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Literature summary extracted from

  • Steffen-Munsberg, F.; Matzel, P.; Sowa, M.; Berglund, P.; Bornscheuer, U.; Hoehne, M.
    Bacillus anthracis omega-amino acid pyruvate transaminase employs a different mechanism for dual substrate recognition than other amine transaminases (2016), Appl. Microbiol. Biotechnol., 100, 4511-4521 .
No PubMed abstract available

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.6.1.B16 recombinant expression of C-terminally His6-tagged wild-type and mutant enzymes in transformed phage-resistant Escherichia coli strain BL21(DE3) uncultured bacterium
2.6.1.B16 recombinant expression of C-terminally His6-tagged wild-type and mutant enzymes in transformed phage-resistant Escherichia coli strain BL21(DE3) Bacillus anthracis

Protein Variants

EC Number Protein Variants Comment Organism
2.6.1.B16 G51S site-directed mutagenesis, the ATA-v2 mutant shows superior operational, temperature and solvent stability as well as improved activity for the pharmaceutical relevant amine product 4-phenyl-2-butanamine appears to be specific for thermal tolerance, compared to wild-type, mutant ATA-v1 shows increased temperature optimum, and features excellent operational stability in biphasic (aqueous/nonpolar solvent) reaction systems at 45°C, when the aqueous phase contained an approximate amine donor-to-acceptor ratio uncultured bacterium
2.6.1.B16 additional information comparison of pH-profile and amino acceptor substrate specificity of mutants R162A and R410A and wild-type Ban-TA Bacillus anthracis
2.6.1.B16 additional information semi-rational mutagenesis study for enzymes with higher activity and higher selectivity, employing the proprietary multi-dimensional-mutagenesis (MDM) and automated generation of mutant (AGM) technologies (c-LEcta) uncultured bacterium
2.6.1.B16 N161I/Y164L site-directed mutagenesis, double mutant ATA-v1 with two point mutations in the cofactor-ring motif shows superior operational, temperature and solvent stability as well as improved activity for the pharmaceutical relevant amine product 4-phenyl-2-butanamine, while the enantioselectivity for (S)-PBA is raised from 59 to 96%, compared to the wild-type enzyme. Compared to wild-type, mutant ATA-v1 shows increased temperature optimum, and features excellent operational stability in biphasic (aqueous/nonpolar solvent) reaction systems at 45°C, when the aqueous phase contained an approximate amine donor-to-acceptor ratio of 50:1. In contrast to wild-type ATA, ATA-v1 seems to resist denaturation upon interfacial contact under turbulent conditions. ATA-v1 appears to slightly gain activity with time in the presence of n-heptane and toluene uncultured bacterium
2.6.1.B16 R162A site-directed mutagenesis, compared to wild-type, activities of the R162 mutant drop around ten times if the reaction comprises carboxylic substrates, e.g. when (S)-PEA is employed with pyruvate or succinic semialdehyde as substrates Bacillus anthracis
2.6.1.B16 R410A site-directed mutagenesis, the mutant converts all tested substrate combinations with similar or slightly higher activity compared to wild-type Bacillus anthracis

Inhibitors

EC Number Inhibitors Comment Organism Structure
2.6.1.B16 isopropyl acetate
-
 uncultured bacterium

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
2.6.1.B16 additional information
-
 additional information reaction kinetics and thermodynamics of wild-type and mutant enzymes uncultured bacterium

Organic Solvent Stability

EC Number Organic Solvent Comment Organism
2.6.1.B16 isopropyl acetate the wild-type enzyme rapidly inactivates in isopropyl acetate, while enzyme mutants ATA-v1 and ATA-v2 are more stable, overview uncultured bacterium
2.6.1.B16 toluene the wild-type enzyme rapidly inactivates in toluene, while enzyme mutants ATA-v1 and ATA-v2 are more stable, overview uncultured bacterium

Organism

EC Number Organism UniProt Comment Textmining
2.6.1.B16 Bacillus anthracis Q81SL2
-
-
2.6.1.B16 uncultured bacterium
-
 sequence from a a metagenomic library
-

