Application | Comment | Organism |
---|---|---|
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 (Comment) | Organism |
---|---|
a synthetic PsBCAT gene is cloned and heterologously expressed in Escherichia coli strain BL21(DE3) as His-tagged fusion protein | Pseudomonas sp. |
Crystallization (Comment) | Organism |
---|---|
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] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | steady-state kinetic analysis, recombinant enzyme | Pseudomonas sp. |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
L-isoleucine + 2-oxoglutarate | Pseudomonas sp. | - |
3-methyl-2-oxopentanoate + L-glutamate | - |
r | |
L-leucine + 2-oxoglutarate | Pseudomonas sp. | - |
4-methyl-2-oxopentanoate + L-glutamate | - |
r | |
L-valine + 2-oxoglutarate | Pseudomonas sp. | - |
3-methyl-2-oxobutanoate + L-glutamate | - |
r |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Pseudomonas sp. | - |
- |
- |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chrormatography | Pseudomonas sp. |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
L-alanine + 2-oxoglutarate | - |
Pseudomonas sp. | pyruvate + L-glutamate | - |
r | |
L-allo-isoleucine + 2-oxoglutarate | - |
Pseudomonas sp. | ? + L-glutamate | - |
r | |
L-isoleucine + 2-oxoglutarate | - |
Pseudomonas sp. | 3-methyl-2-oxopentanoate + L-glutamate | - |
r | |
L-leucine + 2-oxoglutarate | - |
Pseudomonas sp. | 4-methyl-2-oxopentanoate + L-glutamate | - |
r | |
L-norvaline + 2-oxoglutarate | - |
Pseudomonas sp. | ? + L-glutamate | - |
r | |
L-phenylalanine + 2-oxoglutarate | - |
Pseudomonas sp. | phenylpyruvate + L-glutamate | - |
r | |
L-phenylglycine + 2-oxoglutarate | - |
Pseudomonas sp. | ? + L-glutamate | - |
r | |
L-tert-leucine + 2-oxoglutarate | - |
Pseudomonas sp. | ? + L-glutamate | - |
r | |
L-valine + 2-oxoglutarate | - |
Pseudomonas sp. | 3-methyl-2-oxobutanoate + L-glutamate | - |
r | |
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 | Comment | Organism |
---|---|---|
BcaT | - |
Pseudomonas sp. |
branched-chain amino acid aminotransferase | - |
Pseudomonas sp. |
PsBCAT | - |
Pseudomonas sp. |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
40 | - |
- |
Pseudomonas sp. |
Temperature Minimum [°C] | Temperature Maximum [°C] | Comment | Organism |
---|---|---|---|
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 Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
---|---|---|---|
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 Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8.5 | - |
recombinant enzyme | Pseudomonas sp. |
pH Minimum | pH Maximum | Comment | Organism |
---|---|---|---|
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 | Comment | Organism | Structure |
---|---|---|---|
pyridoxal 5'-phosphate | PLP, dependent on, activates, Km is 0.0034 mM, no inhibition at higher concentrations | Pseudomonas sp. |
General Information | Comment | Organism |
---|---|---|
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. |