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An enzyme cascade was established previously consisting of a recycling system with an l-amino acid oxidase (hcLAAO4) and a catalase (hCAT) for different α-keto acid co-substrates of (S)-selective amine transaminases (ATAs) in kinetic resolutions of racemic amines. Only 1 mol % of the co-substrate was required and l-amino acids instead of α-keto acids could be applied. However, soluble enzymes cannot be reused easily. Immobilization of hcLAAO4, hCAT and the (S)-selective ATA from Vibrio fluvialis (ATA-Vfl) was addressed here. Immobilization of the enzymes together rather than on separate beads showed higher reaction rates most likely due to fast co-substrate channeling between ATA-Vfl and hcLAAO4 due to their close proximity. Co-immobilization allowed further reduction of the co-substrate amount to 0.1 mol % most likely due to a more efficient H 2 O 2 -removal caused by the stabilized hCAT and its proximity to hcLAAO4. Finally, the co-immobilized enzyme cascade was reused in 3 cycles of preparative kinetic resolutions to produce (R)-1-PEA with high enantiomeric purity (97.3 %ee). Further recycling was inefficient due to the instability of ATA-Vfl, while hcLAAO4 and hCAT revealed high stability. An engineered ATA-Vfl-8M was used in the co-immobilized enzyme cascade to produce (R)-1-(3-ethoxy-4-methoxyphenyl)-2-(methylsulfonyl)ethanamine, an apremilast-intermediate, with a 1,000 fold lower input of the co-substrate., (© 2023 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Amine transaminases (ATAs) are powerful biocatalysts for the stereoselective synthesis of chiral amines. Machine learning provides a promising approach for protein engineering, but activity prediction models for ATAs remain elusive due to the difficulty of obtaining high-quality training data. Thus, we first created variants of the ATA from Ruegeria sp. (3FCR) with improved catalytic activity (up to 2000-fold) as well as reversed stereoselectivity by a structure-dependent rational design and collected a high-quality dataset in this process. Subsequently, we designed a modified one-hot code to describe steric and electronic effects of substrates and residues within ATAs. Finally, we built a gradient boosting regression tree predictor for catalytic activity and stereoselectivity, and applied this for the data-driven design of optimized variants which then showed improved activity (up to 3-fold compared to the best variants previously identified). We also demonstrated that the model can predict the catalytic activity for ATA variants of another origin by retraining with a small set of additional data., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Amine transaminases (ATAs) are powerful biocatalysts for the stereoselective synthesis of chiral amines. However, wild-type ATAs usually show pH optima at slightly alkaline values and exhibit low catalytic activity under physiological conditions. For efficient asymmetric synthesis ATAs are commonly used in combination with lactate dehydrogenase (LDH, optimal pH: 7.5) and glucose dehydrogenase (GDH, optimal pH: 7.75) to shift the equilibrium towards the synthesis of the target chiral amine and hence their pH optima should fit to each other. Based on a protein structure alignment, variants of ( R )-selective transaminases were rationally designed, produced in E. coli , purified and subjected to biochemical characterization. This resulted in the discovery of the variant E49Q of the ATA from Aspergillus fumigatus , for which the pH optimum was successfully shifted from pH 8.5 to 7.5 and this variant furthermore had a two times higher specific activity than the wild-type protein at pH 7.5. A possible mechanism for this shift of the optimal pH is proposed. Asymmetric synthesis of ( R )-1-phenylethylamine from acetophenone in combination with LDH and GDH confirmed that the variant E49Q shows superior performance at pH 7.5 compared to the wild-type enzyme.

Chiral and enantiopure amines can be produced by enantioselective transaminases via kinetic resolution of amine racemates. This transamination reaction requires stoichiometric amounts of co-substrate. A dual-enzyme recycling system overcomes this limitation: l-amino acid oxidases (LAAO) recycle the accumulating co-product of (S)-selective transaminases in the kinetic resolution of racemic amines to produce pure (R)-amines. However, availability of suitable LAAOs is limited. Here we use the heterologously produced, highly active fungal hcLAAO4 with broad substrate spectrum. H 2 O 2 as byproduct of hcLAAO4 is detoxified by a catalase. The final system allows using sub-stoichiometric amounts of 1 mol% of the transaminase co-substrate as well as the initial application of l-amino acids instead of α-keto acids. With an optimized protocol, the synthetic potential of this kinetic resolution cascade was proven at the preparative scale (>90 mg) by the synthesis of highly enantiomerically pure (R)-methylbenzylamine (>99 %ee) at complete conversion (50 %)., (© 2022 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