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
2.6.1.B16 0.0016
-
 purified recombinant wild-type enzyme, pH 10.0, 30°C, substrates (S)-1-phenylethylamine and 4-phenyl-2-butanone Bacillus anthracis
2.6.1.B16 0.0018
-
 purified recombinant wild-type enzyme, pH 10.0, 30°C, substrates (S)-1-phenylethylamine and rac-2-methylcyclohexanone Bacillus anthracis
2.6.1.B16 0.0019
-
 purified recombinant wild-type enzyme, pH 10.0, 30°C, substrates (S)-1-phenylethylamine and 2-heptanone Bacillus anthracis
2.6.1.B16 0.002
-
 purified recombinant wild-type enzyme, pH 10.0, 30°C, substrates (S)-1-phenylethylamine and cyclohexanone Bacillus anthracis
2.6.1.B16 0.0021
-
 purified recombinant wild-type enzyme, pH 10.0, 30°C, substrates (S)-1-phenylethylamine and 2-butanone Bacillus anthracis
2.6.1.B16 0.0028
-
 purified recombinant wild-type enzyme, pH 10.0, 30°C, substrates (S)-1-phenylethylamine and methoxyacetone Bacillus anthracis
2.6.1.B16 0.0038
-
 purified recombinant wild-type enzyme, pH 10.0, 30°C, substrates (S)-1-phenylethylamine and cyclooctanone Bacillus anthracis
2.6.1.B16 0.0067
-
 purified recombinant wild-type enzyme, pH 10.0, 30°C, substrates (S)-1-phenylethylamine and beta-tetralone Bacillus anthracis
2.6.1.B16 0.007
-
 purified recombinant wild-type enzyme, pH 10.0, 30°C, substrates (S)-1-phenylethylamine and oxaloacetate Bacillus anthracis
2.6.1.B16 0.94
-
 purified recombinant wild-type enzyme, pH 10.0, 30°C, substrates (S)-1-phenylethylamine and pyruvate or methylpyruvate Bacillus anthracis
2.6.1.B16 1.3
-
 purified recombinant wild-type enzyme, pH 10.0, 30°C, substrates (S)-1-phenylethylamine and 2-oxoglutarate Bacillus anthracis
2.6.1.B16 2.5
-
 purified recombinant wild-type enzyme, pH 10.0, 30°C, substrates beta-alanine and pyruvate Bacillus anthracis

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.6.1.B16 (S)-1-phenylethylamine + 2-butanone
-
 Bacillus anthracis acetophenone + ?
-
 r
2.6.1.B16 (S)-1-phenylethylamine + 2-heptanone
-
 Bacillus anthracis acetophenone + ?
-
 r
2.6.1.B16 (S)-1-phenylethylamine + 2-oxoglutarate
-
 Bacillus anthracis acetophenone + L-glutamate
-
 r
2.6.1.B16 (S)-1-phenylethylamine + 4-phenyl-2-butanone
-
 Bacillus anthracis acetophenone + ?
-
 r
2.6.1.B16 (S)-1-phenylethylamine + beta-tetralone
-
 Bacillus anthracis acetophenone + ?
-
 r
2.6.1.B16 (S)-1-phenylethylamine + cyclohexanone
-
 Bacillus anthracis acetophenone + ?
-
 r
2.6.1.B16 (S)-1-phenylethylamine + cyclooctanone
-
 Bacillus anthracis acetophenone + ?
-
 r
2.6.1.B16 (S)-1-phenylethylamine + methoxyacetone
-
 Bacillus anthracis acetophenone + ?
-
 r
2.6.1.B16 (S)-1-phenylethylamine + methylpyruvate
-
 Bacillus anthracis acetophenone + methylalanine
-
 r
2.6.1.B16 (S)-1-phenylethylamine + oxaloacetate
-
 Bacillus anthracis acetophenone + L-aspartate
-
 r
2.6.1.B16 (S)-1-phenylethylamine + pyruvate
-
 Bacillus anthracis acetophenone + L-alanine
-
 r
2.6.1.B16 (S)-1-phenylethylamine + rac-2-methylcyclohexanone
-
 Bacillus anthracis acetophenone + ?
-
 r
2.6.1.B16 beta-alanine + pyruvate high activity Bacillus anthracis 4-hydroxybutyrate + L-alanine
-
 r
2.6.1.B16 isopropylamine + 4-phenylbutan-2-one high activity, two half reaction steps, overview uncultured bacterium acetone + (S)-4-phenyl-2-butanamine
-
 r
2.6.1.B16 additional information substrate specificity of Ban-TA, overview. Even though enzyme Ban-TA shows a relatively narrow amine substrate scope within the tested substrates, it accepts 2-propylamine, which is a prerequisite for industrial asymmetric amine synthesis. Structural information imply that the so-called dual substrate recognition of chemically different substrates (i.e. amines and amino acids) differs from that in formerly known enzymes. It lacks the normally conserved flipping arginine, which enables dual substrate recognition by its side chain flexibility in other omega-amino acid:pyruvate transaminases. Molecular dynamics studies suggest that another arginine (R162) binds omega-amino acids in Ban-TA, but no side chain movements are required for amine and amino acid binding Bacillus anthracis ?
-
-

