Activating Compound | Comment | Organism | Structure |
---|---|---|---|
additional information | the enzyme activity is unaffected by 1 mM of EDTA, iodoacetic acid, and L-(+)-tartaric acid | Pyrobaculum calidifontis |
Cloned (Comment) | Organism |
---|---|
recombinant overexpression of His-tagged and selenomethionyl-labeled wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) codon plus RIPL, expression of the enzyme from the L-SerDH-pET-15b plasmid (pET0699) in a modified M9 medium containing selenomethionine | Pyrobaculum calidifontis |
Crystallization (Comment) | Organism |
---|---|
purified recombinant wild-type and mutant enzymes in complex with NADP+/sulfate ion and with NADP+/L-tartrate (substrate analogue), sitting-drop vapor diffusion method, mixing of 0.001 ml of 13 mg/mL protein in 10 mM Tris-HCl, pH 8.0, 200 mM NaCl, and 5 mM 2-mercaptoethanol, with 0.001 ml of mother liquor containing 30% w/v PEG 2000 MME, 0.2 M ammonium sulfate, and 100 mM acetate buffer, pH 4.6, for complex crystals 0.5 mM NADP+ is added to the protein solution, soaking of the crystals in mother liquor containing 0.45 M L-(+)-tartaric acid for 10 min, X-ray diffraction structure determination and analysis at 1.18 and 1.57 A resolution, respectively. Based on the model of the selenomethionyl enzyme (PDB entry, 3W6U), the structure of the NADP+/ sulfate ion-bound and NADP+/ tartrate-bound enzymes is determined using the molecular replacement method, modeling | Pyrobaculum calidifontis |
Protein Variants | Comment | Organism |
---|---|---|
K170M | site-directed mutagenesis | Pyrobaculum calidifontis |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
CdSO4 | 21% inhibition at 1 mM | Pyrobaculum calidifontis | |
D-cysteine | 59.2% inhibition at 1 mM | Pyrobaculum calidifontis | |
HgCl2 | 31.6% inhibition at 1 mM | Pyrobaculum calidifontis | |
L-cysteine | 53.3% inhibition at 1 mM | Pyrobaculum calidifontis | |
additional information | the enzyme activity is unaffected by 1 mM of EDTA, LiSO4, MgCl2, MnCl2, CaCl2, NiCl2, CoCl2, BaCl2, CuSO4, ZnCl2, iodoacetic acid, and L-(+)-tartaric acid | Pyrobaculum calidifontis | |
NADP+ | 92.2% inhibition at 1 mM | Pyrobaculum calidifontis |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | Michaelis-Menten type kinetics | Pyrobaculum calidifontis | |
0.033 | - |
NADP+ | pH 10.0, 50°C, recombinant wild-type enzyme | Pyrobaculum calidifontis | |
0.38 | - |
NAD+ | pH 10.0, 50°C, recombinant wild-type enzyme | Pyrobaculum calidifontis | |
2.16 | - |
DL-glycerate | pH 10.0, 50°C, recombinant wild-type enzyme | Pyrobaculum calidifontis | |
4.33 | - |
L-serine | pH 10.0, 50°C, recombinant wild-type enzyme | Pyrobaculum calidifontis | |
6.09 | - |
D-serine | pH 10.0, 50°C, recombinant wild-type enzyme | Pyrobaculum calidifontis | |
34.57 | - |
DL-3-hydroxyisobutyrate | pH 10.0, 50°C, recombinant wild-type enzyme | Pyrobaculum calidifontis | |
69.04 | - |
3-hydroxypropionate | pH 10.0, 50°C, recombinant wild-type enzyme | Pyrobaculum calidifontis |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
additional information | the enzyme activity is unaffected by 1 mM of LiSO4, MgCl2, MnCl2, CaCl2, NiCl2, CoCl2, BaCl2, CuSO4, and ZnCl2 | Pyrobaculum calidifontis |
Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
51000 | - |
recombinant enzyme, gel filtration | Pyrobaculum calidifontis |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Pyrobaculum calidifontis | - |
- |
- |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged and selenomethionyl-labeled wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) codon plus RIPL by nickel affinity chromatography, removal of the first 17 amino acids of the N-terminal region of the His-tagged enzyme by thrombin | Pyrobaculum calidifontis |
Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|
3.48 | - |