The enzymatic transamination of ketones into ( R )-amines represents an important route for accessing a range of pharmaceuticals or building blocks. Although many publications have dealt with enzyme discovery, protein engineering, and the application of ( R )-selective amine transaminases [( R )-ATA] in biocatalysis, little is known about the actual in vivo role and how these enzymes have evolved from the ubiquitous α-amino acid transaminases (α-AATs). Here, we show the successful introduction of an ( R )-transaminase activity in an α-amino acid aminotransferase with one to six amino acid substitutions in the enzyme's active site. Bioinformatic analysis combined with computational redesign of the d-amino acid aminotransferase (DATA) led to the identification of a sextuple variant having a specific activity of 326 milliunits mg -1 in the conversion of ( R )-phenylethylamine and pyruvate to acetophenone and d-alanine. This value is similar to those of natural ( R )-ATAs, which typically are in the range of 250 milliunits mg -1 . These results demonstrate that ( R )-ATAs can evolve from α-AAT as shown here for the DATA scaffold.

Agar plate assays represent a useful method for high-throughput prescreening of larger enzyme libraries derived from for example error-prone PCR or multiple site-saturation mutagenesis to decrease screening effort by separating promising variants from less active, inactive, or neutral variants. In order to do so, colonies are directly applied for enzyme expression and screening on adsorbent and microporous membranes instead of elaborately preparing cell lysates in 96-well plates. This way, 400-800 enzyme variants can be prescreened on a single membrane, 10,000-20,000 variants per week and per single researcher respectively (25 membranes per week).The following chapter gives a detailed protocol of how to screen transaminase libraries in solid phase, but it also intends to provide inspiration to establish a direct or coupled agar plate assay for screening variable enzymatic activities by interchanging assay enzymes and adapting assay conditions to individual needs.

Pyridoxal-phosphate (PLP)-dependent enzymes catalyse a remarkable diversity of chemical reactions in nature. A1RDF1 from Arthrobacter aurescens TC1 is a fold type I, PLP-dependent enzyme in the class III transaminase (TA) subgroup. Despite sharing 28 % sequence identity with its closest structural homologues, including β-alanine:pyruvate and γ-aminobutyrate:α-ketoglutarate TAs, A1RDF1 displayed no TA activity. Activity screening revealed that the enzyme possesses phospholyase (E.C. 4.2.3.2) activity towards O-phosphoethanolamine (PEtN), an activity described previously for vertebrate enzymes such as human AGXT2L1, enzymes for which no structure has yet been reported. In order to shed light on the distinctive features of PLP-dependent phospholyases, structures of A1RDF1 in complex with PLP (internal aldimine) and PLP⋅PEtN (external aldimine) were determined, revealing the basis of substrate binding and the structural factors that distinguish the enzyme from class III homologues that display TA activity., (© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Understanding the metabolic potential of organisms or a bacterial community based on their (meta) genome requires the reliable prediction of an enzyme's function from its amino acid sequence. Besides a remarkable development in prediction algorithms, the substrate scope of sequences with low identity to well-characterized enzymes remains often very elusive. From a recently conducted structure function analysis study of PLP-dependent enzymes, we identified a putative transaminase from Bacillus anthracis (Ban-TA) with the crystal structure 3N5M (deposited in the protein data bank in 2011, but not yet published). The active site residues of Ban-TA differ from those in related (class III) transaminases, which thereby have prevented function predictions. By investigating 50 substrate combinations its amine and ω-amino acid:pyruvate transaminase activity was revealed. Even though Ban-TA showed 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 implied 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 ω-amino acid:pyruvate transaminases. Molecular dynamics studies suggested that another arginine (R162) binds ω-amino acids in Ban-TA, but no side chain movements are required for amine and amino acid binding. These results, supported by mutagenesis studies, provide functional insights for the B. anthracis enzyme, enable function predictions of related proteins, and broadened the knowledge regarding ω-amino acid and amine converting transaminases.

