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This thesis investigates the biocatalytic synthesis of amines and amino alcohols. The applicability and economic feasibility of biocatalysis for chiral amine synthesis is reviewed and the findings were compared to established chemical processes using relevant process parameters (TON, TOF and STY). This review clearly showcases the potential of biocatalysis for the synthesis of chiral amines and provides a valuable guide for synthetic chemists who want to benefit from these new opportunities. Next, biocatalysis is applied for the synthesis of an amino alcohol with two stereocentres: A novel route for the synthesis of all four stereoisomers of 4-amino-1-phenylpentane-2-ol is presented. Enzymes were applied to install both stereocentres successively, which allowed the selective synthesis with high yields and optical purities. A small scale preparative asymmetric transamination yielded one amino alcohol stereoisomer selectively. The approach presented in this thesis provides a valuable option for the synthesis of this compound class as it is highly selective, step efficient and circumvents the need for protecting groups as well as transition-metal catalysis. The substrate scope of an (S)-selective amine transaminase (ATA) was altered in order to expand the applicability for amino alcohol synthesis. Protein engineering was conducted to enlarge the small binding pocket. Small scale preparative synthesis of the 1,2-amino alcohol (R)-phenylglycinol exemplifies the applicability of the evolved variants for the asymmetric synthesis of this compound. The designed variants expand the collection of ATAs that are suitable for the synthesis of amino alcohols with bulkier substituents. To deepen the understanding of ATAs further, a class III TA family wide analysis (which includes (S)-selective ATAs) is presented. After comparing the active site architectures and performing literature research amino acids were identified that correlate with the reaction- and substrate specificity of the enzymes within this family. This information is compiled in a sequence-function matrix, which allows the prediction of the main activity of biochemically uncharacterised enzymes from their sequence. These insights provide a better understanding of the activity determining residues in (S)-ATAs and class III TAs in general.
Structure– and sequence–function relationships in (S)-amine transaminases and related enzymes
(2015)
Chiral primary amines are valuable building blocks for many biologically active compounds. Environmentally friendlier alternatives to the classical methods for α-chiral primary amine synthesis are highly desired. A biocatalytic alternative that recently proved beneficial for industrial applications is asymmetric synthesis utilising (S)-selective amine transaminases (S-ATAs). These enzymes can be utilized to transaminate a prochiral ketone with an amino donor (e.g. isopropylamine), to achieve a chiral amine and a carbonyl product (e.g. acetone). However, for several potential applications protein engineering is required to fit (S)-ATAS to the demands of an industrial process. Since no (S)-ATA crystal structure required for understanding the substrate recognition and thus protein engineering was available, we first aimed at obtaining structural data. Instead of solving crystal structures ourselves, we took advantage of structural genomics projects and discovered, that the protein data bank (PDB) already contained crystal structures of four enzymes with unknown function that we hypothesised to possess (S)-ATA activity. After developing a screening method, the four enzymes could be characterized as ω-amino acid:pyruvate transaminases (ωAA:pyr TAs). (S)-amine conversion was suggested to be a ‘substrate-promiscuous’ activity of these enzymes, as it is pronounced differently in the four investigated ones. By comparing the active sites of the highly and poorly active (S)-ATAs, the residues that determine the ability of amine conversion in these enzymes were discovered. Furthermore, the mechanism for dual substrate recognition, the binding of both, carboxyl and bulky hydrophobic substrates in the same active site, could be elucidated with the crystal structures. A flexible arginine side chain is able to adopt various positions thus enabling carboxylate binding and by ‘flipping’ out of the active site, to create space for amine binding. Then, a limitation of these enzymes, the restricted substrate scope caused by a small binding pocket was addressed. First, a rational protein engineering approach was set up to create more space. The tested mutations, however, destroyed most of the activity for both regular and more bulky substrates. We thus learned that the structural requirements for (S)-ATA activity are more complex than initially anticipated and a semi-rational approach was applied to broaden the substrate scope. By systematic saturation of active site positions, substantially improved mutants for bulkier amine synthesis could be obtained. As this study highlighted a lack of understanding of (S)-ATA, the functional important residues in the enzymes belonging to the class III TA family were surveyed. This family is defined by common sequence and structure features and besides (S)-ATAs mainly comprises TAs of various substrate scopes but also a few phospholyases, racemases and decarboxylases. To enable the comparison of active site residues among them, a commercial bioinformatics tool was used to create a family wide structure-based alignment of around 13,000 sequences. Based on statistical analyses of this alignment, structural inspections and literature evaluation, active site residues crucial for certain specificities within this family have been identified. By investigating the ingenious active site designs that enable such a plethora of reactions, and by identifying sets of functional important residues termed ‘active site fingerprints’, the understanding of catalysis in this enzyme family could be broadened. Furthermore, these functional important residues can on the one hand be applied to predict the specificity of uncharacterised enzymes, if a fingerprint is matched. On the other hand, if no fingerprint is matched, they can help to discover yet unknown activities or mechanisms to achieve a known specificity. We exemplified the latter case by functionally characterising a Bacillus anthracis enzyme with the crystal structure 3N5M, whose substrate specificity was unknown and could not be predicted. The 3N5M enzyme was found to possess ωAA:pyr TA and (S)-ATA activity even though it lacks the above-mentioned ‘flipping’ arginine. Based on molecular dynamics simulations we were able to propose an alternative mechanism for dual substrate recognition in the B. anthracis ωAA:pyr TA. By these findings the understanding of the requirements for (S)-ATA activity could be further broadened and a functional knowledge gap within the class III TA family was closed. The active site residue composition in 3N5M is now connected to enzymatic function and may be applied for future specificity predictions.