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Literature summary extracted from

  • Astegno, A.; Maresi, E.; Bertoldi, M.; La Verde, V.; Paiardini, A.; Dominici, P.
    Unique substrate specificity of ornithine aminotransferase from Toxoplasma gondii (2017), Biochem. J., 474, 939-955 .
    View publication on PubMed

Application

EC Number Application Comment Organism
2.6.1.13 drug development because TgOAT possesses parasite-specific structural features as well as differing substrate specificity from its human homologue, it is an attractive target for anti-toxoplasmosis inhibitor design that can be exploited for chemotherapeutic intervention Toxoplasma gondii

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.6.1.13 recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) Codon Plus Toxoplasma gondii

Protein Variants

EC Number Protein Variants Comment Organism
2.6.1.13 additional information generation of an N-terminally truncated TgOAT variant lacking the first 16 residues Toxoplasma gondii
2.6.1.13 V79Y site-directed mutagenesis, the mutation in analogy to the human enzyme causes an inversion of the substrate preference with L-ornithine becoming the preferred substrate of the V79Y variant. The V79Y mutation leads to a 6fold decrease in kcat for 2-oxoglutarate which is countered by a 6fold decrease in Km, such that kcat/Km is reduced only 1.3fold Toxoplasma gondii

Inhibitors

EC Number Inhibitors Comment Organism Structure
2.6.1.13 2-oxoglutarate substrate inhibition Toxoplasma gondii
2.6.1.13 putrescine a non-competitive inhibitor Toxoplasma gondii
2.6.1.13 spermidine a non-competitive inhibitor Toxoplasma gondii

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
2.6.1.13 additional information
-
 additional information steady-state and pre-steady state Michaelis-Menten kinetics, rapid-scanning stopped-flow kinetics, and transamination half-reaction kinetic parameters, overview Toxoplasma gondii
2.6.1.13 0.056
-
 2-oxoglutarate pH 8.0, 37°C, recombinant mutant V79Y Toxoplasma gondii
2.6.1.13 0.31
-
 2-oxoglutarate pH 8.0, 37°C, recombinant wild-type enzyme Toxoplasma gondii
2.6.1.13 4.2
-
 D-ornithine pH 8.0, 37°C, recombinant wild-type enzyme Toxoplasma gondii
2.6.1.13 6.9
-
 L-ornithine pH 8.0, 37°C, recombinant wild-type enzyme Toxoplasma gondii
2.6.1.13 7.4
-
 acetylornithine pH 8.0, 37°C, recombinant wild-type enzyme Toxoplasma gondii
2.6.1.13 496
-
 acetylornithine pH 8.0, 37°C, recombinant mutant V79Y Toxoplasma gondii

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
2.6.1.13 77000
-
 recombinant N-terminal deletion variant of TgOAT, gel filtration Toxoplasma gondii

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2.6.1.13 L-ornithine + 2-oxoglutarate Toxoplasma gondii
-
 L-glutamate 5-semialdehyde + L-glutamate
-
 r
2.6.1.13 L-ornithine + 2-oxoglutarate Toxoplasma gondii ATCC 50611
-
 L-glutamate 5-semialdehyde + L-glutamate
-
 r
2.6.1.13 L-ornithine + 2-oxoglutarate Toxoplasma gondii ME49
-
 L-glutamate 5-semialdehyde + L-glutamate
-
 r
2.6.1.13 additional information Toxoplasma gondii the enzyme from Toxoplasma gondii does not show a specific ornithine aminotransferase activity like its human homologue, but exhibits both N-acetylornithine and gamma-aminobutyric acid (GABA) transaminase activity (EC 2.6.1.19) in vitro, suggesting a role in both arginine and GABA metabolism in vivo ?
-
-
2.6.1.13 additional information Toxoplasma gondii ATCC 50611 the enzyme from Toxoplasma gondii does not show a specific ornithine aminotransferase activity like its human homologue, but exhibits both N-acetylornithine and gamma-aminobutyric acid (GABA) transaminase activity (EC 2.6.1.19) in vitro, suggesting a role in both arginine and GABA metabolism in vivo ?
-
-
2.6.1.13 additional information Toxoplasma gondii ME49 the enzyme from Toxoplasma gondii does not show a specific ornithine aminotransferase activity like its human homologue, but exhibits both N-acetylornithine and gamma-aminobutyric acid (GABA) transaminase activity (EC 2.6.1.19) in vitro, suggesting a role in both arginine and GABA metabolism in vivo ?
-
-

