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Literature summary for 2.6.1.21 extracted from
- Creation of (R)-amine transaminase activity within an alpha-amino acid transaminase scaffold (2020), ACS Chem. Biol., 15, 416-424 .
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| recombinant expression of C-terminally His-tagged wild-type DATA enzyme and mutant enzymes in Escherichia coli strain BL21(DE3) | Bacillus sp. (in: Bacteria) |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| H86F | site-directed mutagenesis, the mutant shows no activity with (R)-PEA and no wild-type DATA activity | Bacillus sp. (in: Bacteria) |
| additional information | creation of an (R)-amine transaminase activity within an alpha-amino acid transaminase scaffold via one to six amino acid substitutions in the enzyme's active site. The final sextuple variant M2-6 shows almost complete depletion of the native DATA activity but high activity toward (R)-phenylethylamine ((R)-PEA) and has a specific activity of 326 milliunits/mg in the conversion of (R)-PEA and pyruvate to acetophenone and D-alanine. The crystal structure of the D-amino acid aminotransferase (DATA, UniProt ID P19938, EC 2.6.1.21) from Bacillus sp. strain YM-1 (PDB entry 3DAA) is used as a scaffold for the computational enzyme redesign. Molecular dynamics simulations and molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) calculations | Bacillus sp. (in: Bacteria) |
| S180A | site-directed mutagenesis, the mutant shows no activity with (R)-PEA and no wild-type DATA activity | Bacillus sp. (in: Bacteria) |
| Y31F | site-directed mutagenesis, the mutant shows low activity with (R)-PEA and good wild-type DATA activity | Bacillus sp. (in: Bacteria) |
| Y31F/H86F | site-directed mutagenesis, variant M2 exhibited some (R)-PEA acceptance when PMP formation after incubating the purified variant with (R)-PEA is assayed, which indicates a completed first half-reaction. Lack of activity in the acetophenone assay and with wild-type substrate | Bacillus sp. (in: Bacteria) |
| Y31F/H86F/S180A/T242I | site-directed mutagenesis, variant M2-4 shows significant activity toward (R)-PEA, and retains no native DATA activity | Bacillus sp. (in: Bacteria) |
| Y31F/H86F/Y88F | site-directed mutagenesis, variant M2-3 shows significant activity toward (R)-PEA and retains 34% of the native DATA activity | Bacillus sp. (in: Bacteria) |
| Y31F/H86F/Y88F/H100L/S180A/T242I | site-directed mutagenesis, variant M2-6 shows almost complete depletion of the native DATA activity but high activity toward (R)-phenylethylamine ((R)-PEA) and has a specific activity of 326 milliunits/mg in the conversion of (R)-PEA and pyruvate to acetophenone and D-alanine | Bacillus sp. (in: Bacteria) |
| Y31F/H86F/Y88F/S180A/T242I | site-directed mutagenesis, variant M2-5 shows significant activity toward (R)-PEA and partially retains native DATA activity | Bacillus sp. (in: Bacteria) |
| Y88E | site-directed mutagenesis, the mutant shows no activity with (R)-PEA and no wild-type DATA activity | Bacillus sp. (in: Bacteria) |
| Y88F | site-directed mutagenesis, the mutant shows low activity with (R)-PEA and no wild-type DATA activity | Bacillus sp. (in: Bacteria) |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Bacillus sp. (in: Bacteria) | P19938 | - |
- |
| Bacillus sp. (in: Bacteria) YM-1 | P19938 | - |
- |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| recombinant C-terminally His-tagged wild-type DATA enzyme and mutant enzymes from Escherichia coli strain BL21(DE3) by immobilized metal affinity chromatography and desalting gel filtration | Bacillus sp. (in: Bacteria) |
Specific Activity [micromol/min/mg]
| Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
|---|---|---|---|
| 0.009 | - |
pH 9.0, 30°C, substrate D-glutamate, recombinant enzyme mutant M2-6 | Bacillus sp. (in: Bacteria) |
| 0.017 | - |
pH 9.0, 30°C, substrate D-glutamate, recombinant enzyme mutant M2-5 | Bacillus sp. (in: Bacteria) |
| 0.208 | - |
pH 9.0, 30°C, substrate D-glutamate, recombinant enzyme mutant Y31F | Bacillus sp. (in: Bacteria) |
| 0.21 | - |
pH 9.0, 30°C, substrate D-glutamate, recombinant enzyme mutant M2-3 | Bacillus sp. (in: Bacteria) |
| 0.61 | - |
pH 9.0, 30°C, substrate D-glutamate, recombinant wild-type enzyme | Bacillus sp. (in: Bacteria) |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| D-glutamate + pyruvate | - |
Bacillus sp. (in: Bacteria) | 2-oxoglutarate + D-alanine | - |
r |  |
| D-glutamate + pyruvate | - |
Bacillus sp. (in: Bacteria) YM-1 | 2-oxoglutarate + D-alanine | - |
r | |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| D-amino acid aminotransferase | - |
Bacillus sp. (in: Bacteria) |
| DAT | - |
Bacillus sp. (in: Bacteria) |
| DatA | - |
Bacillus sp. (in: Bacteria) |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 30 | - |
assay at | Bacillus sp. (in: Bacteria) |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 9 | - |
assay at | Bacillus sp. (in: Bacteria) |
Cofactor
| Cofactor | Comment | Organism | Structure |
|---|---|---|---|
| pyridoxal 5'-phosphate | - |
Bacillus sp. (in: Bacteria) | |
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