Application | Comment | Organism |
---|---|---|
synthesis | pyridoxal-5'-phosphate (PLP)-dependent transaminases are industrially important enzymes catalyzing the stereoselective amination of ketones and keto acids. Transaminases of PLP fold type IV are characterized by (R)- or (S)-stereoselective transfer of amino groups, depending on the substrate profile of the enzyme | Haliangium ochraceum |
Cloned (Comment) | Organism |
---|---|
recombinant expression of His-tagged enzyme in Escherichia coli strain Rosetta (DE3)pLysS | Haliangium ochraceum |
Crystallization (Comment) | Organism |
---|---|
purified native enzyme, hanging drop vapor diffusion technique, 20°C, mixing of 10 mg/ml protein in 15 mM Tris, pH 8.0, 50 mM NaCl, and 0.02 mM PLP, with reservoir solution containing 0.1 M MES, pH 6.0, 0.2 M CaCl2, and 50% methyl-2,4-pentanediol, method screening and optimization, X-ray diffraction structure determination and analysis at 2.35 A resolution, molecular replacement method using the structure of the branched-chain amino acid aminotransferase from Burkholderia pseudomallei (PDB ID 3U0G) as a template, modeling | Haliangium ochraceum |
purified recombinant detagged enzyme, hanging drop vapor diffusion method, mixing of 10 mg/ml protein solution with reservoir solution containing 0.1 M MES, pH 6.0, 0.2 M CaCl2, and 50% methyl-2,4-pentanediol, 20°C, method optimization, X-ray diffraction structure determination and analysis at 2.35 A resolution, molecular replacement method using the structure of the branched-chain amino acid aminotransferase from Burkholderia pseudomallei (PDB ID 3U0G) as a template, modelling | Haliangium ochraceum |
Protein Variants | Comment | Organism |
---|---|---|
H106Y/Y108R | site-directed mutagenesis | Haliangium ochraceum |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
acetonitril | over 90% inhibition at 20-30%; strong inhibition at 20-30% | Haliangium ochraceum | |
DMSO | 40% inhibition at 20%, 70% inhibition at 30%; slight to moderate inhibition at 20-30% | Haliangium ochraceum | |
methanol | 65% inhibition at 20%, 85% inhibition at 30%; moderate inhibition at 20-30% | Haliangium ochraceum |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | steady-state kinetics | Haliangium ochraceum | |
additional information | - |
additional information | Michaelis-Menten steady-state kinetics of the first half-reaction | Haliangium ochraceum | |
0.55 | - |
3-methyl-2-oxovalerate | pH 10.0, 40°C | Haliangium ochraceum | |
0.55 | - |
3-methyl-2-oxovalerate | recombinant enzyme, pH 10.0, 40°C, with (R)-alpha-methylbenzylamine | Haliangium ochraceum | |
2 | 3 | (R)-alpha-methylbenzylamine | pH 10.0, 40°C, with 2-oxoglutarate | Haliangium ochraceum | |
6 | - |
(R)-alpha-methylbenzylamine | pH 10.0, 40°C, with 5 mM 3-methyl-2-oxovalerate | Haliangium ochraceum | |
6 | - |
(R)-alpha-methylbenzylamine | recombinant enzyme, pH 10.0, 40°C, with 3-methyl-2-oxovalerate | Haliangium ochraceum | |
10.4 | - |
(R)-alpha-ethylbenzylamine | pH 10.0, 40°C | Haliangium ochraceum | |
11.8 | - |
(R)-alpha-methylbenzylamine | pH 10.0, 40°C, with 1.5 mM 3-methyl-2-oxovalerate | Haliangium ochraceum | |
11.8 | - |
(R)-alpha-methylbenzylamine | recombinant enzyme, pH 9.0, 40°C, with 3-methyl-2-oxovalerate | Haliangium ochraceum |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