Organism

EC Number Organism UniProt Comment Textmining
2.6.1.13 Toxoplasma gondii S8EY38
-
-
2.6.1.13 Toxoplasma gondii ATCC 50611 S8EY38
-
-
2.6.1.13 Toxoplasma gondii ME49 S8EY38
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.6.1.13 recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) Codon Plus by nickel affinity chromatography and ultarfiltration Toxoplasma gondii

Reaction

EC Number Reaction Comment Organism Reaction ID
2.6.1.13 L-ornithine + a 2-oxo carboxylate = L-glutamate 5-semialdehyde + an L-amino acid reaction mechanism and kinetics, overview Toxoplasma gondii
a

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
2.6.1.13 2.6
-
 purified recombinant His-tagged wild-type enzyme, pH 8.0, 37°C Toxoplasma gondii

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.6.1.13 acetylornithine + 2-oxoglutarate
-
 Toxoplasma gondii ? + L-glutamate
-
 r
2.6.1.13 acetylornithine + 2-oxoglutarate
-
 Toxoplasma gondii ATCC 50611 ? + L-glutamate
-
 r
2.6.1.13 acetylornithine + 2-oxoglutarate
-
 Toxoplasma gondii ME49 ? + L-glutamate
-
 r
2.6.1.13 D-ornithine + 2-oxoglutarate
-
 Toxoplasma gondii D-glutamate 5-semialdehyde + L-glutamate
-
 r
2.6.1.13 D-ornithine + 2-oxoglutarate
-
 Toxoplasma gondii ATCC 50611 D-glutamate 5-semialdehyde + L-glutamate
-
 r
2.6.1.13 D-ornithine + 2-oxoglutarate
-
 Toxoplasma gondii ME49 D-glutamate 5-semialdehyde + L-glutamate
-
 r
2.6.1.13 L-ornithine + 2-oxoglutarate
-
 Toxoplasma gondii L-glutamate 5-semialdehyde + L-glutamate
-
 r
2.6.1.13 L-ornithine + 2-oxoglutarate
-
 Toxoplasma gondii ATCC 50611 L-glutamate 5-semialdehyde + L-glutamate
-
 r
2.6.1.13 L-ornithine + 2-oxoglutarate
-
 Toxoplasma gondii ME49 L-glutamate 5-semialdehyde + L-glutamate
-
 r
2.6.1.13 additional information the enzyme from Toxoplasma gondii does not show a specific ornithine aminotransferase activity like its human homologue, but exhibits both N-acetylornithine and gamma-aminobutyric acid (GABA) transaminase activity (EC 2.6.1.19) in vitro, suggesting a role in both arginine and GABA metabolism in vivo Toxoplasma gondii ?
-
-
2.6.1.13 additional information the external aldimine hydrolysis is rate-limiting, and not its formation. The enzyme TgOAT also shows lyase activity with beta-chloro-L-alanine (BCA) performin g a beta-elimination reaction Toxoplasma gondii ?
-
-
2.6.1.13 additional information the enzyme from Toxoplasma gondii does not show a specific ornithine aminotransferase activity like its human homologue, but exhibits both N-acetylornithine and gamma-aminobutyric acid (GABA) transaminase activity (EC 2.6.1.19) in vitro, suggesting a role in both arginine and GABA metabolism in vivo Toxoplasma gondii ATCC 50611 ?
-
-
2.6.1.13 additional information the external aldimine hydrolysis is rate-limiting, and not its formation. The enzyme TgOAT also shows lyase activity with beta-chloro-L-alanine (BCA) performin g a beta-elimination reaction Toxoplasma gondii ATCC 50611 ?
-
-
2.6.1.13 additional information the enzyme from Toxoplasma gondii does not show a specific ornithine aminotransferase activity like its human homologue, but exhibits both N-acetylornithine and gamma-aminobutyric acid (GABA) transaminase activity (EC 2.6.1.19) in vitro, suggesting a role in both arginine and GABA metabolism in vivo Toxoplasma gondii ME49 ?
-
-
2.6.1.13 additional information the external aldimine hydrolysis is rate-limiting, and not its formation. The enzyme TgOAT also shows lyase activity with beta-chloro-L-alanine (BCA) performin g a beta-elimination reaction Toxoplasma gondii ME49 ?
-
-

Subunits

EC Number Subunits Comment Organism
2.6.1.13 homodimer 2 * 48000, about, SDS-PAGE Toxoplasma gondii

