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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.
Geopolymers (GPs) are inorganic binders created by adding alkaline solution (e.g. KOH) to silicates such as furnace slag, fly ash or clay to dissolve Si and Al that polymerises and precipitates to form an inorganic binder material while hardening. GP properties are similar to ordinary Portland cement regarding their high compressive strength or low shrinkage but they are particularly notable for a high resistance to acid and fire. However, the most significant advantage of GP cements is their low CO2 footprint. The most common clay used as GP raw material is kaolin. The aim of this study is to investigate the suitability of illitic clays as a cheaper alternative to kaolin and determine the necessary preparation steps required to produce effective GP binder materials. Three clays dominated by dioctahedral 2:1 layer silicates, in particular interstratifications of mica and smectite were investigated: (1) Illitic clay from Friedland, Northern Germany, containing an irregularly stacked illite-smectite interstratification (R0 I-S), (2) rectorite from Arkansas, USA, as a regular interstratification of mica and smectite, and (3) clay stated as “sárospatakite” from Füzérradvány clay deposit, Northern Hungary, containing a long range ordered I-S (R3). The three types of I-S interstratification-rich clays were extensively characterised and the Friedland clay, as the most probable raw material for GP production, was studied in more detail including several size fraction analyses. These results are used to investigate and determine the parameters necessary to produce suitable precursors for GP binders. Different approaches of clay activation to yield a highly reactive material by milling and heating were examined. Milling was found to be suitable as a preparation step after heating breaking up sintering aggregates to create pathways for the alkaline solution, but not as a substitute for heating. Important parameters for the precursor design such as temperature, time, and heating rate are determined and discussed. Geopolymerisation is considered to be a multi-parameter system and is influenced strongly by the degree of dehydroxylation, Si:Al ratio, or amount of 5-fold coordinated Al. However, in contrast to kaolin-based systems, none of these parameters explain why the illitic Friedland clay heated to 875 °C was found to be most suitable for GP binders. Based on leaching experiments and specific surface area (AS) measurements of the heated Friedland clay, a conceptual model is presented to explain the observed relationship between the heating temperature and the subsequent compressive strength of the GP cement. An optimum between the counteracting reactions of decreasing AS (fewer particles must be covered with GP phase) and decreasing Si+Al dissolved (less GP phase created) is necessary, which exists at 875 °C for the Friedland clay. In this state enough GP phase is created to bind all remaining sintering aggregates to form a cement with high compressive strength. This relationship can be expressed as (Si+Al) / AS (sum of dissolved Si and Al divided by the surface area of grains that must be covered with GP phase), and can be used as a predictive tool for determining the optimal heating temperature. The results presented in this thesis indicate that illitic clays are suitable raw materials as GP binders if the necessary preparation steps of dehydroxylation, sintering and grinding are made. Proxies used to evaluate the optimal conditions for making GP binders are determined including the (Si+Al) / AS ratio as a key relationship that controls the cementation process and determines its ultimate hardness.