Universität Stuttgart
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Item Open Access Studien zur biotechnologischen Anwendung und ökologischen Funktion von Pyrrolochinolinchinon(PQQ)-abhängigen Alkoholdehydrogenasen(2021) Wehrmann, Matthias; Hauer, Bernhard (Prof. Dr.)Item Open Access Development of artificial single and double reading domains to analyze chromatin modification patterns(2018) Mauser, Rebekka; Jeltsch, Albert (Prof. Dr.)The unstructured N-terminal tails of histone proteins carry many different post-translational modifications (PTMs), like methylation, acetylation or phosphorylation. These PTMs can alter the chromatin structure, influence the interaction of adjacent nucleosomes and serve as specific binding sites for histone interacting domains. Currently, the investigation of histone tail PTMs is mainly based on antibodies, however concerns about the specificity of these antibodies and reproducibility of data arouse. Therefore, it was one aim of this thesis to develop alternative approaches to histone tail PTM antibodies. Previous studies already showed that histone modification interacting domains (HiMIDs) can replace histone tail antibodies in a highly effective manner. As part of this work, the TAF3 PHD domain was established as new H3K4me3 specific HiMID. In peptide array binding and Far-western blot assays, the domain showed a specific interaction with H3K4me3 modifications. Also in ChIP like experiments (CIDOP: Chromatin Interacting Domain Precipitation) coupled to qPCR and next generation sequencing, the domain showed a similar performance as validated H3K4me3 antibodies. With the proposal of the histone code hypothesis the question was raised if combinations of histone modifications carry specific biological functions. However, so far, the experimental analysis of the co-occurrence of histone modification on the same nucleosome in a genome-wide manner is a challenging task. For this reason, the main aim of this work was to develop double reading domains in which two histone reading domains are fused together with a flexible linker to achieve simultaneously readout of dual histone tail modifications in a single CIDOP experiment. To validate the concept, the Dnmt3a PWWP domain and the MPP8 Chromo domain were fused together and their specific recognitions of H3K36me2/3 and H3K9me3 histone tail modifications were analyzed. Biochemical investigations like peptide arrays, Far-western blot and western blot experiments showed that both domains specifically interact with their targets and preferentially interact with double modified chromatin. Additionally, the preferred interaction with double modified chromatin could be further verified with binding pocket mutants and methyl-lysine analogues. The newly generated double domain was used in chromatin precipitation experiments to identify genome regions where both modifications are present. The genome-wide distribution of the H3K36me2/3-H3K9me3 showed that this combination of histone marks represents a novel bivalent chromatin state, which is associated with weakly transcribed genes and is enriched for binding sites of ZNF274 and SetDB1. Also in this work, mixed peptide arrays were introduced as new screening method for the efficient analysis of double reading domains. The naturally occurring double reading domain of the BPTF protein was used to demonstrate the capability of this new screening tool. BPTF contains a PHD domain, which binds to H3K4me3 and a Bromo domain, which interacts with acetyl groups of the H4 tail. Synergistic binding to both peptides was shown using the newly developed mixed peptide arrays. Additionally, in the course of this work mixed peptide arrays were used to optimize several of the designed double reading domains. Furthermore, some other double reading domains were generated in this work, like PWWP-ATRX, MPP8 Chromo domain-L-double Tudor and CBX7 Chromo domain-L-MPP8 Chromo domain and analyzed for specific dual readout. Also double reading domains with dual specificity for DNA methylation and histone marks were generated. The firstly used methyl-DNA binding domain of the MBD2 protein showed a strong binding, dominating the effect of the HiMIDs. Therefore, the weaker but still specific methyl-DNA binding domain of the MBD1 protein was used. First experiments with this new fusion constructs showed a simultaneously interaction with chromatin which is associated with DNA methylation and histone PTMs. In summary, the studies with double reading domains showed that with this novel method precipitation of double modified chromatin is possible and that the genome-wide investigation of newly studied bivalent chromatin states is feasible. Therefore, this novel approach makes it possible to analyze many different combinations of histone modifications, investigate their influence on chromatin and gain a deeper understanding of the biological role behind histone tail modification patterns.Item Open Access Identifizierung und Charakterisierung von Influenza-A-Virus Fusionsinhibitoren, die aus einem Doppelmyxovirus Hochdurchsatz-Screen hervorgingen(2019) Weißhaar, Marco; Wolf, Dieter