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Literature summary extracted from

  • Hayashi, J.; Inoue, S.; Kim, K.; Yoneda, K.; Kawarabayasi, Y.; Ohshima, T.; Sakuraba, H.
    Crystal structures of a hyperthermophilic archaeal homoserine dehydrogenase suggest a novel cofactor binding mode for oxidoreductases (2015), Sci. Rep., 5, 11674.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.1.1.3 gene PH1075, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21 (DE3) codon plus-RIPL Pyrococcus horikoshii

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.1.1.3 for the purified ligand-free recombinant wild-type enzyme: sitting drop vapor diffusion method, mixing of 0.001 ml of 12.0 mg/ml protein solution with an equal volume of mother liquor composed of 0.1 M potassium phosphate, pH 6.2, and 20% MPD, 2 days, 20°C, for the homoserine-bound K57A mutant enzyme: sitting drop vapor diffusion method, mixing of 0.002 ml of 10 mg/ml protein solution containing 1 mM homoserine with 0.002 ml of mother liquor containing 16% polyethylene glycol monomethyl ether 2000 and 0.1 M citrate buffer, pH 6.5, 7 days, 20°C, X-ray diffraction structure determination and analysis at 2.3 A resolution, molecular replacement and modelling Pyrococcus horikoshii

Protein Variants

EC Number Protein Variants Comment Organism
1.1.1.3 K57Aa site-directed mutagenesis, in contrast to the wild-type enzyme, the mutant enzyme shows catalytic activity with NADP+, the activity with NAD+ is increased compared to the wild-type enzyme Pyrococcus horikoshii
1.1.1.3 R40A site-directed mutagenesis, in contrast to the wild-type enzyme, the mutant enzyme shows catalytic activity with NADP+, the activity with NAD+ is decreased compared to the wild-type enzyme Pyrococcus horikoshii

Inhibitors

EC Number Inhibitors Comment Organism Structure
1.1.1.3 NADP+ NADP+ does not act as a cofactor for this enzyme, but as a strong inhibitor of NAD+-dependent oxidation of Hse, evaluation of the factors responsible for the NADP+-mediated inhibition Pyrococcus horikoshii

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.1.1.3 additional information
-
 additional information enzyme HseDH shows typical Michaelis-Menten kinetics for oxidation Pyrococcus horikoshii
1.1.1.3 0.04
-
 NADP+ pH 9.0, 50°C, recombinant mutant R40A Pyrococcus horikoshii
1.1.1.3 0.05
-
 NAD+ pH 9.0, 50°C, recombinant mutant K57A Pyrococcus horikoshii
1.1.1.3 0.06
-
 NADP+ pH 9.0, 50°C, recombinant mutant K57A Pyrococcus horikoshii
1.1.1.3 0.32
-
 NAD+ pH 9.0, 50°C, recombinant wild-type enzyme Pyrococcus horikoshii
1.1.1.3 0.95
-
 NAD+ pH 9.0, 50°C, recombinant mutant R40A Pyrococcus horikoshii
1.1.1.3 6.1
-
 L-homoserine pH 9.0, 50°C, recombinant enzyme Pyrococcus horikoshii

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
1.1.1.3 40000
-
-
 Pyrococcus horikoshii
1.1.1.3 92000
-
 gel filtration, recombinant His-tagged enzyme HseDH Pyrococcus horikoshii

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.1.1.3 L-homoserine + NAD+ Pyrococcus horikoshii
-
 L-aspartate 4-semialdehyde + NADH + H+
-
 r
1.1.1.3 L-homoserine + NAD+ Pyrococcus horikoshii ATCC 700860
-
 L-aspartate 4-semialdehyde + NADH + H+
-
 r

Organism

EC Number Organism UniProt Comment Textmining
1.1.1.3 Pyrococcus horikoshii O58802
-
-
1.1.1.3 Pyrococcus horikoshii ATCC 700860 O58802
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.1.1.3 recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21 (DE3) codon plus-RIPL by heat treatment at 90°C for 10 min, nickel affinity chromatography, dialysis, gel filtration, and ultrafiltration Pyrococcus horikoshii

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.1.1.3 L-homoserine + NAD+
-
 Pyrococcus horikoshii L-aspartate 4-semialdehyde + NADH + H+
-
 r
1.1.1.3 L-homoserine + NAD+
-
 Pyrococcus horikoshii ATCC 700860 L-aspartate 4-semialdehyde + NADH + H+
-
 r
1.1.1.3 L-homoserine + NADP+ no activity of the wild-type enzyme with NADP+, but only with enzyme mutants R40A and K57A Pyrococcus horikoshii L-aspartate 4-semialdehyde + NADPH + H+
-
 r
1.1.1.3 L-homoserine + NADP+ no activity of the wild-type enzyme with NADP+, but only with enzyme mutants R40A and K57A Pyrococcus horikoshii ATCC 700860 L-aspartate 4-semialdehyde + NADPH + H+
-
 r

