus jannaschii acts on multiple (S)-2-hydroxycarboxylates including (2R)-3-sulfolactate, (S)-malate, (S)-lactate, and (S)-2-hydroxyglutarate [3]. Note that (2R)-3-sulfolactate has the same stereo configuration as (2S)-2-hydroxycarboxylates.
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
Paracoccus pantotrophus NKNCYSA / DSM 12449
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Manually annotated by BRENDA team
i.e. Lactobacillus confusus
SwissProt
Manually annotated by BRENDA team
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
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SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(2R)-3-sulfolactate + NAD+
3-sulfopyruvate + NADH + H+
show the reaction diagram
(S)-2-hydroxyglutarate + NAD+
2-oxoglutarate + NADH + H+
show the reaction diagram
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-
-
r
(S)-lactate + NAD+
pyruvate + NADH + H+
show the reaction diagram
1-hydroxy-1,3,4,6-hexanetetracarboxylate + NAD+
1-oxo-1,3,4,6-hexanetetracarboxylate + NADH + H+
show the reaction diagram
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-
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r
2-hydroxycaproate + NAD+
2-oxocaproate + NADH + H+
show the reaction diagram
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r
2-hydroxyisocaproate + NAD+
2-oxoisocaproate + NADH + H+
show the reaction diagram
the initial rate of the reduction of 2-oxoisocaproate is about seven times faster than the reverse reaction, the dehydrogenation of L-2-hydroxyisocaproate, measured in buffers at the respective pH optima
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r
2-hydroxyisovalerate + NAD+
2-oxoisovalerate + NADH + H+
show the reaction diagram
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r
2-hydroxyoctanoate + NAD+
2-oxooctanoate + NADH + H+
show the reaction diagram
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-
-
?
2-hydroxyvalerate + NAD+
2-oxovalerate + NADH + H+
show the reaction diagram
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-
-
r
2-oxobutyrate + NADH + H+
2-hydroxybutyrate + NAD+
show the reaction diagram
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r
2-oxocaproate + NADH + H+
2-hydroxycaproate + NAD+
show the reaction diagram
2-oxoglutarate + NADH + H+
(S)-2-hydroxyglutarate + NAD+
show the reaction diagram
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-
-
-
?
2-oxoisocaproate + NADH + H+
2-hydroxyisocaproate + NAD+
show the reaction diagram
2-oxoisovalerate + NADH + H+
2-hydroxyisovalerate + NAD+
show the reaction diagram
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r
2-oxooctanoate + NADH + H+
2-hydroxyoctanoate + NAD+
show the reaction diagram
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r
2-oxopentanedioic acid + NADH + H+
2-hydroxypentandioic acid + NAD+
show the reaction diagram
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-
-
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?
2-oxovalerate + NADH + H+
2-hydroxyvalerate + NAD+
show the reaction diagram
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r
3-phenyllactate + NAD+
pyruvate + NADH + H+
show the reaction diagram
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r
3-sulfopyruvate + NADH + H+
(S)-3-sulfolactate + NAD+
show the reaction diagram
3-sulfopyruvate + NADH + H+
3-sulfolactate + NAD+
show the reaction diagram
4-methyl-2-oxopentanoate + NADH + H+
(S)-2-hydroxy-4-methylpentanoate + NAD+
show the reaction diagram
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r
a 2-oxocarboxylate + NADH + H+
(2S)-2-hydroxycarboxylate + NAD+
show the reaction diagram
glyoxylate + NADH + H+
2-hydroxypropanoate + NAD+
show the reaction diagram
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?
L-lactate + NAD+
pyruvate + NADH + H+
show the reaction diagram
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r
oxaloacetate + NADH + H+
(S)-malate + NAD+
show the reaction diagram
oxaloacetate + NADH + H+
malate + NAD+
show the reaction diagram
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-
-
-
?
oxo-tert-leucine + NADH + H+
? + NAD+
show the reaction diagram
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r
oxopropandioic acid + NADH + H+
2-hydroxypropanedioic acid + NAD+
show the reaction diagram
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?
phenylglyoxylate + NADH + H+
? + NAD+
show the reaction diagram
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r
phenylpyruvate + NADH + H+
3-phenyllactate + NAD+
show the reaction diagram
pyruvate + NADH + H+
(S)-lactate + NAD+
show the reaction diagram
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?