purified recombinant wild-type enzyme, pH 10.0, 50°C | Pyrobaculum calidifontis |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
2-aminomalonate semialdehyde | spontaneous | Pyrobaculum calidifontis | 2-aminoacetaldehyde + CO2 | - |
? | |
3-hydroxypropionate + NAD+ | - |
Pyrobaculum calidifontis | ? + NADH + H+ | - |
? | |
D-serine + NAD+ | overall reaction, 5% of the relative activity observed with L-serine | Pyrobaculum calidifontis | 2-aminoacetaldehyde + CO2 + NADH + H+ | - |
? | |
DL-3-hydroxyisobutyrate + NAD+ | - |
Pyrobaculum calidifontis | ? + NADH + H+ | - |
? | |
DL-glycerate + NAD+ | - |
Pyrobaculum calidifontis | ? + NADH + H+ | - |
? | |
L-serine + NAD(P)+ | - |
Pyrobaculum calidifontis | 2-aminomalonate semialdehyde + NAD(P)H + H+ | - |
? | |
L-serine + NAD+ | overall reaction, NAD+ is the preferred cofactor | Pyrobaculum calidifontis | 2-aminoacetaldehyde + CO2 + NADH + H+ | - |
? | |
L-serine + NADP+ | overall reaction, low activity with NADP+ | Pyrobaculum calidifontis | 2-aminoacetaldehyde + CO2 + NADPH + H+ | - |
? | |
additional information | modeling of the D-serine and 3-hydroxypropionate molecules into the enzyme's active site. No steric hindrance is observed between D-serine and the side chains of the active site residues. D-serine is placed at the position through interactions similar to the L-serine binding model. The C3 hydrogen of D-serine is located at the same position as that of L-serine. This may explain the high reactivity toward D-serine exhibited by Pyrobaculum calidifontis L-SerDH | Pyrobaculum calidifontis | ? | - |
- |
Subunits | Comment | Organism |
---|---|---|
homodimer | 2 * 27000, recombinant enzyme, SDS-PAGE | Pyrobaculum calidifontis |
Synonyms | Comment | Organism |
---|---|---|
L-SerDH | - |
Pyrobaculum calidifontis |
L-serine 3-dehydrogenase | - |
Pyrobaculum calidifontis |
L-serine dehydrogenase | - |
Pyrobaculum calidifontis |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
50 | - |
assay at | Pyrobaculum calidifontis |
Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
---|---|---|---|
100 | - |
recombinant enzyme, no loss of activity is observed by incubation for 30 min at temperatures up to 100°C | Pyrobaculum calidifontis |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
10 | - |
assay at | Pyrobaculum calidifontis |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
additional information | both NAD+ and NADP+ are capable of serving as cofactors for the enzyme, but the reaction rate with NADP+ is 7.9% of that observed with NAD+ with L-serine as substrate. Deamido-NAD+ is not able to serve as cofactor. Cofactor binding mechanism, overview | Pyrobaculum calidifontis | |
NAD+ | - |
Pyrobaculum calidifontis | |
NADH | - |
Pyrobaculum calidifontis | |
NADP+ | - |
Pyrobaculum calidifontis | |
NADPH | - |
Pyrobaculum calidifontis |
General Information | Comment | Organism |
---|---|---|
additional information | catalytic domain fold of the Pyrobaculum calidifontis enzyme shows similarity with that of Pseudomonas aeruginosa L-SerDH, but the active site structure significantly differs between the two enzymes. Based on the structure of the tartrate, L- and D-serine and 3-hydroxypropionate molecules are modeled into the active site and the substrate binding modes are estimated. The wide cavity at the substrate binding site is likely responsible for the high reactivity of the enzyme toward both L- and D-serine enantiomers. Analysis of the substrate binding mechanism of L-SerDH, and detailed enzyme structure analysis, overview | Pyrobaculum calidifontis |
physiological function | L-serine 3-dehydrogenase acts at the beta-carbon (C3) position of L-serine. The product of this reaction is supposed to be 2-aminomalonate semialdehyde, which nonenzymatically decomposes into 2-aminoacetaldehyde and CO2 | Pyrobaculum calidifontis |