Synonyms

EC Number Synonyms Comment Organism
2.6.1.13 OAT
-
 Toxoplasma gondii
2.6.1.13 ornithine aminotransferase
-
 Toxoplasma gondii
2.6.1.13 TgOAT
-
 Toxoplasma gondii

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
2.6.1.13 37
-
 assay at Toxoplasma gondii

Temperature Range [°C]

EC Number Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
2.6.1.13 15 75 temperature range, profile overview Toxoplasma gondii

Temperature Stability [°C]

EC Number Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
2.6.1.13 55
-
 purified recombinant enzyme, pH 8.0, 10 min, stable up to Toxoplasma gondii
2.6.1.13 65
-
 purified recombinant enzyme, pH 8.0, 10 min, loss of 50% activity Toxoplasma gondii
2.6.1.13 80
-
 purified recombinant enzyme, pH 8.0, 10 min, inactivation Toxoplasma gondii

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
2.6.1.13 0.41
-
 2-oxoglutarate pH 8.0, 37°C, recombinant mutant V79Y Toxoplasma gondii
2.6.1.13 0.42
-
 D-ornithine pH 8.0, 37°C, recombinant wild-type enzyme Toxoplasma gondii
2.6.1.13 2.1
-
 L-ornithine pH 8.0, 37°C, recombinant wild-type enzyme Toxoplasma gondii
2.6.1.13 2.5
-
 acetylornithine pH 8.0, 37°C, recombinant mutant V79Y Toxoplasma gondii
2.6.1.13 2.51
-
 2-oxoglutarate pH 8.0, 37°C, recombinant wild-type enzyme Toxoplasma gondii
2.6.1.13 4.6
-
 acetylornithine pH 8.0, 37°C, recombinant wild-type enzyme Toxoplasma gondii

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
2.6.1.13 8
-
 assay at Toxoplasma gondii

Cofactor

EC Number Cofactor Comment Organism Structure
2.6.1.13 pyridoxal 5'-phosphate PLP, dependent on Toxoplasma gondii

Ki Value [mM]

EC Number Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
2.6.1.13 17
-
 2-oxoglutarate pH 8.0, 37°C, recombinant wild-type enzyme Toxoplasma gondii
2.6.1.13 20
-
 2-oxoglutarate pH 8.0, 37°C, recombinant mutant V79Y Toxoplasma gondii
2.6.1.13 24
-
 spermidine pH 8.0, 37°C, recombinant wild-type enzyme Toxoplasma gondii
2.6.1.13 93
-
 putrescine pH 8.0, 37°C, recombinant wild-type enzyme Toxoplasma gondii

General Information

EC Number General Information Comment Organism
2.6.1.13 malfunction mutation of Val79 to Tyr results in a change of substrate preference between GABA, N-acetylornithine and L-ornithine, suggesting a key role of Val79 in defining substrate specificity Toxoplasma gondii
2.6.1.13 additional information the presence of Val79 in the active site of TgOAT in place of Tyr, as in its human counterpart, provides the necessary room to accommodate N-acetylornithine and GABA, resembling the active site arrangement of GABA transaminases. Contribution of active site Val79 to the specificity of the TgOAT enzym. The crystal structure of TgOAT in its internal aldimine form (PDB ID 4ZLV) is used to generate a structrue model of the V79Y mutant enzyme form, molecular modelling Toxoplasma gondii
2.6.1.13 physiological function the enzyme from Toxoplasma gondii does not show a specific ornithine aminotransferase activity like its human homologue, but exhibits both N-acetylornithine and ?-aminobutyric acid (GABA) transaminase activity in vitro, suggesting a role in both arginine and GABA metabolism in vivo Toxoplasma gondii

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
2.6.1.13 0.005
-
 acetylornithine pH 8.0, 37°C, recombinant mutant V79Y Toxoplasma gondii
2.6.1.13 0.1
-
 D-ornithine pH 8.0, 37°C, recombinant wild-type enzyme Toxoplasma gondii
2.6.1.13 0.304
-
 L-ornithine pH 8.0, 37°C, recombinant wild-type enzyme Toxoplasma gondii
2.6.1.13 0.62
-
 acetylornithine pH 8.0, 37°C, recombinant wild-type enzyme Toxoplasma gondii
2.6.1.13 7.321
-
 2-oxoglutarate pH 8.0, 37°C, recombinant mutant V79Y Toxoplasma gondii
2.6.1.13 8.097
-
 2-oxoglutarate pH 8.0, 37°C, recombinant wild-type enzyme Toxoplasma gondii