H. (Prof. Dr.)Item Open Access Data integration and data mining for the exploration of enzymatic sequence-structure-function relationships(2018) Buchholz, Patrick C. F.; Pleiss, Jürgen (Prof. Dr.)Item Open Access Enzymatische Hydratisierung kurzkettiger Fettsäuren und Alkene(2018) Demming, Rebecca M.; Hauer, Bernhard (Prof. Dr.)Item Open Access Biochemical characterization and identification of novel substrates of protein lysine methyltransferases(2019) Schuhmacher, Maren Kirstin; Jeltsch, Albert (Prof. Dr.)The methylation of lysine side chains is a prevalent post-translational modification (PTM) of proteins, which is introduced by protein lysine methyltransferases (PKMTs). Histone methylation can have different effects on chromatin structure, lysine methylation of non-histone proteins can regulate protein/protein interactions and protein stability. For most PKMTs currently not all methylation sites are known which limits our understanding of the regulatory role of these enzymes in cells. Therefore, it is an important research aim to gain more information about the substrate spectrum of PKMTs. The identification of the substrate specificity of a PKMT is a very important step on the way to identify new PKMT methylation sites. The focus of this study was the analysis of the substrate specificity of different PKMTs by SPOT peptide arrays and based on this on the identification and validation of possible new methylation substrates. The analysis of the substrate specificity of human SUV39H2 revealed significant differences to its human homolog SUV39H1, although both enzymes methylate the same histone substrate (H3K9). SUV39H2 is more stringent than the SUV39H1, which could be demonstrated by the lack of methylation of SUV39H1 non-histone targets by SUV39H2 and by the fact that it was not possible in this study to identify non-histone substrates for SUV39H2. Kinetic studies showed that SUV39H2 prefers the unmethylated H3K9 as substrate. Moreover, it was shown that the N324K mutation of SUV39H2 which leads to a genetic disease in Labrador retrievers causes a change in folding finally leading to the inactivation of the enzyme. It had been reported by another group that the histone variant H2AX is methylated by SUV39H2. However, the sequence of H2AX K134 does not fit to the substrate specificity profile of SUV39H2 determined in the present work. Follow-up in vitro peptide and protein methylation studies indeed showed that H2AX K134 is not methylated by SUV39H2. This indicates that H2AX methylation by SUV39H2 is most probably a wrong assignment of a substrate to a PKMT. Based on already available specificity data for the SUV39H1 PKMT, the SET8 protein was validated as novel substrate in cellular studies. SET8 is a PKMT itself and it could be shown in this thesis that methylation of SET8 at residue K210 by SUV39H1 stimulated the SET8 activity. In humans, there exist different PKMTs, which methylate H3K36. For example, NSD1, NSD2 and SETD2 which were investigated in this thesis. In literature, it was shown that the oncohistone mutation K36M inactivates NSD2 and SETD2. Steady-state methylation kinetics using a peptide substrate and a K36M peptide as inhibitor revealed that NSD1 is inhibited by this histone oncomutation as well. The steady-state inhibition parameters for all enzymes showed a better binding of the PKMTs to the inhibitor peptide than to the substrate, suggesting some mechanistic similarities in target peptide interaction. The SETD2 is a methyltransferase, which is able to introduce trimethylation of H3K36. During this thesis two substrate specificity motifs of SETD2 were determined using peptide array methylation experiments. Additionally, based on the substrate specificity investigations a super-substrate at peptide and protein level was determined. Furthermore, one novel substrate (FBN1) for SETD2 was discovered and validated. The Legionella pneumophila RomA PKMT was shown previously by our collaborators to methylate H3 at K14. Based on the specificity profile of RomA determined in this study it could be shown that this enzyme methylates seven additional human non-histone proteins. Collaborators tested the methylation of one of the non-histone targets (AROS) and could demonstrate its methylation during the infection of human cells with L. pneumophila. The role of these methylation events in the infection process must be studied in future experiments.Item Open Access Alternative active site confinement by enforcing substrate pre-organization in cyclases(2023) Schell, Kristina; Hauer, Bernhard (Prof. Dr.)Confinement of an enzyme’s active site is critical to the efficiency of chemical reactions and has been recognized as an important tool for catalysis. Confined active sites facilitate the pre-organization of substrates and intermediates to control the reaction course, protect against premature quenching and provide unique products. The catalytic center activates the substrate, and its activity can be enhanced by residues surrounding the substrate in the active site, changing the local catalyst geometry, and maintaining a