pyruvate + NADH + H+
3-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
a 2-oxocarboxylate + NADH + H+
(2S)-2-hydroxycarboxylate + NAD+
show the reaction diagram
P14295
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r
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
NAD+
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the enzyme requires NAD+ as a cofactor, no reaction with NADP+ in concentrations up to 3 mM
NADPH
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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at 1 mM no influence is observed with Mg2+, Ca2+, Co2+, Ni2+, and Cd2+ and very weak effects with Mn2+, Cu2+, and Zn2+
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INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1,10-phenanthroline
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10 mM, 15% inhibition
4-chloromercuribenzoate
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weak inhibition
iodoacetamide
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weak inhibition
iodoacetate
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weak inhibition
KCN
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weak inhibition
NADH
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inhibition above 0.24 mM
additional information
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reduced glutathione (20 mM) has no effect. EDTA, 2,2'-bipyridyl in 1 mM and 10 mM final concentrations have no effect on enzymatic activity
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
15
1-oxo-1,3,4,6-hexanetetracarboxylate
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cosubstrate NADH, pH 8.0, 70°C
2.2
2-hydroxycaproate
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pH 8.0, 30°C
0.62
2-hydroxyisocaproate
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pH 8.0, 30°C
0.6
2-Hydroxyisovalerate
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pH 8.0, 30°C
1.9
2-Hydroxyoctanoate
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pH 8.0, 30°C
1.8
2-hydroxyvalerate
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pH 8.0, 30°C
0.45
2-oxobutyrate
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pH 7.0, 30°C
0.071 - 62
2-oxocaproate
1.9
2-oxoglutarate
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cosubstrate NADH, pH 8.0, 70°C
0.06
2-oxoisocaproate
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pH 7.0, 30°C
0.065
2-oxoisovalerate
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pH 7.0, 30°C
0.17
2-oxooctanoate
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pH 7.0, 30°C
1.9
2-oxopentanedioic acid
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pH 8.0, 70°C
0.1
2-oxovalerate
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pH 7.0, 30°C
0.64
3-Phenyllactate
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pH 8.0, 30°C
0.04 - 0.21
3-sulfopyruvate
0.067 - 110
4-methyl-2-oxopentanoate
46
glyoxylate
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pH 8.0, 70°C
100
L-lactate
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pH 8.0, 30°C
0.11 - 5.32
oxaloacetate
3.4
oxopropandioic acid
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pH 8.0, 70°C
0.026 - 19
phenylpyruvate
2 - 200
pyruvate
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.63 - 360000
2-oxocaproate
2 - 13000
2-oxoisocaproate
1.7 - 520000
4-methyl-2-oxopentanoate
0.07 - 52
oxo-tert-leucine
0.1 - 18000
phenylglyoxylate
8 - 89000
phenylpyruvate
0.004 - 340
pyruvate
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.035 - 3300000
2-oxocaproate
3.84 - 203000
2-oxoisocaproate
0.092 - 190000
4-methyl-2-oxopentanoate
0.0469 - 133
oxo-tert-leucine
0.00526 - 1880
phenylglyoxylate
1.4 - 281000
phenylpyruvate
0.00013 - 26
pyruvate
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
2.52
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pH 7.5, 30°C
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8 - 8.5
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dehydrogenation of L-2-hydroxyisocaproate
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30 - 50
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the maximal temperature is around 50°C. At this temperature the reaction proceeded at about 3fold increase of the rate compared to 30°C
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pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.06
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calculated from sequence
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SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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maximum specific and volume activities are found at the end of the exponential growth phase, during further fermentation enzyme activity declines rapidly and is no longer detectable after 15 h
Manually annotated by BRENDA team
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
75000
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2 * 75000, calculated
125000
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gel filtration
150000
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gel filtration and velocity sedimentation data
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SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dimer
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2 * 75000, calculated
homotetramer
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4 * 33000
tetramer
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4 * 33000, SDS-PAGE
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Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
in complex with NADH, to 2.5 A resolution. The asymmetric unit contains a tetramer of tight dimers. Structure does not contain a cofactor-binding domain with Rossmann-fold topology. The NADH is bound in an extended conformation in each active site, in a manner that explains the pro-S specificity. Cofactor binding involves residues belonging to both subunits within the tight dimers. The results indicate the existence of a substrate discrimination mechanism, which involves electrostatic interactions
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recombinant enzyme is crystallized with ammonium sulfate as precipitant in the presence of NAD+. The crystals belong to the trigonal space group P3(2)21, with a = 135.9 A and c = 205.9 A, and diffract X-rays to 2.2 A resolution
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.5 - 9
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4°C, 24 h, stable
726750
6.5
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4°C, 6 months, concentrated enzyme solutions, 10% loss of activity
726750
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
40
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1 h, stable up to
52
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heat capacity function of the enzyme shows a single peak with the T(m) values between 52.14° C and 55.89°C at pH 7.0 at different scan rates
additional information
