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Literature summary for 2.6.1.78 extracted from

  • Holland, C.K.; Berkovich, D.A.; Kohn, M.L.; Maeda, H.; Jez, J.M.
    Structural basis for substrate recognition and inhibition of prephenate aminotransferase from Arabidopsis (2018), Plant J., 94, 304-314 .
    View publication on PubMed
Search for more literature for 2.6.1.78

Crystallization (Commentary)

Crystallization (Comment) Organism
AtPAT wild-type enzyme and K306A, T84V, and T84V/K169V mutant enzymes in complex with either 2-oxoglutarate or pyridoxamine 5'-phosphate and glutamate, X-ray diffraction structure determination and analysis at 1.4-3.0 A resolution, molecular replacement Arabidopsis thaliana

Protein Variants

Protein Variants Comment Organism
A168G site-directed mutagenesis, altered substrate binding kinetics compared to wild-type Arabidopsis thaliana
E108K site-directed mutagenesis, altered substrate binding kinetics compared to wild-type Arabidopsis thaliana
K169S site-directed mutagenesis, inactive with aspartate and glutamate Arabidopsis thaliana
K169V site-directed mutagenesis, inactive with glutamate, altered substrate binding kinetics compared to wild-type Arabidopsis thaliana
K306A site-directed mutagenesis, structure comparison with wild-type, overview. The alanine substitution of Lys306 prevents Schiff base formation with the cofactor, inactive mutant Arabidopsis thaliana
R445G site-directed mutagenesis, inactive mutant Arabidopsis thaliana
T84V site-directed mutagenesis, altered substrate binding kinetics compared to wild-type Arabidopsis thaliana
T84V/K169V site-directed mutagenesis, altered substrate binding kinetics compared to wild-type Arabidopsis thaliana

Inhibitors

Inhibitors Comment Organism Structure
cysteine docking study with wild-type and mutant enzymes Arabidopsis thaliana

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
 additional information steady-state kinetics analysis Arabidopsis thaliana
0.17
-
 prephenate pH 7.5, 25°C, recombinant wild-type enzyme Arabidopsis thaliana
0.48
-
 prephenate pH 7.5, 25°C, recombinant mutant A168G Arabidopsis thaliana
0.55
-
 prephenate pH 7.5, 25°C, recombinant mutant T84V Arabidopsis thaliana
2
-
 prephenate pH 7.5, 25°C, recombinant mutant E108K Arabidopsis thaliana

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
L-arogenate + oxaloacetate Arabidopsis thaliana
-
 L-aspartate + prephenate
-
 r

Organism

Organism UniProt Comment Textmining
Arabidopsis thaliana Q9SIE1
-
-

Reaction

Reaction Comment Organism Reaction ID
L-arogenate + oxaloacetate = prephenate + L-aspartate catalytic reaction mechanism, overview Arabidopsis thaliana
a

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
L-arogenate + oxaloacetate
-
 Arabidopsis thaliana L-aspartate + prephenate
-
 r
L-aspartate + prephenate
-
 Arabidopsis thaliana L-arogenate + oxaloacetate
-
 r

Subunits

Subunits Comment Organism
homodimer AtPAT crystallizes as a homodimer. Each monomer of AtPAT consists of 15 alpha-helices and 9 beta-strands divided between two structural domains. The N-terminal domain (Lys115-Leu353) contains two sets of three a-helices surrounding six parallel and one anti-parallel beta-strand. Two additional alpha-helices in the PLP-binding pocket, as well as the a-helix connecting the N- and C-terminal domains, complete the N-terminal domain. The smaller C-terminal domain contains part of the N-terminal region (Ser71-Pro114) and residues Gly354 through Leu469, totaling five alpha-helices and two beta-strands. The N-terminal flexible loop (Ser71-Ser82) and features of the N-terminal domain form the dimer interface Arabidopsis thaliana

Synonyms

Synonyms Comment Organism
AtPAT
-
 Arabidopsis thaliana
bifunctional aspartate aminotransferase and glutamate/aspartate-prephenate aminotransferase UniProt Arabidopsis thaliana
class Ibeta AAT
-
 Arabidopsis thaliana
More cf. EC 2.6.1.79 and EC 2.6.1.1 Arabidopsis thaliana
Pat
-
 Arabidopsis thaliana
prephenate aminotransferase
-
 Arabidopsis thaliana

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25
-
 assay at Arabidopsis thaliana

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.18
-
 prephenate pH 7.5, 25°C, recombinant mutant T84V Arabidopsis thaliana
2.67
-
 prephenate pH 7.5, 25°C, recombinant mutant E108K Arabidopsis thaliana
3.83
-
 prephenate pH 7.5, 25°C, recombinant wild-type enzyme Arabidopsis thaliana
6.67
-
 prephenate pH 7.5, 25°C, recombinant mutant A168G Arabidopsis thaliana

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5
-
 assay at Arabidopsis thaliana

Cofactor

Cofactor Comment Organism Structure
pyridoxal 5'-phosphate PLP, dependent on, PLP is bound in the active site of each chain in the wild-type structure. In the AtPAT crystal structure, PLP is covalently linked to the epsilon-nitrogen of Lys306 to form the internal aldimine (i.e. Schiff base). Trp193 and Ile274 position the ring of PLP through pi-pi stacking and van der Waals interactions, respectively. The pyridine ring nitrogen of PLP forms a charge-charge interaction with the side chain of Asp272 Arabidopsis thaliana

IC50 Value

IC50 Value IC50 Value Maximum Comment Organism Inhibitor Structure
0.56
-
 versus 2-oxoglutarate, pH 7.5, 25°C, recombinant wild-type enzyme Arabidopsis thaliana cysteine
0.75
-
 versus prephenate, pH 7.5, 25°C, recombinant wild-type enzyme Arabidopsis thaliana cysteine

General Information

General Information Comment Organism
additional information key residues, such as Glu108, are involved in both keto acid and amino acid substrate specificities and probably contribute to the evolution of PAT activity among class Ibeta AAT enzymes, ligand binding study, molecular mechanisms underlying recognition of keto acid and amino acid substrates, overview. Lys306 is the catalytic Lys in AtPAT Arabidopsis thaliana

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.34
-
 prephenate pH 7.5, 25°C, recombinant mutant E108K Arabidopsis thaliana
2.15
-
 prephenate pH 7.5, 25°C, recombinant mutant T84V Arabidopsis thaliana
13.9
-
 prephenate pH 7.5, 25°C, recombinant mutant A168G Arabidopsis thaliana
22.53
-
 prephenate pH 7.5, 25°C, recombinant wild-type enzyme Arabidopsis thaliana