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Literature summary for 1.1.1.272 extracted from

  • Domenech, J.; Ferrer, J.
    A new D-2-hydroxyacid dehydrogenase with dual coenzyme-specificity from Haloferax mediterranei, sequence analysis and heterologous overexpression (2006), Biochim. Biophys. Acta, 1760, 1667-1674.
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
gene ddh, DNA and amino acid sequence determination and analysis, sequence comparison, subcloning and expression in Escherichia coli strains XL1 Blue and BL21(DE3) in inclusion bodies Haloferax mediterranei

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.031
-
NADPH pH 8.5, 40°C, recombinant enzyme, substrate 2-oxoisocaproate Haloferax mediterranei
0.046
-
NADPH pH 8.5, 40°C, recombinant enzyme, substrate 2-oxoisoleucine Haloferax mediterranei
0.054
-
NADPH pH 8.5, 40°C, recombinant enzyme, substrate 2-oxobutyrate Haloferax mediterranei
0.233
-
NADH pH 8.5, 40°C, recombinant enzyme, substrate 2-oxoisocaproate Haloferax mediterranei
0.33
-
NADH pH 8.5, 40°C, recombinant enzyme, substrate 2-oxoisoleucine Haloferax mediterranei
0.5
-
NADH pH 8.5, 40°C, recombinant enzyme, substrate 2-oxobutyrate Haloferax mediterranei
1.31
-
2-oxoisoleucine pH 8.5, 40°C, recombinant enzyme, cofactor NADPH Haloferax mediterranei
3.77
-
2-oxoisocaproate pH 8.5, 40°C, recombinant enzyme, cofactor NADPH Haloferax mediterranei
11.9
-
2-oxoisoleucine pH 8.5, 40°C, recombinant enzyme, cofactor NADH Haloferax mediterranei
13.45
-
2-oxobutyrate pH 8.5, 40°C, recombinant enzyme, cofactor NADPH Haloferax mediterranei
13.9
-
2-oxoisocaproate pH 8.5, 40°C, recombinant enzyme, cofactor NADH Haloferax mediterranei
21.96
-
pyruvate pH 5.0, 40°C, recombinant enzyme, cofactor NADPH Haloferax mediterranei
106
-
2-oxobutyrate pH 8.5, 40°C, recombinant enzyme, cofactor NADH Haloferax mediterranei

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
29500
-
4 * 33336, sequence calculation, 4 * 29500, recombinant enzyme, CTAB-PAGE, 4 * 47000, SDS-PAGE Haloferax mediterranei
33336
-
4 * 33336, sequence calculation, 4 * 29500, recombinant enzyme, CTAB-PAGE, 4 * 47000, SDS-PAGE Haloferax mediterranei
47000
-
4 * 33336, sequence calculation, 4 * 29500, recombinant enzyme, CTAB-PAGE, 4 * 47000, SDS-PAGE Haloferax mediterranei
111000
-
recombinant enzyme, gel filtration Haloferax mediterranei

Organism

Organism UniProt Comment Textmining
Haloferax mediterranei Q2VEQ7 gene ddh; strain R4, gene ddh
-
Haloferax mediterranei R-4 / ATCC 33500 Q2VEQ7 gene ddh; strain R4, gene ddh
-

Purification (Commentary)

Purification (Comment) Organism
recombinant enzyme partially from Escherichia coli strain BL21(DE3) inclusion bodies by solubilization with 8 M urea in DTT-containing buffer and refolding by rapid 10fold dilution Haloferax mediterranei

Reaction

Reaction Comment Organism Reaction ID
an (R)-2-hydroxycarboxylate + NADP+ = a 2-oxocarboxylate + NADPH + H+ the catalytic triad is probably formed by Arg226, Glu255, and His274 Haloferax mediterranei

Renatured (Commentary)

Renatured (Comment) Organism
refolding of recombinant enzyme from Escherichia coli strain BL21(DE3) inclusion bodies, solubilized by 8 M urea in DTT-containing buffer, by rapid 10fold dilution, optimally in presence of 4 M NaCl at pH 8.0 and 37°C Haloferax mediterranei

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
additional information
-
substrate and cofactor specificity, overview Haloferax mediterranei

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2-oxo-4-methylpentanoate + NAD(P)H
-
Haloferax mediterranei (2R)-2-hydroxy-4-methylpentanoate + NAD(P)+
-
ir
2-oxo-4-methylpentanoate + NAD(P)H
-
Haloferax mediterranei R-4 / ATCC 33500 (2R)-2-hydroxy-4-methylpentanoate + NAD(P)+
-
ir
2-oxobutyrate + NAD(P)H
-
Haloferax mediterranei (2R)-2-hydroxybutyrate + NAD(P)+
-
ir
2-oxobutyrate + NAD(P)H
-
Haloferax mediterranei R-4 / ATCC 33500 (2R)-2-hydroxybutyrate + NAD(P)+
-
ir
2-oxobutyrate + NADH + H+
-
Haloferax mediterranei (2R)-2-hydroxybutyrate + NAD+
-
r
2-oxoisocaproate + NADH + H+
-
Haloferax mediterranei 2-D-hydroxyisocaproate + NAD+
-
r
2-oxoisoleucine + NAD(P)H
-
Haloferax mediterranei 2-D-hydroxyisoleucine + NAD(P)+
-
ir
2-oxoisoleucine + NAD(P)H
-
Haloferax mediterranei R-4 / ATCC 33500 2-D-hydroxyisoleucine + NAD(P)+
-
ir
2-oxoisoleucine + NADH + H+
-
Haloferax mediterranei 2-D-hydroxyisoleucine + NAD+
-
r
additional information no activity with 2-D-hydroxyacids and L-2-hydroxyisocaproic acid in oxidation reaction Haloferax mediterranei ?
-
?
additional information no activity with 2-D-hydroxyacids and L-2-hydroxyisocaproic acid in oxidation reaction Haloferax mediterranei R-4 / ATCC 33500 ?
-
?
pyruvate + NADPH + H+
-
Haloferax mediterranei D-lactate + NADP+
-
ir
pyruvate + NADPH + H+
-
Haloferax mediterranei R-4 / ATCC 33500 D-lactate + NADP+
-
ir

Subunits

Subunits Comment Organism
tetramer 4 * 33336, sequence calculation, 4 * 29500, recombinant enzyme, CTAB-PAGE, 4 * 47000, SDS-PAGE Haloferax mediterranei

Synonyms

Synonyms Comment Organism
2-D-hydroxyacid dehydrogenase
-
Haloferax mediterranei
D-2-hydroxyacid dehydrogenase
-
Haloferax mediterranei
D2-HDH
-
Haloferax mediterranei

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
40
-
assay at, recombinant refolded enzyme Haloferax mediterranei

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
5
-
assay at, with substrate pyruvate Haloferax mediterranei
8.5
-
assay at, all substrates, except for pyruvate Haloferax mediterranei

Cofactor

Cofactor Comment Organism Structure
NADH
-
Haloferax mediterranei
NADPH preferred cofactor Haloferax mediterranei