NaCl | the enzyme shows tolerance to high salt concentrations up to 2 M NaCl. Addition of up to 400 mM NaCl in the reaction mixture results in a slight increase in the activity of enzyme Hoch3033 | Haliangium ochraceum | |
NaCl | the enzyme shows tolerance to high salt concentrations, up to 2 M NaCl | Haliangium ochraceum |
Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
70000 | - |
gel filtration | Haliangium ochraceum |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Haliangium ochraceum | - |
- |
- |
Haliangium ochraceum | D0LR31 | - |
- |
Haliangium ochraceum DSM 14365 | D0LR31 | - |
- |
Haliangium ochraceum JCM 11303 | D0LR31 | - |
- |
Haliangium ochraceum SMP-2 | D0LR31 | - |
- |
Purification (Comment) | Organism |
---|---|
native enzyme to homogeneity | Haliangium ochraceum |
recombinant His-tagged enzyme from Escherichia coli strain Rosetta (DE3)pLysS by nickel affinity chromatgraphy, tag cleavage through TEV protease, another step of nickel affinity chromatgraphy, followed by gel filtration | Haliangium ochraceum |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
(R)-alpha-methylbenzylamine + 2-oxo acid = acetophenone + L-amino acid | first transaminase half-reaction via internal aldimine, external aldimine, ketimine, and pyridoxamine (PMP)-bound form | Haliangium ochraceum |
Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|
0.0042 | - |
purified enzyme, pH 10.0, 40°C, substrate 2-oxoglutarate | Haliangium ochraceum |
0.0042 | - |
purified recombinant enzyme, pH 10.0, 40°C, substrate (R)-alpha-methylbenzylamine and 2-oxoglutarate | Haliangium ochraceum |
0.007 | - |
purified enzyme, pH 10.0, 40°C, substrate pyruvate | Haliangium ochraceum |
0.007 | - |
purified recombinant enzyme, pH 10.0, 40°C, substrate (R)-alpha-methylbenzylamine and pyruvate | Haliangium ochraceum |
0.013 | - |
purified recombinant enzyme, pH 10.0, 40°C, substrate (R)-alpha-ethylbenzylamine and 3-methyl-2-oxovalerate | Haliangium ochraceum |
0.025 | - |
purified enzyme, pH 10.0, 40°C, substrate 2-oxobutyrate | Haliangium ochraceum |
0.025 | - |
purified recombinant enzyme, pH 10.0, 40°C, substrate (R)-alpha-methylbenzylamine and 2-oxobutyrate | Haliangium ochraceum |
0.12 | - |
purified enzyme, pH 10.0, 40°C, substrate 3-methyl-2-oxobutyrate | Haliangium ochraceum |
0.12 | - |
purified recombinant enzyme, pH 10.0, 40°C, substrate (R)-alpha-methylbenzylamine and 3-methyl-2-oxobutyrate | Haliangium ochraceum |
0.135 | - |
purified enzyme, pH 10.0, 40°C, substrate 2-oxohexanoate | Haliangium ochraceum |
0.135 | - |
purified recombinant enzyme, pH 10.0, 40°C, substrate (R)-alpha-methylbenzylamine and 2-oxohexanoate | Haliangium ochraceum |
0.15 | - |
purified enzyme, pH 10.0, 40°C, substrate 4-methyl-2-oxovalerate | Haliangium ochraceum |
0.15 | - |
purified recombinant enzyme, pH 10.0, 40°C, substrate (R)-alpha-methylbenzylamine and 4-methyl-2-oxovalerate | Haliangium ochraceum |
0.19 | - |
purified enzyme, pH 10.0, 40°C, substrate 3-methyl-2-oxovalerate | Haliangium ochraceum |
0.19 | - |
purified recombinant enzyme, pH 10.0, 40°C, substrate (R)-alpha-methylbenzylamine and 3-methyl-2-oxovalerate | Haliangium ochraceum |
0.23 | - |
purified enzyme, pH 10.0, 40°C, substrate 2-oxovalerate | Haliangium ochraceum |