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.

In this review we analyse structure/sequence-function relationships for the superfamily of PLP-dependent enzymes with special emphasis on class III transaminases. Amine transaminases are highly important for applications in biocatalysis in the synthesis of chiral amines. In addition, other enzyme activities such as racemases or decarboxylases are also discussed. The substrate scope and the ability to accept chemically different types of substrates are shown to be reflected in conserved patterns of amino acids around the active site. These findings are condensed in a sequence-function matrix, which facilitates annotation and identification of biocatalytically relevant enzymes and protein engineering thereof., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Amine transaminases (ATAs) are powerful enzymes for the stereospecific production of chiral amines. However, the synthesis of amines incorporating more than one stereocenter is still a challenge. We developed a cascade synthesis to access optically active 3-alkyl-substituted chiral amines by combining two asymmetric synthesis steps catalyzed by an enoate reductase and ATAs. The ATA wild type from Vibrio fluvialis showed only modest enantioselectivity (14 % de) in the amination of (S)-3-methylcyclohexanone, the product of the enoate-reductase-catalyzed reaction step. However, by protein engineering we created two variants with substantially improved diastereoselectivities: variant Leu56Val exhibited a higher R selectivity (66 % de) whereas the Leu56Ile substitution caused a switch in enantiopreference to furnish the S-configured diastereomer (70 % de). Addition of 30 % DMSO further improved the selectivity and facilitated the synthesis of (1R,3S)-1-amino-3-methylcyclohexane with 89 % de at 87 % conversion., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Chiral amines are important precursors for the pharmaceutical and fine-chemical industries. Because of this, the demand for enantiopure amines is currently increasing. Amine transaminases can produce a large spectrum of chiral amines in the (R)- or (S)-configuration, depending on their substrate scope and stereo-preference, by converting a prochiral ketone into the chiral amine while using alanine as the amine donor producing pyruvate as an α-keto acid product. In order to guide the protein engineering of transaminases to improve substrate specificity and enantioselectivity, we carried out a crystal structure analysis at 1.6 Å resolution of the (R)-amine transaminase from Aspergillus fumigatus with the bound inhibitor gabaculine. This revealed that Arg126 has an important role in the dual substrate recognition of this enzyme because mutating this residue to alanine reduced substantially the ability of the enzyme to use pyruvate as an amino acceptor., (© 2014 FEBS.)

Transaminases from Aspergillus fumigatus ((R)-selective, AspFum), Ruegeria pomeroyi ((S)-selective, 3HMU) and Rhodobacter sphaeroides 2.4.1 ((S)-selective, 3I5T) were immobilized on chitosan with specific activities of 99, 157, and 163U/g and acceptable yields (54, 21, and 23%, respectively) for glutaraldehyde (GA) immobilization. Besides GA, also divinylsulfone was used as linker molecule leading to a similar efficient immobilization for two enzymes, GibZea and NeoFis, whereas GA was superior in the other cases. Storage of the GA-immobilized enzymes for one month resulted in increased relative activities between 120 and 180%. The thermal stability was improved, especially for the GA-immobilized AspFum compared to the free enzyme after incubation for 4h at 60°C (10% vs. 235% residual activity). Especially after incubation of AspFum (free or immobilized) for 2h at 50°C a strongly increased activity was observed (up to 359% of the initial activity). This effect was studied in more detail, revealing that one heat activation prior and one after immobilization increased the overall immobilization efficiency. Recycling of the immobilized ATAs resulted only in a small reduction of activity after four batches. Asymmetric synthesis of (R)- or (S)-1-methyl-3-phenylpropylamine from the prostereogenic ketone using isopropylamine (IPA) as amino donor was applied with conversions up to 50% (AspFum) or 75% (3HMU). Except for NeoFis, all immobilized ATAs showed higher conversions compared to the free enzyme., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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.

