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In this work, the regioselectivity of different Baeyer-Villiger monooxygenases (BVMOs) for the conversion of selected substrates was reversed or improved by protein engineering. These studies highlight the importance of substrate positioning for the regioselectivity and that the position of the substrate can be efficiently influenced by introducing proper mutations. It was shown that the beneficial mutations for all BVMOs were partly in corresponding positions. Additionally, the sulfoxidation activity and the stability of BVMOs were targeted and improved by applying protein engineering.
In this work, the discovery, expression and characterization of new eukaryotic Baeyer-Villiger monooxygenases (BVMOs) from yeasts has been shown. A rational design of one of these enzymes led to the identification of key residues to alter the sulfoxidation activity of this group of enzymes. Additionally, in another rational design approach, the cofactor specificity of the BVMO cyclohexanone monooxygenase from Acinetobacter calcoaceticus could be substantially altered to accept the much cheaper and therefore industrially more relevant cofactor NADH.
The four stranded G-quadruplexes are important secondary structures of nucleic acids formed by guanosine-rich sequences. Besides the application as scaffold for technological applications, they are involved in many cellular processes such as gene regulation, replication, or maintenance of chromosomal ends. Characteristically, a large diversity of quadruplex structures is observed, whereas the correlation between sequence and structure is still not fully understood. In this thesis, the effects of modified nucleotides on G-quadruplexes were analyzed using NMR-spectroscopy to gain insight into driving forces determining the folding process. Contrary to DNA quadruplexes, the folding landscape of RNA structures is mostly restricted to parallel topologies. Therefore, ribose moieties were introduced into DNA sequences to isolate the effect of the additional hydroxy group. In this way, sequential CHO hydrogen bonds between the 2′-OH and the H8 of the 3′-neighbored anti conformer were identified and subsequently detected within RNA structures. In a second part, 2′-fluoro-2′-deoxyribose was incorporated at positions with guanosine in unfavored syn orientation. Instead of a changed global fold, the direction of the hydrogen bond network in the modified tetrad was reversed. This first example of tetrad inversion within a unimolecular quadruplex yielded a unique (3+1)-hybrid topology with only homopolar stacking interactions. Additionally, the effect was reproduced for another sequence and high-resolution structures were determined. Unfavored interactions between the 2′-fluorine and the narrow groove of the quadruplex were identified as a reason for different sugar conformations and consequent structural rearrangements.
The overarching goal of this work was to develop a biosensor based on functional nucleic acids. The biosensor should be modular, such that by exchange of the recognition unit, tailored biosensors could be created, allowing detecting a variety of analytes on demand. In the context of the cooperation with a company, initially, TNFalpha was chosen as an analyte. In a previous work, it was tried to build a modular aptazyme for TNFalpha that was based on four aptamers that were developed by SELEX. Here, these aptamers were investigated more closely by different methods (SPR, QCM). In the present work, it was proven beyond doubt that this attempt was not feasible. The aptamers were not able to bind the biologically active form of TNFalpha. An even more interesting finding was that a common tool to immobilize molecules to investigate their interactions with a binding partner, namely the streptavidin-biotin interaction, can strongly influence the result of the assay and causing false-positive results. Afterwards, it was decided to continue the work with a DNAzyme and modular approach was strictly refrained. It was tried to build aptazymes for TNFa or creatinine by in vitro selection, which failed. Most likely, the crucial factors were the ligands itself and the high demand on in vitro selection to select two functionalities (aptamer and catalytic activity) in parallel. This was the reason, to develop a new and a different method with streptavidin as a model analyte. The new strategy was to combine in vitro selection and rational design. The 17E-DNAzyme was chosen as catalytically active module. In preparation of the in vitro selection work, its properties were analyzed. An oligo-based inhibitor of the 17E-DNAzyme was rationally designed and its functionality was experimentally evaluated. Then, a library was designed which contained the 17E-DNAzyme, a randomized domain, and the inhibitor and its functionality was experimentally proven. The in vitro selection for the aptamer and the catalytic function were separated in two steps where the substrate strand was introduced in the second step. The knowledge about in vitro selection procedures, which was gained in the first trials with TNFalpha and creatinine was applied and could be substantially broadened. The crucial factors for the success of this process were identified. Most important steps are the amplification steps between the rounds and the in vitro selection