constrained structural and/or electronic configuration of the catalytic center. These properties are characteristic of confinement, resulting in the generation of proximity between the substrate and the catalytic center, as well as complementary binding of the substrate into the active site. Effectively, this accelerates the reaction, controls the progress of the reaction, and positions the substrate in a productive conformation. The reaction course is selectively controlled by the stabilization of intermediates and by the interaction of electron-rich residues with electron-poor molecules and vice versa. Despite these benefits, a strongly confined active site is inherently limited to compounds that resemble the native substrate, with only small deviations tolerated. This restricts the applications for new reactivities and prevents broad substrate scopes. In this work, analysis of the enzyme structure in combination with iterative saturation mutagenesis were employed for the development of biocatalysts with alternative confinement and productive substrate pre-organization. To unlock the potential of confined Brønsted acid catalysts this approach was applied on terpene synthases. These enzymes form several carbocations as transition states and intermediates, which can be selectively converted by confinement of the active site. Exploiting the potential of terpene synthases to convert modified terpene scaffolds could provide interesting building blocks with branched isoprene/terpene motifs. In addition, the control of the reaction progression to specific products rather than a mixture of products could be targeted by stabilizing carbocation intermediates or transition states. The present work demonstrates a structure-guided strategy to create an alternative confinement in the squalene hopene cyclase from Alicyclobacillus acidocaldarius (AacSHC). This strategy aims to create proximity between substrate and catalytic center and complementarity between substrate and active site to obtain productive pre organization of the substrate. This may allow the conversion of geranylacetone (GA), farnesol and farnesylacetone analogs as substrates with modified isoprene patterns. Among different rational and semi-rational approaches only variant G600M (VD1), in which the substrate tunnel was modified, yielded starting activity. Structural analysis of VD1 led to the identification of a bottleneck in the tunnel. This resulted presumably in steric interactions and proximity by decreasing average distances between the double bond of the substrate’s terminal isoprene unit and the catalytic center. Furthermore, a lower fluctuation of these distances around this mean value was observed in VD1 compared to the wild type (WT). These observations support the hypothesis of improved substrate pre-organization and confirm the creation of proximity between the substrate and the catalytic center. The development of a screening method and optimization of reaction conditions facilitated iterative saturation mutagenesis to investigate the evolvability and the potential of the approach. The positions for saturation mutagenesis targeted the shape complementarity of the active site to the GA analog dihydropseudoirone, and the finally developed variant (VD5) showed an 1174-fold increase of the total turnover number and 111-fold increase in catalytic efficiency compared to the WT. Creation of alternative active site confinement demonstrates evolvability and great potential to overcome limitations in the engineering of biocatalysts and allowed the generation of novel building blocks in preparative mg scale. Limitations in the generation of alternative confinement were approached by using lycopene cyclases. These catalysts convert linear lycopene to carotenes under physiological conditions and were mainly studied for the conversion of pseudoionones in this work. The latter substrate could not be converted by AacSHC through generation of alternative confinement. Three different lycopene cyclases were tested, of which CanLCY B showed immediate activity in converting pseudoionone to a monocyclic product. AthLCY-B and AthLCY-E initially showed no conversion of selected terpenes. After optimizing the reaction conditions, a multiple sequence alignment (MSA) was performed to identify non-conserved positions around the catalytic acid of LCY-B. It was hypothesized that these amino acids influence the confinement and the pre-organization of the substrate. Saturation mutagenesis at the identified positions improved β-Ionone formation by 4-fold and conversion by 5% with variant V335L compared to the WT. The applicability of this MSA-based alternative confinement strategy for engineering of further lycopene cyclases was demonstrated by using saturation mutagenesis of the respective residues in AthLCY-E. Under optimized conditions α-Ionone product formation increased 4.5-fold with the best performing variant AthLCY-E S359F compared to WT AthLCY-E. Application of lycopene cyclases to complement activities with challenging steric interactions demonstrated the successful expansion of the diversity for