The importance of amine transaminases for producing optically pure chiral precursors for pharmaceuticals and chemicals has substantially increased in recent years. The X-ray crystal structure of the (R)-selective amine transaminase from the fungus Aspergillus fumigatus was solved by S-SAD phasing to 1.84 Å resolution. The refined structure at 1.27 Å resolution provides detailed knowledge about the molecular basis of substrate recognition and conversion to facilitate protein-engineering approaches. The protein forms a homodimer and belongs to fold class IV of the pyridoxal-5'-phosphate-dependent enzymes. Both subunits contribute residues to form two active sites. The structure of the holoenzyme shows the catalytically important cofactor pyridoxal-5'-phosphate bound as an internal aldimine with the catalytically responsible amino-acid residue Lys179, as well as in its free form. A long N-terminal helix is an important feature for the stability of this fungal (R)-selective amine transaminase, but is missing in branched-chain amino-acid aminotransferases and D-amino-acid aminotransferases.

The (R)-selective amine transaminase from Aspergillus fumigatus was expressed in Escherichia coli and purified to homogeneity. Bright yellow crystals appeared while storing the concentrated solution in the refrigerator and belonged to space group C222(1). X-ray diffraction data were collected to 1.27 Å resolution, as well as an anomalous data set to 1.84 Å resolution that was suitable for S-SAD phasing.

Biocatalysis has emerged as a powerful alternative to traditional chemistry, especially for asymmetric synthesis. One key requirement during process development is the discovery of a biocatalyst with an appropriate enantiopreference and enantioselectivity, which can be achieved, for instance, by protein engineering or screening of metagenome libraries. We have developed an in silico strategy for a sequence-based prediction of substrate specificity and enantiopreference. First, we used rational protein design to predict key amino acid substitutions that indicate the desired activity. Then, we searched protein databases for proteins already carrying these mutations instead of constructing the corresponding mutants in the laboratory. This methodology exploits the fact that naturally evolved proteins have undergone selection over millions of years, which has resulted in highly optimized catalysts. Using this in silico approach, we have discovered 17 (R)-selective amine transaminases, which catalyzed the synthesis of several (R)-amines with excellent optical purity up to >99% enantiomeric excess.

Amine-transaminases (ATAs, omega-transaminases, omega-TA) are PLP-dependent enzymes that catalyze amino group transfer reactions. In contrast to the widespread and well-known amino acid-transaminases, ATAs are able to convert substrates lacking an alpha-carboxylic functional group. They have gained increased attention because of their potential for the asymmetric synthesis of optically active amines, which are frequently used as building blocks for the preparation of numerous pharmaceuticals. Having already introduced a fast kinetic assay based on the conversion of the model substrate alpha-methylbenzylamine for the characterization of the amino acceptor specificity, we now report on a kinetic conductivity assay for investigating the amino donor specificity of a given ATA. The course of an ATA-catalyzed reaction can be followed conductometrically since the conducting substrates, a positively charged amine and a negatively charged keto acid, are converted to nonconducting products, a noncharged ketone and a zwitterionic amino acid. The decrease of conductivity for the investigated reaction systems were determined to be 33-52 microS mM(-1). In contrast to other ATA-assays previously described, with this approach all transamination reactions between any amine and any keto acid can be monitored without the need for an additional enzyme or staining solutions. The assay was used for the characterization of a ATA from Rhodobacter sphaeroides, and the data obtained were in excellent agreement with gas chromatography analysis.

For the biocatalytic preparation of optically active amines, omega-transaminases (omega-TA) are of special interest since they allow the asymmetric synthesis starting from prostereogenic ketones with 100% yield. To facilitate the purification and characterization of novel omega-TA, a fast kinetic assay was developed based on the conversion of the widely used model substrate alpha-methylbenzylamine, which is commonly accepted by most of the known omega-TAs. The product from this reaction, acetophenone, can be detected spectrophotometrically at 245 nm with high sensitivity (epsilon = 12 mM(-1) cm(-1)), since the other reactants show only a low absorbance. Besides the standard substrate pyruvate, all low-absorbing ketones, aldehydes, or keto acids can be used as cosubstrates, and thus the amino acceptor specificity of a given omega-TA can be obtained quickly. Furthermore, the assay allows the fast investigation of enzymatic properties like pH and temperature optimum and stability. This method was used for the characterization of a novel omega-TA cloned from Rhodobacter sphaeroides, and the data obtained were in excellent accordance with a standard capillary electrophoresis assay.