pressure. The template concentration in the PCR has to be very low; the selection pressure has to be high. However, in fact, the exact quantity of "low" and "high" is difficult to determine exactly, it has to be individually evaluated for every amplification step, and this makes in vitro selection a method that requires a lot of experimental skills, optimization procedures, and experience. An EMSA was established and performed to qualitatively prove the affinity of the library for streptavidin in the first step of the in vitro selection method. For the second step, the in vitro selection of the catalytic function, considerable effort was done, but the in vitro selection did not succeed. Using the Biacore, the dissociation constant of the pool, which was applied in the second step of in vitro selection, was determined to be KD = 38 nM. This is very low, and by sequencing the pool it was found that the sequence variability was too low. The sequences share a cramp-like stem-loop structure, which hold the DNAzyme in an inactive conformation. This work presents valuable results for the development of biosensors based on nucleic acids, applying in vitro selection and rational design. Aptamers for streptavidin were selected. The library, which was used for this in vitro selection was structurally constrained. This obviously, represented an exceptionally good starting point for the in vitro selection. In this work, a lot of information about the development of in vitro selection systems was gained. Important work was done on establishing a click chemistry-based immobilization strategy. This work is going to fundamentally facilitate a new in vitro selection approach based on this immobilization strategy.
Investigation on the primary and secondary metabolism of marine and terrestrial endosymbionts
(2017)
Ph.D. thesis describes the metabolism of marine fungus and isolation of natural product for human use in part I and also describes earthworm endosymbiosis mechanism in part II. From the marine fungus project, three new producers have been identified for the previously reported bioactive secondary metabolites. And, from the Earthworm endosymbiosis project, the role of primary metabolites in the host fitness has been partially studied. the results outcome will be a partial contribution to microbial symbiosis.
Chiral amines represent high-value fine chemicals serving as key intermediate products in pharmaceutical, chemical and agrochemical industries. In the past decades, application of amine transaminases (ATAs) for stereoselective amination of prochiral ketones emerged to an environmentally benign and economically attractive alternative to transition metal-catalyzed asymmetric synthesis to afford optically pure amines at industrial scale. However, the restricted substrate scope of wild-type transaminases prohibited the conversion of particularly sterically demanding substrates, making protein engineering indispensable. The following thesis covers elaboration of a novel assay for transaminases (Article I) and identification and development of transaminase variants in order to achieve biocatalytic preparation of a set of pharmaceutically relevant model amines, ideally in optically pure form for both stereoisomers, preferentially using asymmetric synthesis and most preferably using isopropylamine as cost-efficient amine donor co-substrate (Article II-IV). The aforementioned target amines and the corresponding precursor ketones (see Scheme 4.1) were conceived and provided by the company F. Hoffmann-La Roche to attain suitable biocatalysts for a variety of potential intermediates for active pharmaceutical ingredients. Protein engineering of the transaminase scaffolds investigated in this thesis comprised: Initial screening for suitable starting enzyme scaffolds, structure-guided rational design of these scaffolds to enable bulky planar substrate acceptance, elaboration of a sequence motif, verification of the motif and preparative-scale asymmetric synthesis reactions (Article II). For non-planar and structurally different target substrates, namely spatially bulky or bi-cyclic bridged substrates, the transaminase variants were specifically refined and a different evolutionary route had to be pursued (Article III and Article IV). These results (Article II) represent not only the first successful endeavor to engineer a PLP-fold type I amine transaminase (commonly denoted as (S)-selective) for the conversion of highly sterically demanding substrates, but also generally expanded the scope of available fold type I amine transaminases by enzymes having a novel and exceptionally broad substrate spectrum. Aside from structure-guided rational protein engineering, as well non-rational methods, such as site-specific saturation mutagenesis or directed evolution, were applied for protein-engineering. In order to do so for all of the target compounds, a novel high-throughput solid phase activity assay for transaminases that was actually developed during the master thesis, was refined and published (Article I). In the context of this thesis, the same assay principle was as well adapted for quantification of specific activities in liquid phase (Article III). A comparison of different methodologies for developing agar plate assays and a detailed step by step protocol of our transaminase assay are illustrated in a book chapter.