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the thermal denaturation of the enzyme is studied by Differential Scanning Calorimetry and Circular Dichroism spectroscopy. The thermal denaturation is pH dependent. The thermal denaturation is irreversible and the T(m) is dependent on the scan-rate. Stabilizing effect of NADH binding. The denaturation process of L-HicDH is kinetically determined
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4°C, pH 6.5, concentrated enzyme solution, 10% loss of activity after 6 months
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
single-step purification of hexahistidine-tagged enzyme. The tag removal is accomplished by a derivative of recombinant tobacco etch virus protease
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
high level expression of hexahistidine-tagged enzyme
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the L-HicDH gene is expressed under control of phage lambda Leftward and rightward promoters in Escherichia coli up to 35% of total cell protein. The enzyme produced under these conditions exhibits full specific activity and is found exclusively in soluble form
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
del I81/delK82
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phenylpyruvate is the only substrate which is converted at a significant catalytic rate by the mutant enzyme. In the publication this mutant is referred to as Ile100ADELTA/Lys100BDELTA, according to the numbering system of the dogfish L-lactate dehydrogenase coenzyme loop region
del I81/delK82/delN87/delP88
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specific modifications in catalytic rates and substrate recognition
del L 83
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deletion mutant shows an altered substrate specificity. In the publication this mutant is referred to as Leu101DELTA, according to the numbering system of the dogfish L-lactate dehydrogenase coenzyme loop region
delI81
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drastic reductions in the catalytic activity for all tested substrates. In the publication this mutant is referred to as Ile100ADELTA, according to the numbering system of the dogfish L-lactate dehydrogenase coenzyme loop region
delK82
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drastic reductions in the catalytic activity for all tested substrates. In the publication this mutant is referred to as Lys100BDELTA, according to the numbering system of the dogfish L-lactate dehydrogenase coenzyme loop region
delN87
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deletion mutant shows an altered substrate specificity. In the publication this mutant is referred to as Asn105ADELTA, according to the numbering system of the dogfish L-lactate dehydrogenase coenzyme loop region
delN87/delP88
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for the deletion mutant enzyme 2-oxo carboxylic acids branched at C4 are better substrates than 2-oxocaproate, the substrate with the best kcat,/KM ratio known for the wild-type enzyme.The mutation results in a 5.2fold increased catalytic efficiency towards 4-methyl-2-oxopentanoate compared to the wild-type enzyme. In the publication this mutant is referred to as Asn105ADELTA/Pro105BDELTA, according to the numbering system of the dogfish L-lactate dehydrogenase coenzyme loop region
delP88
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deletion mutant shows an altered substrate specificity. In the publication this mutant is referred to as Pro105BDELTA, according to the numbering system of the dogfish L-lactate dehydrogenase coenzyme loop region
F236S
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phenylpyruvate displays the largest kcat of the tested substrates. All kcat values, with the exception of phenylpyruvate, are reduced, but the relative acceptance of phenylglyoxylate is greatly increased
F236V
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decrease in the second-order rate constants for all tested substrates. The kcat values for the smaller substrates decrease drastically. Phenylpyruvate is the favourite substrate (2-oxocaproate is the favourite substrate of the wild-type enzyme)
G234V/G235D
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the second-order rate constant decreased for most substrates with the exception of pyruvate, which reacts 2.5times faster in the mutant enzyme than in the wild-type enzyme. The catalysis of 2-oxoisocaproate is unaffected
L239A
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shift of enzyme specificity towards the substrates branched at C3 corresponding to an increase in the turnover numbers for 2-oxoisocaproate
L239F
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slight decrease in the KM values for phenylglyoxylate combined with a dramatic decrease in the kcat values
L239M
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KM values of all substrates of the enzyme variant are very similar to those of the wild-type enzyme, large improvement in the kcat values of the C4-branched substrates 2-oxoisocaproate and phenylpyruvate, decrease in the turnover number of 2-oxocaproate (the substrate favoured by the wild-type enzyme). Whereas in the wild-type enzyme the second-order rate constant for 2-oxoisocaproate is only 1% of that for the unbranched substrate, in the mutant enzyme the rate constant of 2-oxocaproate is only 18% of that for 2-oxoisocaproate
L239M/T245A
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the catalytic rates are reduced by several orders of magnitude and the KM values shows at least a 100fold increase for most substrates (with the exception of phenylglyoxylate). With respect to L239M the substrate specificity shifts towards keto-tert-leucine and with respect to T245A 2-oxoisocaproate and phenylpyruvate are more favoured
L239W
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slight decrease in the KM values for phenylglyoxylate combined with a dramatic decrease in the kcat values
T245A
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the catalytic rates are reduced by several orders of magnitude, relative shift of substrate specificity for keto-tert-leucine of more than 17 000 compared with the 2-oxocaproate (kcat/KM)
additional information
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the coenzyme loop, a functional element which is essential for catalysis and substrate specificity, is modified in order to identify the residues involved in the catalytic reaction and substrate specificity
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
synthesis
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the enzyme can be applied in an industrial process for the production of L-amino acids
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LINKS TO OTHER DATABASES (specific for EC-Number 1.1.1.337)
ExplorEnz
ExPASy
KEGG
MetaCyc
SABIO-RK
NCBI: PubMed, Protein, Nucleotide, Structure, Genome, OMIM
IUBMB Enzyme Nomenclature
PROSITE Database of protein families and domains
InterPro (database of protein families, domains and functional sites)