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 |
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 |
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 |
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 |
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 |
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 |
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 |
EC Number | Organism | UniProt | Comment | Textmining |
---|---|---|---|---|
1.1.1.3 | Pyrococcus horikoshii | O58802 | - |
- |
1.1.1.3 | Pyrococcus horikoshii ATCC 700860 | O58802 | - |
- |
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 |
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 |
EC Number | Subunits | Comment | Organism |
---|---|---|---|
1.1.1.3 | homodimer | 2 * 36925, sequence calculation, 2 * 40000, SDS-PAGE | Pyrococcus horikoshii |
EC Number | Synonyms | Comment | Organism |
---|---|---|---|
1.1.1.3 | HseDH | - |
Pyrococcus horikoshii |
EC Number | Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|---|
1.1.1.3 | 50 | - |
assay at | Pyrococcus horikoshii |
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 |
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 |
EC Number | pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|---|
1.1.1.3 | 11 | - |
recombinant enzyme | Pyrococcus horikoshii |
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 |
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 |
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 |
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 |
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 |