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Literature summary for 1.1.1.103 extracted from

  • Higashi, N.; Tanimoto, K.; Nishioka, M.; Ishikawa, K.; Taya, M.
    Investigating a catalytic mechanism of hyperthermophilic L-threonine dehydrogenase from Pyrococcus horikoshii (2008), J. Biochem., 144, 77-85.
    View publication on PubMed
Search for more literature for 1.1.1.103

Cloned(Commentary)

Cloned (Comment) Organism
orf PH0655, overexpression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) Pyrococcus horikoshii

Protein Variants

Protein Variants Comment Organism
E152A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Pyrococcus horikoshii
E152C site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Pyrococcus horikoshii
E152D site-directed mutagenesis, the E152D mutant shows 3-fold higher turnover rate and reduced affinities toward L-threonine and NAD+ compared to wild-type TDH, Glu152 to Asp substitution causes the enhancement of deprotonation of His47 or ionization of zinc-bound water and threonine in the enzyme-NAD+ complex Pyrococcus horikoshii
E152K site-directed mutagenesis, inactive mutant Pyrococcus horikoshii
E152Q site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Pyrococcus horikoshii
E152S site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Pyrococcus horikoshii
E152T site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Pyrococcus horikoshii

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
 additional information kinetic analysis of Glu152 mutants, overview Pyrococcus horikoshii
0.0099
-
 NAD+ pH 7.5, 65°C, recombinant wild-type enzyme Pyrococcus horikoshii
0.0615
-
 NAD+ pH 7.5, 65°C, recombinant mutant E152A Pyrococcus horikoshii
0.152
-
 NAD+ pH 7.5, 65°C, recombinant mutant E152T Pyrococcus horikoshii
0.163
-
 NAD+ pH 7.5, 65°C, recombinant mutant E152C Pyrococcus horikoshii
0.216
-
 NAD+ pH 7.5, 65°C, recombinant mutant E152Q Pyrococcus horikoshii
0.399
-
 NAD+ pH 7.5, 65°C, recombinant mutant E152D Pyrococcus horikoshii
0.607
-
 NAD+ pH 7.5, 65°C, recombinant mutant E152S Pyrococcus horikoshii

Metals/Ions

Metals/Ions Comment Organism Structure
Zn2+ 1.22 mol of Zn2+ per mol of enzyme subunit, the catalytic zinc atom at the active center of TDH is coordinated by the highly conserved four residues Cys42, His67, Glu68 and Glu152 with low affinity Pyrococcus horikoshii

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
38000
-
 x * 38000, recombinant wild-type and mutant enzymes, SDS-PAGE Pyrococcus horikoshii

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
L-threonine + NAD+ Pyrococcus horikoshii
-
 (2S)-2-amino-3-oxobutanoate + NADH + H+
-
 ?

Organism

Organism UniProt Comment Textmining
Pyrococcus horikoshii O58389
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by heat treatment at 85°C for 30 min, anion exchange and hydrophobic interaction chromatography Pyrococcus horikoshii

Reaction

Reaction Comment Organism Reaction ID
L-threonine + NAD+ = L-2-amino-3-oxobutanoate + NADH + H+ catalytic mechanism, the carboxyl group of Glu152 is important for expressing the catalytic activity, the proton relay system works as a catalytic mechanism of TDH Pyrococcus horikoshii
a

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
L-threonine + NAD+
-
 Pyrococcus horikoshii (2S)-2-amino-3-oxobutanoate + NADH + H+
-
 ?
L-threonine + NAD+ active site structure, overview Pyrococcus horikoshii (2S)-2-amino-3-oxobutanoate + NADH + H+
-
 ?

Subunits

Subunits Comment Organism
? x * 38000, recombinant wild-type and mutant enzymes, SDS-PAGE Pyrococcus horikoshii

Synonyms

Synonyms Comment Organism
L-threonine dehydrogenase
-
 Pyrococcus horikoshii
TDH
-
 Pyrococcus horikoshii

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
65
-
 assay at Pyrococcus horikoshii

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.009
-
 NAD+ pH 7.5, 65°C, recombinant mutant E152A Pyrococcus horikoshii
0.01
-
 NAD+ pH 7.5, 65°C, recombinant mutant E152Q Pyrococcus horikoshii
0.08
-
 NAD+ pH 7.5, 65°C, recombinant mutant E152T Pyrococcus horikoshii
0.085
-
 NAD+ pH 7.5, 65°C, recombinant mutant E152S Pyrococcus horikoshii
0.24
-
 NAD+ pH 7.5, 65°C, recombinant mutant E152C Pyrococcus horikoshii
1.11
-
 NAD+ pH 7.5, 65°C, recombinant wild-type enzyme Pyrococcus horikoshii
3.5
-
 NAD+ pH 7.5, 65°C, recombinant mutant E152D Pyrococcus horikoshii

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5
-
 assay at Pyrococcus horikoshii

pH Range

pH Minimum pH Maximum Comment Organism
additional information
-
 pH dependencies of wild-type enzyme and E152D mutant, overview Pyrococcus horikoshii

Cofactor

Cofactor Comment Organism Structure
NAD+
-
 Pyrococcus horikoshii