protonation reactions by biocatalysts and the successful application of an MSA-based approach to generate alternative confinement. To further investigate the generation of alternative confinement and expand the toolbox of Brønsted acid catalysis, the acid isomerization of monoterpenes catalyzed by squalene hopene cyclases (SHCs) was investigated. To access selective product formation with monoterpenes a strategy based on cation stabilization was applied to overcome the challenging pre-organization of the cyclic C10 compounds. The focus was on aromatic residues with high electron density and residues surrounding the carbocation to direct the reaction course toward a single monoterpene product. In an initial screening, four monoterpenes were converted by AacSHC, resulting in complex product mixtures. Of these, one monocyclic and one bicyclic substrate were selected for further engineering. The goal here was to increase the formation of terpinen-4-ol, a hydrated monoterpene. In addition, limonene that was not converted by AacSHC was tested. Optimization of reaction conditions and semi-rational engineering to stabilize the carbocation intermediate produced improved variants in terms of selectivity and terpinen-4-ol formation. Saturation mutagenesis of hydrophobic amino acids that interact with the docked substrate and surrounding residues resulted in variants VT3 and VS2, which had the best selectivity and the highest measured terpinene 4-ol formation, respectively. VT3 converted monocyclic terpinolene with a selectivity of 64% and with a 3.4-fold increase in total turnover number (TTN) compared to the WT. The highest terpinene-4-ol formation of 219 µM and a 2-fold increase in TTN compared to the WT was measured with the bicyclic compound sabinene. Features, such as bulkier residues at position 600, found in VT3 and VS2 are likely responsible for generation of alternative confinement by the positioning of aromatic residues, which stabilize cationic intermediates along the reaction trajectory towards terpinene-4-ol formation. Creating alternative confinement shows great potential for overcoming limitations in biocatalyst engineering. Interesting building blocks were generated and new reactivities with improved selectivity in protonation reactions have been discovered. Moreover, this strategy could be used for predicting potential hot spots in enzyme engineering campaigns and data-driven predictions of enzyme functions to decipher the catalytic potential of enzyme scaffolds.Item Open Access The PedS2/PedR2 two-component system is crucial for the rare earth element switch in Pseudomonas putida KT2440(2018) Wehrmann, Matthias; Berthelot, Charlotte; Billard, Patrick; Klebensberger, JanoschABSTRACT In Pseudomonas putida KT2440, two pyrroloquinoline quinone-dependent ethanol dehydrogenases (PQQ-EDHs) are responsible for the periplasmic oxidation of a broad variety of volatile organic compounds (VOCs). Depending on the availability of rare earth elements (REEs) of the lanthanide series (Ln3+), we have recently reported that the transcription of the genes encoding the Ca2+-utilizing enzyme PedE and the Ln3+-utilizing enzyme PedH are inversely regulated. With adaptive evolution experiments, site-specific mutations, transcriptional reporter fusions, and complementation approaches, we now demonstrate that the PedS2/PedR2 (PP_2671/PP_2672) two-component system (TCS) plays a central role in the observed REE-mediated switch of PQQ-EDHs in P. putida. We provide evidence that in the absence of lanthanum (La3+), the sensor histidine kinase PedS2 phosphorylates its cognate LuxR-type response regulator PedR2, which in turn not only activates pedE gene transcription but is also involved in repression of pedH. Our data further suggest that the presence of La3+ lowers kinase activity of PedS2, either by the direct binding of the metal ions to the periplasmic region of PedS2 or by an uncharacterized indirect interaction, leading to reduced levels of phosphorylated PedR2. Consequently, the decreasing pedE expression and concomitant alleviation of pedH repression causes - in conjunction with the transcriptional activation of the pedH gene by a yet unknown regulatory module - the Ln3+-dependent transition from PedE- to PedH-catalyzed oxidation of alcoholic VOCs. IMPORTANCE The function of lanthanides for methanotrophic and methylotrophic bacteria is gaining increasing attention, while knowledge about the role of rare earth elements (REEs) in nonmethylotrophic bacteria is still limited. The present study investigates the recently described differential expression of the two PQQ-EDHs of P. putida in response to lanthanides. We demonstrate that a specific TCS is crucial for their inverse regulation and provide evidence for a dual regulatory function of the LuxR-type response regulator involved. Thus, our study represents the first detailed characterization of the molecular mechanism underlying the REE switch of PQQ-EDHs in a nonmethylotrophic bacterium and stimulates subsequent investigations for the identification of additional genes or phenotypic traits that might be coregulated