Any feedback?
Please rate this page
(literature.php)
(0/150)

BRENDA support

Literature summary extracted from

  • Voss, M.; Xiang, C.; Esque, J.; Nobili, A.; Menke, M.J.; Andre, I.; Hoehne, M.; Bornscheuer, U.T.
    Creation of (R)-amine transaminase activity within an alpha-amino acid transaminase scaffold (2020), ACS Chem. Biol., 15, 416-424 .
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.6.1.B21 recombinant expression of C-terminally His-tagged (R)-amine transaminase, a engineered modified DATA mutant enzyme, in Escherichia coli strain BL21(DE3) Bacillus sp. YM-1
2.6.1.21 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

EC Number Protein Variants Comment Organism
2.6.1.21 H86F site-directed mutagenesis, the mutant shows no activity with (R)-PEA and no wild-type DATA activity Bacillus sp. (in: Bacteria)
2.6.1.B21 H86F site-directed mutagenesis, the mutant shows no activity with (R)-PEA and no wild-type DATA activity Bacillus sp. YM-1
2.6.1.21 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)
2.6.1.B21 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 high specific activity 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. YM-1
2.6.1.21 S180A site-directed mutagenesis, the mutant shows no activity with (R)-PEA and no wild-type DATA activity Bacillus sp. (in: Bacteria)
2.6.1.B21 S180A site-directed mutagenesis, the mutant shows no activity with (R)-PEA and no wild-type DATA activity Bacillus sp. YM-1
2.6.1.21 Y31F site-directed mutagenesis, the mutant shows low activity with (R)-PEA and good wild-type DATA activity Bacillus sp. (in: Bacteria)
2.6.1.B21 Y31F site-directed mutagenesis, the mutant shows low activity with (R)-PEA and good wild-type DATA activity Bacillus sp. YM-1
2.6.1.21 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)
2.6.1.B21 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 Bacillus sp. YM-1
2.6.1.21 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)
2.6.1.B21 Y31F/H86F/S180A/T242I site-directed mutagenesis, variant M2-4 shows significant activity toward (R)-PEA, and retains no native DATA activity Bacillus sp. YM-1
2.6.1.21 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)
2.6.1.B21 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. YM-1
2.6.1.21 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)
2.6.1.B21 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. YM-1
2.6.1.21 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)
2.6.1.B21 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. YM-1
2.6.1.21 Y88E site-directed mutagenesis, the mutant shows no activity with (R)-PEA and no wild-type DATA activity Bacillus sp. (in: Bacteria)
2.6.1.B21 Y88E site-directed mutagenesis, the mutant shows no activity with (R)-PEA and no wild-type DATA activity Bacillus sp. YM-1
2.6.1.21 Y88F site-directed mutagenesis, the mutant shows low activity with (R)-PEA and no wild-type DATA activity Bacillus sp. (in: Bacteria)
2.6.1.B21 Y88F site-directed mutagenesis, the mutant shows low activity with (R)-PEA and no wild-type DATA activity Bacillus sp. YM-1

Organism

EC Number Organism UniProt Comment Textmining
2.6.1.21 Bacillus sp. (in: Bacteria) P19938
-
-
2.6.1.21 Bacillus sp. (in: Bacteria) YM-1 P19938
-
-
2.6.1.B21 Bacillus sp. YM-1
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.6.1.21 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)
2.6.1.B21 recombinant His-tagged engineered (R)-amine transaminase from Escherichia coli strain BL21(DE3) by immobilized metal affinity chromatography and desalting gel filtration Bacillus sp. YM-1

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
2.6.1.B21 0.008
-
 pH 9.0, 30°C, substrate (R)-1-phenylethylamine, recombinant enzyme mutant Y31F Bacillus sp. YM-1
2.6.1.21 0.009
-
 pH 9.0, 30°C, substrate D-glutamate, recombinant enzyme mutant M2-6 Bacillus sp. (in: Bacteria)
2.6.1.B21 0.012
-
 pH 9.0, 30°C, substrate (R)-1-phenylethylamine, recombinant enzyme mutant Y88E Bacillus sp. YM-1
2.6.1.21 0.017
-
 pH 9.0, 30°C, substrate D-glutamate, recombinant enzyme mutant M2-5 Bacillus sp. (in: Bacteria)
2.6.1.B21 0.0183
-
 pH 9.0, 30°C, substrate (R)-1-phenylethylamine, recombinant enzyme mutant M2-4 Bacillus sp. YM-1
2.6.1.B21 0.041
-
 pH 9.0, 30°C, substrate (R)-1-phenylethylamine, recombinant enzyme mutant M2-3 Bacillus sp. YM-1
2.6.1.B21 0.185
-
 pH 9.0, 30°C, substrate (R)-1-phenylethylamine, recombinant enzyme mutant M2-5 Bacillus sp. YM-1
2.6.1.21 0.208
-
 pH 9.0, 30°C, substrate D-glutamate, recombinant enzyme mutant Y31F Bacillus sp. (in: Bacteria)
2.6.1.21 0.21
-
 pH 9.0, 30°C, substrate D-glutamate, recombinant enzyme mutant M2-3 Bacillus sp. (in: Bacteria)
2.6.1.B21 0.326
-
 pH 9.0, 30°C, substrate (R)-1-phenylethylamine, recombinant enzyme mutant M2-6 Bacillus sp. YM-1
2.6.1.21 0.61
-
 pH 9.0, 30°C, substrate D-glutamate, recombinant wild-type enzyme Bacillus sp. (in: Bacteria)

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.6.1.B21 (R)-1-phenylethylamine + 2-oxoisocaproate
-
 Bacillus sp. YM-1 acetophenone + D-leucine
-
 r
2.6.1.B21 (R)-1-phenylethylamine + pyruvate
-
 Bacillus sp. YM-1 acetophenone + D-alanine
-
 r
2.6.1.21 D-glutamate + pyruvate
-
 Bacillus sp. (in: Bacteria) 2-oxoglutarate + D-alanine
-
 r
2.6.1.21 D-glutamate + pyruvate
-
 Bacillus sp. (in: Bacteria) YM-1 2-oxoglutarate + D-alanine
-
 r

Synonyms

EC Number Synonyms Comment Organism
2.6.1.B21 (R)-amine transaminase
-
 Bacillus sp. YM-1
2.6.1.B21 (R)-amine:pyruvate transaminase
-
 Bacillus sp. YM-1
2.6.1.B21 (R)-ATA
-
 Bacillus sp. YM-1
2.6.1.B21 (R)-selective amine transaminase
-
 Bacillus sp. YM-1
2.6.1.B21 (R)-transaminase
-
 Bacillus sp. YM-1
2.6.1.21 D-amino acid aminotransferase
-
 Bacillus sp. (in: Bacteria)
2.6.1.21 DAT
-
 Bacillus sp. (in: Bacteria)
2.6.1.21 DatA
-
 Bacillus sp. (in: Bacteria)

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
2.6.1.21 30
-
 assay at Bacillus sp. (in: Bacteria)
2.6.1.B21 30
-
 assay at Bacillus sp. YM-1

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
2.6.1.21 9
-
 assay at Bacillus sp. (in: Bacteria)
2.6.1.B21 9
-
 assay at Bacillus sp. YM-1

Cofactor

EC Number Cofactor Comment Organism Structure
2.6.1.21 pyridoxal 5'-phosphate
-
 Bacillus sp. (in: Bacteria)
2.6.1.B21 pyridoxal 5'-phosphate
-
 Bacillus sp. YM-1