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Literature summary for 2.6.1.52 extracted from

  • Singh, R.K.; Tomar, P.; Dharavath, S.; Kumar, S.; Gourinath, S.
    N-terminal residues are crucial for quaternary structure and active site conformation for the phosphoserine aminotransferase from enteric human parasite E. histolytica (2019), Int. J. Biol. Macromol., 132, 1012-1023 .
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
gene EhPSAT, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) Entamoeba histolytica

Crystallization (Commentary)

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

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

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 [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/ Products (Substrates)

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

Organism UniProt Comment Textmining
Entamoeba histolytica Q60I38
-
-
Entamoeba histolytica HM1:IMSS Q60I38
-
-

Purification (Commentary)

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 and Products (Substrate)

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

Synonyms Comment Organism
EhPSAT
-
Entamoeba histolytica
phosphoserine aminotransferase
-
Entamoeba histolytica
PSAT
-
Entamoeba histolytica

Turnover Number [1/s]

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

pH Optimum Minimum pH Optimum Maximum Comment Organism
8.5
-
-
Entamoeba histolytica

Cofactor

Cofactor Comment Organism Structure
pyridoxal 5'-phosphate PLP Entamoeba histolytica

General Information

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 [mM/s]

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