0.23 | - |
purified recombinant enzyme, pH 10.0, 40°C, substrate (R)-alpha-methylbenzylamine and 2-oxovalerate | Haliangium ochraceum |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
(R)-alpha-ethylbenzylamine + 3-methyl-2-oxovalerate | reaction of (R)-amine:pyruvate transaminase, EC 2.6.1.B21 | Haliangium ochraceum | alpha-ethylbenzaldehyde + L-isoleucine | - |
r | |
(R)-alpha-ethylbenzylamine + pyruvate | R-EtBA | Haliangium ochraceum | alpha-ethylbenzaldehyde + D-alanine | - |
r | |
(R)-alpha-methylbenzylamine + 2-oxobutyrate | - |
Haliangium ochraceum | acetophenone + L-alpha-aminobutyric acid | - |
r | |
(R)-alpha-methylbenzylamine + 2-oxoglutarate | - |
Haliangium ochraceum | acetophenone + L-glutamate | - |
r | |
(R)-alpha-methylbenzylamine + 2-oxoglutarate | reaction of (R)-amine:pyruvate transaminase, EC 2.6.1.B21 | Haliangium ochraceum | acetophenone + L-glutamate | - |
r | |
(R)-alpha-methylbenzylamine + 2-oxohexanoate | - |
Haliangium ochraceum | acetophenone + L-norleucine | - |
r | |
(R)-alpha-methylbenzylamine + 2-oxovalerate | - |
Haliangium ochraceum | acetophenone + L-norvaline | - |
r | |
(R)-alpha-methylbenzylamine + 3-methyl-2-oxobutyrate | - |
Haliangium ochraceum | acetophenone + L-valine | - |
r | |
(R)-alpha-methylbenzylamine + 3-methyl-2-oxovalerate | - |
Haliangium ochraceum | acetophenone + D-isoleucine | - |
r | |
(R)-alpha-methylbenzylamine + 3-methyl-2-oxovalerate | - |
Haliangium ochraceum | acetophenone + L-isoleucine | - |
r | |
(R)-alpha-methylbenzylamine + 3-methyl-2-oxovalerate | reaction of (R)-amine:pyruvate transaminase, EC 2.6.1.B21 | Haliangium ochraceum | acetophenone + L-isoleucine | - |
r | |
(R)-alpha-methylbenzylamine + 4-methyl-2-oxovalerate | - |
Haliangium ochraceum | acetophenone + L-leucine | - |
r | |
(R)-alpha-methylbenzylamine + pyruvate | R-MBA, specific substrate for (R)-amine:pyruvate transaminases | Haliangium ochraceum | acetophenone + D-alanine | - |
r | |
2-oxohexanoate + L-glutamate | reaction of branched-chain amino acid transaminase, EC 2.6.1.42 | Haliangium ochraceum | norleucine + 2-oxoglutarate | - |
r | |
L-alanine + 2-oxoglutarate | reaction of branched-chain amino acid transaminase, EC 2.6.1.42 | Haliangium ochraceum | pyruvate + L-glutamate | - |
r | |
L-isoleucine + 2-oxoglutarate | reaction of branched-chain amino acid transaminase, EC 2.6.1.42 | Haliangium ochraceum | 3-methyl-2-oxopentanoate + L-glutamate | - |
r | |
L-leucine + 2-oxoglutarate | reaction of branched-chain amino acid transaminase, EC 2.6.1.42 | Haliangium ochraceum | 4-methyl-2-oxopentanoate + L-glutamate | - |
r | |
L-valine + 2-oxoglutarate | reaction of branched-chain amino acid transaminase, EC 2.6.1.42 | Haliangium ochraceum | 3-methyl-2-oxobutanoate + L-glutamate | - |
r | |
additional information | enzyme Hoch3033 is a transaminases with a n enzyme with mixed type of activity showing branched-chain amino acid amintransferase activity (EC 2.6.1.42) and (R)-amine:pyruvate transaminase (EC 2.6.1). The mixed type of activity of Hoch3033 is implemented within the BCAT-like active site, but in the active site of Hoch3033, substitutions of specificity-determining residues, that are important for substrate binding in canonical BCATs, are observed. In the standard assay, the specific activity towards (S)-alpha-methylbenzylamine (S-MBA) is only 0.32% of the activity towards R-MBA. A much lower value of activity is obtained with (R)-alpha-ethylbenzylamine (R-EtBA), the closest homologue of R-MBA. No activity towards S-EtBA is observed. Substrate specificity overview | Haliangium ochraceum | ? | - |
