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Literature summary for 2.6.1.13 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

Application Comment Organism
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)

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

Protein Variants

Protein Variants Comment Organism
additional information generation of an N-terminally truncated TgOAT variant lacking the first 16 residues Toxoplasma gondii
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

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

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
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
0.056
-
2-oxoglutarate pH 8.0, 37°C, recombinant mutant V79Y Toxoplasma gondii
0.31
-
2-oxoglutarate pH 8.0, 37°C, recombinant wild-type enzyme Toxoplasma gondii
4.2
-
D-ornithine pH 8.0, 37°C, recombinant wild-type enzyme Toxoplasma gondii
6.9
-
L-ornithine pH 8.0, 37°C, recombinant wild-type enzyme Toxoplasma gondii
7.4
-
acetylornithine pH 8.0, 37°C, recombinant wild-type enzyme Toxoplasma gondii
496
-
acetylornithine pH 8.0, 37°C, recombinant mutant V79Y Toxoplasma gondii

Molecular Weight [Da]

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

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
L-ornithine + 2-oxoglutarate Toxoplasma gondii
-
L-glutamate 5-semialdehyde + L-glutamate
-
r
L-ornithine + 2-oxoglutarate Toxoplasma gondii ATCC 50611
-
L-glutamate 5-semialdehyde + L-glutamate
-
r
L-ornithine + 2-oxoglutarate Toxoplasma gondii ME49
-
L-glutamate 5-semialdehyde + L-glutamate
-
r
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 ?
-
-
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 ?
-
-
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

Organism UniProt Comment Textmining
Toxoplasma gondii S8EY38
-
-
Toxoplasma gondii ATCC 50611 S8EY38
-
-
Toxoplasma gondii ME49 S8EY38
-
-

Purification (Commentary)

Purification (Comment) Organism
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

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

Specific Activity [micromol/min/mg]

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

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
acetylornithine + 2-oxoglutarate
-
Toxoplasma gondii ? + L-glutamate
-
r
acetylornithine + 2-oxoglutarate
-
Toxoplasma gondii ATCC 50611 ? + L-glutamate
-
r
acetylornithine + 2-oxoglutarate
-
Toxoplasma gondii ME49 ? + L-glutamate
-
r
D-ornithine + 2-oxoglutarate
-
Toxoplasma gondii D-glutamate 5-semialdehyde + L-glutamate
-
r
D-ornithine + 2-oxoglutarate
-
Toxoplasma gondii ATCC 50611 D-glutamate 5-semialdehyde + L-glutamate
-
r
D-ornithine + 2-oxoglutarate
-
Toxoplasma gondii ME49 D-glutamate 5-semialdehyde + L-glutamate
-
r
L-ornithine + 2-oxoglutarate
-
Toxoplasma gondii L-glutamate 5-semialdehyde + L-glutamate
-
r
L-ornithine + 2-oxoglutarate
-
Toxoplasma gondii ATCC 50611 L-glutamate 5-semialdehyde + L-glutamate
-
r
L-ornithine + 2-oxoglutarate
-
Toxoplasma gondii ME49 L-glutamate 5-semialdehyde + L-glutamate
-
r
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 ?
-
-
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 ?
-
-
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 ?
-
-
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 ?
-
-
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 ?
-
-
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

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

Synonyms

Synonyms Comment Organism
OAT
-
Toxoplasma gondii
ornithine aminotransferase
-
Toxoplasma gondii
TgOAT
-
Toxoplasma gondii

Temperature Optimum [°C]

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

Temperature Range [°C]

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

Temperature Stability [°C]

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

Turnover Number [1/s]

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

pH Optimum

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

Cofactor

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

Ki Value [mM]

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

General Information

General Information Comment Organism
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
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
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]

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