Subunits

EC Number Subunits Comment Organism
2.6.1.B16 homotetramer
-
 uncultured bacterium

Synonyms

EC Number Synonyms Comment Organism
2.6.1.B16 amine transaminase
-
 uncultured bacterium
2.6.1.B16 ATA
-
 uncultured bacterium
2.6.1.B16 Ban-TA
-
 Bacillus anthracis
2.6.1.B16 omega-amino acid:pyruvate transaminase
-
 Bacillus anthracis

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
2.6.1.B16 30
-
 assay at Bacillus anthracis
2.6.1.B16 40
-
 wild-type ATA uncultured bacterium
2.6.1.B16 50
-
 mutant ATA-v1 uncultured bacterium

Temperature Range [°C]

EC Number Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
2.6.1.B16 30 70 over 50% of maximal activity within this range, mutant enzymes ATA-v1 and ATA-v2 uncultured bacterium
2.6.1.B16 30 55 activity range, inactivation at 60°C, wild-type enzyme uncultured bacterium

Temperature Stability [°C]

EC Number Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
2.6.1.B16 50 75 enzyme mutant ATA-v1, completely stable up to 70°C, loss of 50% activity at 71°C, inactivation at 75°C uncultured bacterium
2.6.1.B16 50 62 wild-type enzyme, completely stable up to 55°C, loss of 50% activity at 60°C, inactivation at 62°C uncultured bacterium
2.6.1.B16 50 78 enzyme mutant ATA-v2, completely stable up to 72°C, loss of 50% activity at 74°C, inactivation at 78°C uncultured bacterium

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
2.6.1.B16 8
-
 assay at uncultured bacterium
2.6.1.B16 10
-
 assay at Bacillus anthracis

Cofactor

EC Number Cofactor Comment Organism Structure
2.6.1.B16 pyridoxal 5'-phosphate dependent on uncultured bacterium
2.6.1.B16 pyridoxal 5'-phosphate dependent on Bacillus anthracis

General Information

EC Number General Information Comment Organism
2.6.1.B16 evolution the enzyme belongs to the class-III pyridoxal-phosphate-dependent aminotransferase family Bacillus anthracis
2.6.1.B16 evolution the enzyme has a high sequence identity to a fold type I class III transaminase from Pseudomonas fluorescens uncultured bacterium
2.6.1.B16 additional information sequence-structure-function relationship uncultured bacterium
2.6.1.B16 additional information structure-function analysis, molecular dynamics simulations, and molecular modeling of substrate recognition. Residue W56 is the only residue in direct contact with alanine's carboxylate. Residue R162, pointing towards the substrate from the active site's entrance is binding the carboxylate indirectly via two water molecules Bacillus anthracis