- |
|
additional information | the bifunctional transaminase from myxobacterium Haliangium ochraceum, encoded by gene Hoch3033, is active towards keto analogues of branched-chain amino acids (specific substrates for BCATs, EC 2.6.1.42) and (R)-alpha-methylbenzylamine (specific substrate for (R)-amine:pyruvate transaminases, EC 2.6.1.B21). The enzyme shows no activity with (S)-2-methylbenzylamine, 2-amino-5-methylhexane, 1-methyl-3-phenyl-propylamine, (R)-2-aminohexane, D-alanine, L-beta-leucine, and beta-alanine | Haliangium ochraceum | ? | - |
- |
|
norvaline + 2-oxoglutarate | reaction of branched-chain amino acid transaminase, EC 2.6.1.42 | Haliangium ochraceum | 2-oxovalerate + L-glutamate | - |
r |
Subunits | Comment | Organism |
---|---|---|
homodimer | - |
Haliangium ochraceum |
Synonyms | Comment | Organism |
---|---|---|
(R)-amine:pyruvate transaminase | - |
Haliangium ochraceum |
halotolerant R-TA | - |
Haliangium ochraceum |
Hoch3033 | - |
Haliangium ochraceum |
IlvE | - |
Haliangium ochraceum |
More | see also branched-chain amino acid transaminase, EC 2.6.1.42 | Haliangium ochraceum |
R-TA | - |
Haliangium ochraceum |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
40 | 45 | - |
Haliangium ochraceum |
45 | - |
- |
Haliangium ochraceum |
Temperature Minimum [°C] | Temperature Maximum [°C] | Comment | Organism |
---|---|---|---|
40 | 60 | maximal activity at 40-45°C, 40% of maximal activity at 60°C | Haliangium ochraceum |
Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
---|---|---|---|
60 | - |
the purified recombinant enzyme retains about 40% of the maximum activity at pH 11.0 | Haliangium ochraceum |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.0113 | - |
(R)-alpha-ethylbenzylamine | pH 10.0, 40°C, with 2-oxoglutarate | Haliangium ochraceum | |
0.15 | - |
3-methyl-2-oxovalerate | pH 10.0, 40°C | Haliangium ochraceum | |
0.15 | - |
3-methyl-2-oxovalerate | recombinant enzyme, pH 10.0, 40°C, with (R)-alpha-methylbenzylamine | Haliangium ochraceum | |
0.19 | - |
(R)-alpha-methylbenzylamine | pH 10.0, 40°C, with 1.5 mM 3-methyl-2-oxovalerate | Haliangium ochraceum | |
0.19 | - |
(R)-alpha-methylbenzylamine | recombinant enzyme, pH 9.0, 40°C, with 3-methyl-2-oxovalerate | Haliangium ochraceum | |
0.22 | - |
(R)-alpha-methylbenzylamine | pH 10.0, 40°C, with 5 mM 3-methyl-2-oxovalerate | Haliangium ochraceum | |
0.22 | - |
(R)-alpha-methylbenzylamine | recombinant enzyme, pH 10.0, 40°C, with 3-methyl-2-oxovalerate | Haliangium ochraceum | |
0.23 | - |
(R)-alpha-methylbenzylamine | pH 10.0, 40°C, with 2-oxoglutarate | Haliangium ochraceum |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
10 | - |
- |
Haliangium ochraceum |
10 | 10.5 | - |
Haliangium ochraceum |
pH Minimum | pH Maximum | Comment | Organism |
---|---|---|---|
10 | 11 | maximal activity at pH 10.0, 70% of maximal activity at pH 11.0 | Haliangium ochraceum |
pH Stability | pH Stability Maximum | Comment | Organism |
