University of Groningen Characterization and computation-supported engineering of an ω-transaminase Meng, Qinglong DOI: 10.33612/diss.172243517 IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2021 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Meng, Q. (2021). Characterization and computation-supported engineering of an ω-transaminase. University of Groningen. https://doi.org/10.33612/diss.172243517 Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 23-09-2021 SUMMARY AND PERSPECTIVES Summary ω-Transaminases (ω-TAs) are PLP-dependent enzymes with growing importance for the conversion of ketones to amines. The latter are used in a diversity of applications, such as the synthesis of fine chemicals. The high enantioselectivity of ω-TAs that is often observed in asymmetric amination reactions makes them especially attractive for the preparation of chiral building blocks for pharmaceutical synthesis. However, the use of ω-TAs in industrial biocatalysis is often hampered by enzyme instability and a rather limited substrate scope. The work described in this thesis is aimed at improving relevant properties of the fold-type-I homodimeric ω-TA from Pseudomonas jessenii (PjTA) via protein engineering, using computational design to find the necessary mutations (Scheme 1). Scheme 1. The framework for making PjTA applicable in the synthesis of chiral amines. 157 The transaminase PjTA was previously discovered and crystallized by our group. It naturally converts 6-aminohexanoic acid to 6-oxohexanoic acid in the caprolactam biodegradation pathway. In Chapter 2 we investigated the substrate scope of PjTA with 34 different amino donors, including aliphatic amines, aromatic amines, and proteinogenic as well as non- proteinogenic amino acids. For this we used a coupled enzyme assay that detects formation of alanine by linking it to NADH-dependent regeneration to pyruvate by alanine dehydrogenase. PjTA displayed decent activities towards aromatic amines such as (S)-1-phenylethylamine, benzylamine, and 4-phenylbutylamine, and also with aliphatic amines like 1-aminoheptane and 1- aminohexane. Non-proteinogenic amino acids that were accepted included 6-aminohexanoic acid, 5-aminopentanoic acid and 4-aminobutanoic acid. The activity differences with various substrates were explored by docking simulations. In comparison the well-studied ω-TAs from Chromobacterium violaceum (CvTA) and Vibrio fluvialis (VfTA), PjTA displayed a preference for 6-aminohexanoic acid and 4-aminobutanoic acid. The modeled structures of the external aldimines of these two amino acids showed that phenylalanine side chains in CvTA (Phe89) and VfTA (Phe85) could restrict space at the active site entrance by pointing towards a conserved arginine (Arg416 in CvTA, Arg415 in VfTA). At the corresponding position of this phenylalanine, PjTA possesses the smaller Ser87 which may provide a more spacious active site entrance allowing Arg417 to form a salt bridge with the carboxylate group of an ω-amino acid. This slight difference in active site geometry allows a dual interaction of Arg417 with the substrate and the presence of Ser87 may play a role in the role of this specific enzyme in the caprolactam biodegradation pathway. In view of the potential applications of PjTA as a biocatalyst for the synthesis of valuable amines and the very modest stability of the wild-type enzyme, PjTA was stabilized by computational protein engineering (FRESCO) as described in Chapter 3. According to previous research, the poor stability of dimeric ω-TAs may be due to loss of the aminated cofactor PMP, which was proposed to diffuse out of the active site after the first half-reaction, which is then followed by irreversible denaturation. Subunit dissociation may facilitate this cofactor release. After computational prediction and experimental verification of mutations that enhance stability, the spatial distribution of the best stabilizing mutations and the extent of stabilization indicated that the subunit interface was critical for stability. After a rational combination of confirmed stabilizing app app mutations, two robust variants called PjTA-R4 (∆Tm = +18 °C) and PjTA-R6 (∆Tm = +23 °C) were obtained. These variants were more active at their respective higher optimum temperatures, more tolerant to cosolvents (DMSO and methanol) present in reaction mixtures, and better accepted high concentrations of the amine donor isopropylamine than the wild-type PjTA. With PjTA-R6, the yield of (S)-1-phenylethylamine in reaction mixtures increased to 92% (ee > 99%) under harsh reaction conditions (1 M isopropylamine as the amino donor, 100 mM acetophenone with 20% DMSO at 56 °C). The crystal structures of the PjTA-R4 and PjTA-R6 variants were solved and mostly confirmed the expected structural changes. A rarely described stabilization 158 mechanism, i.e. removal of steric strain, was identified as the effect of the most stabilizing mutation I154V. In short, this enzyme stability engineering study indicates that computational interface redesign can be a rapid and powerful strategy for the stabilization of an ω-TA. Based on the activity of PjTA, which naturally acts on aliphatic substrates as amine donor (Chapter 2), the catalytic activity of the robust variant PjTA-R6 in the synthesis of optically pure aliphatic amines from ketones was explored using the cheap amino donor isopropylamine (Chapter 4). The results showed that PjTA-R6 displayed better performance in terms of product yield and enantioselectivity (ee > 95%) in the synthesis of ten relevant aliphatic amines than the homologous enzymes CvTA and VfTA. Since a salt bridge between the conserved Arg417 and the carboxylate group of a keto acid acting as amino acceptor is an important and conserved feature of the active site geometry of these transaminases, it was considered that the iminium function of Arg417 could provide repulsive interactions with aliphatic substrates lacking a carboxylate group. Thus, mutant R417L was constructed since it might better accept aliphatic amines. The results showed that the PjTA-R6-R417L mutant displayed a similar performance as PjTA-R6, indicating the switching arginine Arg417 is indeed dispensable when a carboxylate functionality in the substrate is absent. For a set of aliphatic amines, the Rosetta Interface Energies of PjTA-R6 and VfTA by Rosetta docking simulations of the external aldimines exhibited a clear correlation with the yield of amine that was obtained. The docked structures revealed differences between PjTA-R6 and VfTA in switch-on/off positions for the conserved arginine, which explained some of the differences between these enzymes when producing aliphatic amines. After obtaining two highly stable variants of PjTA (Chapter 3), the substrate scope of the most robust variant PjTA-R6 was expanded (Chapter 5). Previous studies described that the substrate scope of ω-TAs in asymmetric amination reactions is limited due to steric hindrance in the active site binding pockets that should accommodate the rest groups connected to the carbonyl carbon of the ketone. A large and a small binding pocket can be distinguished, where in case of PjTA the large binding pocket overlaps with the active site tunnel that is occupied by the carboxyalkyl group in the crystal structure. Many bulky amines cannot be produced by PjTA and related ω-TAs. In Chapter 5, six bulky amines were selected for which PjTA-R6 has no detectable activity. A computational protein engineering strategy was explored to reshape the binding pockets of PjTA-R6. Docking simulations were used to construct models of PjTA-R6 with the external aldimine form of the target substrates bound, and the binding energies were minimized using the Rosetta search algorithm. This algorithm searches for variants with low energy by varying the identities and rotamer conformations of a chosen set of residues surrounding the bound substrate. In total seven residues (Met54, Leu57, Trp58, Tyr151, Ala230, Ile261 and Arg417) in the large binding pocket and one residue in the small binding pocket (Phe86) were included in the search space for all six substrates. The potentially improved variants with low Rosetta interface energy were further filtered through visual inspection and for each target substrate a small library was created for experimental verification. These six small libraries contained 40 unique designs in total, and when tested in the laboratory 38 of these 40 mutants indeed produced the targeted enantiopure 159 amine (ee > 99%). Moreover, the stability of most mutants was not compromised by the introduced mutations. It appeared that the yields of six selected bulky amines were strongly correlated to Rosetta interface