Subunits

EC Number Subunits Comment Organism
1.1.1.3 homodimer 2 * 36925, sequence calculation, 2 * 40000, SDS-PAGE Pyrococcus horikoshii

Synonyms

EC Number Synonyms Comment Organism
1.1.1.3 HseDH
-
 Pyrococcus horikoshii

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.1.1.3 50
-
 assay at Pyrococcus horikoshii

Temperature Stability [°C]

EC Number Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
1.1.1.3 75
-
 purified recombinant His-tagged enzyme, 10 min, retains full activity Pyrococcus horikoshii
1.1.1.3 90
-
 purified recombinant His-tagged enzyme, 10 min, retains 70% activity Pyrococcus horikoshii

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.1.1.3 3.4
-
 NADP+ pH 9.0, 50°C, recombinant mutant R40A Pyrococcus horikoshii
1.1.1.3 26.7
-
 NADP+ pH 9.0, 50°C, recombinant mutant K57A Pyrococcus horikoshii
1.1.1.3 48.5
-
 NAD+ pH 9.0, 50°C, recombinant mutant K57A Pyrococcus horikoshii
1.1.1.3 70.1
-
 NAD+ pH 9.0, 50°C, recombinant wild-type enzyme Pyrococcus horikoshii
1.1.1.3 96.1
-
 NAD+ pH 9.0, 50°C, recombinant mutant R40A Pyrococcus horikoshii

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.1.1.3 11
-
 recombinant enzyme Pyrococcus horikoshii

pH Stability

EC Number pH Stability pH Stability Maximum Comment Organism
1.1.1.3 5 12 purified recombinant His-tagged enzyme, 10 min, 50°C, stable at Pyrococcus horikoshii

Cofactor

EC Number Cofactor Comment Organism Structure
1.1.1.3 additional information NADP does not act as a cofactor for this enzyme, but as a strong inhibitor of NAD+-dependent oxidation of Hse, analysis of the cofactor-binding site of the enzyme, Pyrococcus horikoshii HseDH shows a unique cofactor binding mode, which is not observed in conventional NAD(P)-dependent dehydrogenases. Superposition of the Hse/NADPH-bound K57A structure onto the NADPH-bound wild-type structure shows that the NADPH molecule in the mutant structure is positioned/configured nearly identically to the NADPH molecule in the wild-type structure, except for the positioning of the C2 phosphate group of the adenine ribose. The C2 phosphate is tightly held in position through five surrounding hydrogen bonds in the wild-type enzyme. In K57A mutant the C2 phosphate group is rotates in a clockwise direction around C2B of NADPH by about 30° relative to the wild-type structure. The guanidino group of Arg40 in the mutant is also rotated clockwise by about 90° around the NE atom of Arg40 relative to the wild-type structure Pyrococcus horikoshii
1.1.1.3 NAD+
-
 Pyrococcus horikoshii
1.1.1.3 NADH
-
 Pyrococcus horikoshii
1.1.1.3 NADP+ no activity of the wild-type enzyme with NADP+, but only with enzyme mutants R40A and K57A Pyrococcus horikoshii
1.1.1.3 NADPH no activity of the wild-type enzyme with NADPH, but only with enzyme mutants R40A and K57A Pyrococcus horikoshii

Ki Value [mM]

EC Number Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
1.1.1.3 0.0000052
-
 NADP+ pH 9.0, 50°C, recombinant wild-type enzyme Pyrococcus horikoshii

General Information

EC Number General Information Comment Organism
1.1.1.3 evolution the catalytic region of the enzyme is unique, the nucleotide-binding domain conforms to the Rossmann fold-like conventional NAD(P)-dependent dehydrogenases Pyrococcus horikoshii

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.1.1.3 89.1
-
 NADP+ pH 9.0, 50°C, recombinant mutant R40A Pyrococcus horikoshii
1.1.1.3 102
-
 NAD+ pH 9.0, 50°C, recombinant mutant R40A Pyrococcus horikoshii
1.1.1.3 219
-
 NAD+ pH 9.0, 50°C, recombinant wild-type enzyme Pyrococcus horikoshii
1.1.1.3 518
-
 NADP+ pH 9.0, 50°C, recombinant mutant K57A Pyrococcus horikoshii
1.1.1.3 1020
-
 NAD+ pH 9.0, 50°C, recombinant mutant K57A Pyrococcus horikoshii