Literature summary for 2.6.1.B21 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 .

Search for more literature for 2.6.1.B21

Cloned(Commentary)

Cloned (Comment)	Organism
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

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. YM-1
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
S180A	site-directed mutagenesis, the mutant shows no activity with (R)-PEA and no wild-type DATA activity	Bacillus sp. YM-1
Y31F	site-directed mutagenesis, the mutant shows low activity with (R)-PEA and good wild-type DATA activity	Bacillus sp. YM-1
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
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
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
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
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
Y88E	site-directed mutagenesis, the mutant shows no activity with (R)-PEA and no wild-type DATA activity	Bacillus sp. YM-1
Y88F	site-directed mutagenesis, the mutant shows low activity with (R)-PEA and no wild-type DATA activity	Bacillus sp. YM-1

Organism

Organism	UniProt	Comment	Textmining
Bacillus sp. YM-1	-	-	-

Purification (Commentary)

Purification (Comment)	Organism
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]

Specific Activity Minimum [µmol/min/mg]	Specific Activity Maximum [µmol/min/mg]	Comment	Organism
0.008	-	pH 9.0, 30°C, substrate (R)-1-phenylethylamine, recombinant enzyme mutant Y31F	Bacillus sp. YM-1
0.012	-	pH 9.0, 30°C, substrate (R)-1-phenylethylamine, recombinant enzyme mutant Y88E	Bacillus sp. YM-1
0.0183	-	pH 9.0, 30°C, substrate (R)-1-phenylethylamine, recombinant enzyme mutant M2-4	Bacillus sp. YM-1
0.041	-	pH 9.0, 30°C, substrate (R)-1-phenylethylamine, recombinant enzyme mutant M2-3	Bacillus sp. YM-1
0.185	-	pH 9.0, 30°C, substrate (R)-1-phenylethylamine, recombinant enzyme mutant M2-5	Bacillus sp. YM-1
0.326	-	pH 9.0, 30°C, substrate (R)-1-phenylethylamine, recombinant enzyme mutant M2-6	Bacillus sp. YM-1

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
(R)-1-phenylethylamine + 2-oxoisocaproate	-	Bacillus sp. YM-1	acetophenone + D-leucine	-	r
(R)-1-phenylethylamine + pyruvate	-	Bacillus sp. YM-1	acetophenone + D-alanine	-	r

Synonyms

Synonyms	Comment	Organism
(R)-amine transaminase	-	Bacillus sp. YM-1
(R)-amine:pyruvate transaminase	-	Bacillus sp. YM-1
(R)-ATA	-	Bacillus sp. YM-1
(R)-selective amine transaminase	-	Bacillus sp. YM-1
(R)-transaminase	-	Bacillus sp. YM-1

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
30	-	assay at	Bacillus sp. YM-1

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
9	-	assay at	Bacillus sp. YM-1

Cofactor

Cofactor	Comment	Organism	Structure
pyridoxal 5'-phosphate	-	Bacillus sp. YM-1

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Release 2023.1 (January 2023)