---|---|---|---|
11 | - |
the purified recombinant enzyme retains more than 70% of the maximum activity at pH 11.0 | Haliangium ochraceum |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
pyridoxal 5'-phosphate | PLP, dependent on | Haliangium ochraceum |
General Information | Comment | Organism |
---|---|---|
evolution | the enzyme belongs to the PLP fold type IV transaminases. PLP fold type IV transaminases include branched-chain amino acid transaminases (BCATs), D-amino acid transaminases, and (R)-amine:pyruvate transaminases | Haliangium ochraceum |
evolution | the enzyme is a PLP fold type IV transaminase. PLP fold type IV transaminases include branched-chain amino acid transaminases (BCATs), D-amino acid transaminases, and (R)-amine:pyruvate transaminases. Enzyme Hoch3033 is a transaminases with mixed type of activity. The mixed type of activity of Hoch3033 is implemented within the BCAT-like active site, but in the active site of Hoch3033, substitutions of specificity-determining residues, that are important for substrate binding in canonical BCATs, are observed | Haliangium ochraceum |
additional information | enzyme Hoch3033 active site structure and substrate binding. Hoch3033 has changes in the specificity determining key residues, which constitute characteristic sequence motifs, overview | Haliangium ochraceum |
additional information | the mixed type of activity of the enzyme is implemented within the BCAT-like active site. In the active site of the enzyme, substitutions of specificity-determining residues, that are important for substrate binding in canonical BCATs, are observed. These changes result in the loss of activity towards 2-oxoglutarate and increase the affinity towards (R)-alpha-methylbenzylamine. Active structure analysis | Haliangium ochraceum |
physiological function | enzyme Hoch3033 is a transaminases with mixed type of activity. The mixed type of activity of Hoch3033 is implemented within the BCAT-like active site, but in the active site of Hoch3033, substitutions of specificity-determining residues, that are important for substrate binding in canonical BCATs, are observed. These changes (H106Y/Y108R) result in the loss of activity towards 2-oxoglutarate and increase the affinity towards (R)-alpha-methylbenzylamine | Haliangium ochraceum |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.0011 | - |
(R)-alpha-ethylbenzylamine | pH 10.0, 40°C, with 2-oxoglutarate | Haliangium ochraceum | |
0.01 | - |
(R)-alpha-methylbenzylamine | pH 10.0, 40°C, with 2-oxoglutarate | Haliangium ochraceum | |
0.016 | - |
(R)-alpha-methylbenzylamine | pH 10.0, 40°C, with 1.5 mM 3-methyl-2-oxovalerate | Haliangium ochraceum | |
0.016 | - |
(R)-alpha-methylbenzylamine | recombinant enzyme, pH 9.0, 40°C, with 3-methyl-2-oxovalerate | Haliangium ochraceum | |
0.037 | - |
(R)-alpha-methylbenzylamine | pH 10.0, 40°C, with 5 mM 3-methyl-2-oxovalerate | Haliangium ochraceum | |
0.037 | - |
(R)-alpha-methylbenzylamine | recombinant enzyme, pH 10.0, 40°C, with 3-methyl-2-oxovalerate | Haliangium ochraceum | |
0.27 | - |
3-methyl-2-oxovalerate | pH 10.0, 40°C | Haliangium ochraceum | |
0.27 | - |
3-methyl-2-oxovalerate | recombinant enzyme, pH 10.0, 40°C, with (R)-alpha-methylbenzylamine | Haliangium ochraceum |