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Literature summary extracted from

  • Zheng, X.; Cui, Y.; Li, T.; Li, R.; Guo, L.; Li, D.; Wu, B.
    Biochemical and structural characterization of a highly active branched-chain amino acid aminotransferase from Pseudomonas sp. for efficient biosynthesis of chiral amino acids (2019), Appl. Microbiol. Biotechnol., 103, 8051-8062 .
    View publication on PubMed

Application

EC Number Application Comment Organism
2.6.1.42 synthesis usage of PsBCAT in a coupled reaction with Bacillus subtilis ornithine aminotransferase (BsOrnAT), applied to synthesize L-tert-leucine and L-norvaline. The reaction mixture contains 0.5 M Tris-HCl buffer, pH 8.5, 0.1 M trimethylpyruvate or 2-oxovalerate, 20 mM sodium glutamate, 80 mM L-ornithine monohydrochloride, 0.02 mM PLP, 0.2 mg PsBCAT, and 0.05 mg BsOrnAT enzyme in a total volume of 5 ml at 30°C Pseudomonas sp.

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.6.1.42 a synthetic PsBCAT gene is cloned and heterologously expressed in Escherichia coli strain BL21(DE3) as His-tagged fusion protein Pseudomonas sp.

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
2.6.1.42 purified recombinant enzyme in complex with PLP, hanging drop vapor diffusion method, mixing of equal volumes of 12 mg/ml protein solution with reservoir solution containing 0.25 M ammonium citrate dibasic and 20% w/v PEG 3350, at 16°C, X-ray diffraction structure determination and analysis at 1.70 A resolution Pseudomonas sp.

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
2.6.1.42 additional information
-
 additional information steady-state kinetic analysis, recombinant enzyme Pseudomonas sp.

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2.6.1.42 L-isoleucine + 2-oxoglutarate Pseudomonas sp.
-
 3-methyl-2-oxopentanoate + L-glutamate
-
 r
2.6.1.42 L-leucine + 2-oxoglutarate Pseudomonas sp.
-
 4-methyl-2-oxopentanoate + L-glutamate
-
 r
2.6.1.42 L-valine + 2-oxoglutarate Pseudomonas sp.
-
 3-methyl-2-oxobutanoate + L-glutamate
-
 r

Organism

EC Number Organism UniProt Comment Textmining
2.6.1.42 Pseudomonas sp.
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.6.1.42 recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chrormatography Pseudomonas sp.

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.6.1.42 L-alanine + 2-oxoglutarate
-
 Pseudomonas sp. pyruvate + L-glutamate
-
 r
2.6.1.42 L-allo-isoleucine + 2-oxoglutarate
-
 Pseudomonas sp. ? + L-glutamate
-
 r
2.6.1.42 L-isoleucine + 2-oxoglutarate
-
 Pseudomonas sp. 3-methyl-2-oxopentanoate + L-glutamate
-
 r
2.6.1.42 L-leucine + 2-oxoglutarate
-
 Pseudomonas sp. 4-methyl-2-oxopentanoate + L-glutamate
-
 r
2.6.1.42 L-norvaline + 2-oxoglutarate
-
 Pseudomonas sp. ? + L-glutamate
-
 r
2.6.1.42 L-phenylalanine + 2-oxoglutarate
-
 Pseudomonas sp. phenylpyruvate + L-glutamate
-
 r
2.6.1.42 L-phenylglycine + 2-oxoglutarate
-
 Pseudomonas sp. ? + L-glutamate
-
 r
2.6.1.42 L-tert-leucine + 2-oxoglutarate
-
 Pseudomonas sp. ? + L-glutamate
-
 r
2.6.1.42 L-valine + 2-oxoglutarate
-
 Pseudomonas sp. 3-methyl-2-oxobutanoate + L-glutamate
-
 r
2.6.1.42 additional information evaluation of substrate specificity of PsBCAT. The enzyme is able to catalyze transamination of 19 amino acids, and all 2-oxo acids are capable of serving as amino acceptors. With 2-oxoglutarate as amino acceptor, PsBCAT exhibits maximum activity toward L-allo-isoleucine followed by L-phenylglycine, L-leucine, L-norvaline, L-valine, and L-isoleucine. Despite the considerably lower activity, PsBCAT also shows activities toward L-phenylalanine, L-alanine, and L-tert-leucine, but it is inert to L-tryptophan, L-tyrosine, Lthreonine, L-histidine, and L-lysine. PsBCAT has a broader substrate specificity and prefers to catalyze the transamination of bulked aliphatic amino acids. PsBCAT exhibits no activity with amines in propylamine, aniline, and phenylethylamine, which indicates the carboxyl group is essential for binding in the active site, docking study and analysis of ligand binding structures Pseudomonas sp. ?
-
-

Synonyms

EC Number Synonyms Comment Organism
2.6.1.42 BcaT
-
 Pseudomonas sp.
2.6.1.42 branched-chain amino acid aminotransferase
-
 Pseudomonas sp.
2.6.1.42 PsBCAT
-
 Pseudomonas sp.

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
2.6.1.42 40
-
-
 Pseudomonas sp.

Temperature Range [°C]

EC Number Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
2.6.1.42 22 50 the PsBCAT activity decreases very quickly to approximately 30% at 50°C, maximal activity at about 40°C, active at room temperature (about 22°C) Pseudomonas sp.

Temperature Stability [°C]

EC Number Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
2.6.1.42 40
-
 the apparent melting temperature of the enzyme is 40°C determined by the method of fluorescence-based thermal stability assay Pseudomonas sp.

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
2.6.1.42 8.5
-
 recombinant enzyme Pseudomonas sp.

pH Range

EC Number pH Minimum pH Maximum Comment Organism
2.6.1.42 7.5 10 the highest activity of PsBCAT for L-valine is observed at pH 8.5 in 50 mM Tris-HCl buffer, while it decreases significantly with pH values less than pH 7.5 or greater than pH 10.0 with only approximately 10% activity or even less Pseudomonas sp.

Cofactor

EC Number Cofactor Comment Organism Structure
2.6.1.42 pyridoxal 5'-phosphate PLP, dependent on, activates, Km is 0.0034 mM, no inhibition at higher concentrations Pseudomonas sp.

General Information

EC Number General Information Comment Organism
2.6.1.42 additional information structure-function analysis of pyridoxal 5'-phosphate (PLP)-bound enzyme and structure comparisons, overview. L-Glutamate and L-tert-leucine are docked into the active site of PsBCAT Pseudomonas sp.