during REE-dependent niche adaptation.Item Open Access Development of a chemoenzymatic (-)-menthol synthesis(2018) Kreß, Nico; Hauer, Bernhard (Prof. Dr.)Biocatalysis is an emergent research area for the development of efficient and sustainable synthesis processes. A crucial milestone for the better applicability of biocatalysts thereby consists of the increasing knowledge of the adaptability of enzymes for distinct synthetic needs like the conversion of specific molecular structures with defined selectivity. In addition, it is equally important to demonstrate that such novel catalysts are combinable among themselves and with established non enzymatic catalysts to enable unexplored synthetic routes. Using the example of the chemoenzymatic synthesis of (-)-menthol from citral, this work therefore addresses the development and applicability of such evolved enzyme catalysts for the synthesis of an industrially relevant molecule. In this complementary synthetic route inspired from an existing industrial process, a mixture of citral isomers is reduced to citronellal using an R-selective ene reductase. In a subsequent Prins reaction, the selective cyclization of R-citronellal to (-)-isopulegol is achieved by the application of an engineered squalene hopene cyclase variant. The final reduction to (-)-menthol proceeds by hydrogenation on a palladium catalyst. Especially the first catalytic step enables an immediate synthetic advantage in comparison to the currently performed industrial process. So far, no catalyst is applied converting both isomers of citral R-selectively at the same time. Both isomers have to be separated under high energy expenditure by distillation prior to reduction. No enzymatic catalyst is described displaying this reactivity yet. As, however, the opposite enantioconvergent S-selective citral reduction by ene reductases is known, the development of an enzyme catalyst constituted an attractive solution for this limitation. Hence, a focus of the work laid on the inversion of the S-selectivity of the citral reduction by NCR ene reductase from Zymomonas mobilis by enzyme engineering. The studies started by characterization of the citral reduction by NCR wild type. Next to the determination of the course of the reaction over time, semi empiric quantum mechanics calculations on the oxidative half reaction of this conversion were carried out. The calculations suggest a so far undescribed catalytic role of an arginine at position 224 for a facilitated hydride transfer and a more complex proton shift involving water molecules in the reaction. The subsequently performed engineering comprised the identification of selectivity determining amino acid positions W66, Y177, I231 and F269 in the active site of the enzyme followed by their variation in an iterative combinatorial fashion. In order to enable the analysis of the multitude of generated enzyme variants, a whole cell screening was developed using chiral gas chromatography. Thereby, the triple variant W66A/I231R/F269V was created converting E/Z-citral in the whole system to R-citronellal with an enantiomeric excess of 89 %. It could be determined that a cell induced citral isomerization leads to increased enantioselectivity in comparison to using purified enzyme. Especially for the influence of the selectivity determining positions W66 and I231 an increased understanding of structure function relations was achieved during the course of semi rational enzyme evolution by the separated analysis of single citral isomers and by supportive in silico analyses like docking and molecular dynamics simulations. The subsequent integration of the established variant A419G/Y420C/G600A of the squalene hopene cyclase from Alicyclobacillus acidocaldarius is remarkable catalyzing the Prins cyclization to (-)-isopulegol with an enantiomeric excess of 99 % and a diastereoselectivity of 90 %. In this context, the enzyme’s underlying Brønsted acid chemistry could be evolved towards the in nature unknown Prins reaction reactivity. In this work it could be shown that enzyme catalysts acquired by such chemical inspection can be implemented in application oriented synthetic routes. In combination with the developed selective ene reductase, the bienzymatic cascade to (-)-isopulegol was successfully performed and characterized. For the final reduction to (-)-menthol an established heterogeneous catalyst like palladium on charcoal could be applied under hydrogen atmosphere. This demonstrates nicely that novel biocatalysts can be combined with approved synthetic processes. With the attained insights, highly valuable (-)-menthol was made accessible for the first time by a chemoenzymatic cascade using an isomeric mixture of citral on preparative scale with 7 % isolated yield. This work not only highlights different strategies for the development of novel biocatalysts, but also contributes to their possible synthetic applicability in the synthesis of industrially relevant molecules.Item Open Access Regioselective hydration of terpenoids using cofactor-independent hydratases(2019) Schmid, Jens; Hauer, Bernhard (Prof. Dr.)