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To alter the amine donor/acceptor spectrum of an (S)-selective amine transaminase (ATA), a library based on the Vibrio fluvialis ATA targeting four residues close to the active site (L56, W57, R415 and L417) was created. A 3DM-derived alignment comprising fold class I pyridoxal-5'-phosphate (PLP)-dependent enzymes allowed identification of positions, which were assumed to determine substrate specificity. These positions were targeted for mutagenesis with a focused alphabet of hydrophobic amino acids to convert an amine:α-keto acid transferase into an amine:aldehyde transferase. Screening of 1200 variants revealed three hits, which showed a shifted amine donor/acceptor spectrum towards aliphatic aldehydes (mainly pentanal), as well as an altered pH profile. Interestingly, all three hits, although found independently, contained the same mutation R415L and additional W57F and L417V substitutions.

Transaminases represent one of the most important enzymes of the biocatalytic toolbox for chiral amine synthesis as they allow asymmetric synthesis with quantitative yields and high enantioselectivity. In order to enable substrate profiling of transaminases for acceptance of different amines, a glycine oxidase and horseradish peroxidase coupled assay was developed. Transaminase activity is detected upon transfer of an amine group from an amino donor substrate to glyoxylate, generating glycine, which is subsequently oxidized by glycine oxidase, releasing hydrogen peroxide in turn. Horseradish peroxidase uses the hydrogen peroxide to produce benzoquinone, which forms a red quinone imine dye by a subsequent condensation reaction. As glycine does not carry a chiral center, both (R)- and (S)-selective transaminases accepting glyoxylate as amino acceptor are amenable to screening. The principle has been transferred to establish a high-throughput solid-phase assay which dramatically decreases the screening effort in directed evolution of transaminases, as only active variants are selected for further analysis.

Chiral amines are important building blocks, especially for the pharmaceutical industry. Although amine transaminases (ATAs) are versatile enzymes to synthesize chiral amines, the wildtype enzymes do not accept ketones with two large substituents next to the carbonyl functionality. Using bioinformatic tools to design a seven-site mutant library followed by high-throughput screening, we were able to identify variants of the enzyme from Vibrio fluvialis (VF-ATA) with a widened binding pocket, as exemplified for a range of ketones. Three variants allowed the asymmetric synthesis of 2,2-dimethyl-1-phenylpropan-1-amine-not accessible by any wildtype ATA described so far. The best variant containing four mutations (L56V, W57C, F85V, V153A) gave 100 % conversion of the ketone to yield the amine with an enantiomeric excess value >99 %, notably with preference for the ( R)-enantiomer. In silico modeling enabled the reconstruction of the substrate binding mode to the newly evolved pocket and, hence, allowed explanation of the experimental results. [ABSTRACT FROM AUTHOR]

One lock for different keys: A flexible arginine in the active site allows γ‐aminobutyrate:pyruvate transaminases to bind the chemically different substrates L‐alanine and γ‐aminobutyric acid. Moreover, a flexible arginine residue facilitates the promiscuous conversion of (S)‐amines and ketones. The degree of promiscuity can be related to distinct key amino acids lying at the surface of the active site. [ABSTRACT FROM AUTHOR]

Digging in the database: Four synthetically useful transaminases for which no function was known were studied by evaluating a set of eight amino donors and seven acceptors (see figure). The four enzymes differ substantially in their catalytic properties and substrate preferences. We also used the enzymes in the asymmetric synthesis of a range of chiral amines and observed excellent enantioselectivities and conversions. [ABSTRACT FROM AUTHOR]