Cloned (Comment) | Organism |
---|---|
gene EhPSAT, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) | Entamoeba histolytica |
Crystallization (Comment) | Organism |
---|---|
purified wild-type enzyme and enzyme mutants EhPSAT_DELTA45 and EhPSAT_DELTA4 in complex with cofactor pyridoxal-5'phosphate, hanging drop vapour diffusion method, mixing of protein solution with precipitant solution containing 25-30% PEG 3350 w/v, 100 mM Tris, pH 6.5, 200 mM sodium formate, and 5% glycerol v/v for the wild-type complex, and 25% PEG 2000 MME, and 100 mM potassium thiocyanate for mutant EhPSAT_DELTA45 , and 20% PEG 2000 MME, 100 mM Tris, pH 8.5, and 200 mM TMAO for mutant EhPSAT_DELTA4, all at 16°C and at room temperature, X-ray diffraction structure determination and analysis at 3.0, 1.8, and 2.4 A resolution, respectively, modeling | Entamoeba histolytica |
Protein Variants | Comment | Organism |
---|---|---|
additional information | deletion of the 45 N-terminal residues in mutant EhPSAT_DELTA45 results in an inactive protein, the structure shows a dimeric arrangement drastically different from that of the wild-type protein, with the two monomers translated and rotated by almost 180° with respect to each other, causing a rearrangement of the active site to which cofactor PLP is unable to bind. Deletions of first N-terminal 15 (EhPSAT_DELTA15) and four 11th to 14th residues (EhPSAT_DELTA4) yield up to 98% and 90% decrease in activity, respectively. Absence of aldimine linkage between PLP-Lys in the crystal structure of EhPSAT_DELTA4 mutant explains the decrease in activity and describes the importance of these N-terminal residues | Entamoeba histolytica |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
chloride | the chloride ion in EhPSAT becomes trapped in the space between residues Gly99, Val 100, Ile 149, Thr 148, and Asn 147. The halide-binding site in close proximity to the active site is detected. A stretch of six amino acids (146-NNTIYG-151) only conserved in the Entamoeba genus, contributes to halide binding. Structure comparisons, overview | Entamoeba histolytica |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | Michaelis-Menten kinetics | Entamoeba histolytica | |
0.0118 | - |
O-phospho-L-serine | pH 8.5, temperature not specified in the publication, recombinant wild-type enzyme | Entamoeba histolytica | |
0.0274 | - |
O-phospho-L-serine | pH 8.5, temperature not specified in the publication, recombinant mutant EhPSAT_DELTA4 | Entamoeba histolytica | |
0.0712 | - |
O-phospho-L-serine | pH 8.5, temperature not specified in the publication, recombinant mutant EhPSAT_DELTA15 | Entamoeba histolytica |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
O-phospho-L-serine + 2-oxoglutarate | Entamoeba histolytica | - |
3-phosphooxypyruvate + L-glutamate | - |
r | |
O-phospho-L-serine + 2-oxoglutarate | Entamoeba histolytica HM1:IMSS | - |
3-phosphooxypyruvate + L-glutamate | - |
r |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Entamoeba histolytica | Q60I38 | - |
- |
Entamoeba histolytica HM1:IMSS | Q60I38 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and gel filtration. The N-terminal 45 amino acid residues of wild-type EhPSAT protein can be truncated either at the time of purification or in vivo | Entamoeba histolytica |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
O-phospho-L-serine + 2-oxoglutarate | - |
Entamoeba histolytica | 3-phosphooxypyruvate + L-glutamate | - |
r | |
O-phospho-L-serine + 2-oxoglutarate | - |
Entamoeba histolytica HM1:IMSS | 3-phosphooxypyruvate + L-glutamate | - |
r |
Synonyms | Comment | Organism |
---|---|---|
EhPSAT | - |
Entamoeba histolytica |
phosphoserine aminotransferase | - |
Entamoeba histolytica |
PSAT | - |
Entamoeba histolytica |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.012 | - |
O-phospho-L-serine | pH 8.5, temperature not specified in the publication, recombinant mutant EhPSAT_DELTA15 | Entamoeba histolytica | |
0.054 | - |
O-phospho-L-serine | pH 8.5, temperature not specified in the publication, recombinant mutant EhPSAT_DELTA4 | Entamoeba histolytica | |
0.625 | - |
O-phospho-L-serine | pH 8.5, temperature not specified in the publication, recombinant wild-type enzyme | Entamoeba histolytica |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8.5 | - |
- |
Entamoeba histolytica |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
pyridoxal 5'-phosphate | PLP | Entamoeba histolytica |
General Information | Comment | Organism |
---|---|---|
malfunction | deletion of 45 N-terminal residues (EhPSAT_DELTA45) results in an inactive protein, the structure shows a dimeric arrangement drastically different from that of the wild-type protein | Entamoeba histolytica |
metabolism | L-serine is involved in several important metabolic pathways in the protozoan parasite Entamoeba histolytica. Phosphoserine aminotransferase (PSAT) is a pyridoxal-5'phosphate (PLP)-dependent enzyme that catalyzes the second reversible step in the phosphoserine biosynthetic pathway producing L-serine | Entamoeba histolytica |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.17 | - |
O-phospho-L-serine | pH 8.5, temperature not specified in the publication, recombinant mutant EhPSAT_DELTA15 | Entamoeba histolytica | |
1.97 | - |
O-phospho-L-serine | pH 8.5, temperature not specified in the publication, recombinant mutant EhPSAT_DELTA4 | Entamoeba histolytica | |
53 | - |
O-phospho-L-serine | pH 8.5, temperature not specified in the publication, recombinant wild-type enzyme | Entamoeba histolytica |