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Item Open Access The Bacteroidetes Aequorivita sp. and Kaistella jeonii produce promiscuous esterases with PET-hydrolyzing activity(2022) Zhang, Hongli; Perez-Garcia, Pablo; Dierkes, Robert F.; Applegate, Violetta; Schumacher, Julia; Chibani, Cynthia Maria; Sternagel, Stefanie; Preuss, Lena; Weigert, Sebastian; Schmeisser, Christel; Danso, Dominik; Pleiss, Jürgen; Almeida, Alexandre; Höcker, Birte; Hallam, Steven J.; Schmitz, Ruth A.; Smits, Sander H. J.; Chow, Jennifer; Streit, Wolfgang R.Certain members of the Actinobacteria and Proteobacteria are known to degrade polyethylene terephthalate (PET). Here, we describe the first functional PET-active enzymes from the Bacteroidetes phylum. Using a PETase-specific Hidden-Markov-Model- (HMM-) based search algorithm, we identified several PETase candidates from Flavobacteriaceae and Porphyromonadaceae. Among them, two promiscuous and cold-active esterases derived from Aequorivita sp. (PET27) and Kaistella jeonii (PET30) showed depolymerizing activity on polycaprolactone (PCL), amorphous PET foil and on the polyester polyurethane Impranil® DLN. PET27 is a 37.8 kDa enzyme that released an average of 174.4 nmol terephthalic acid (TPA) after 120 h at 30°C from a 7 mg PET foil platelet in a 200 μl reaction volume, 38-times more than PET30 (37.4 kDa) released under the same conditions. The crystal structure of PET30 without its C-terminal Por-domain (PET30ΔPorC) was solved at 2.1 Å and displays high structural similarity to the IsPETase. PET30 shows a Phe-Met-Tyr substrate binding motif, which seems to be a unique feature, as IsPETase, LCC and PET2 all contain Tyr-Met-Trp binding residues, while PET27 possesses a Phe-Met-Trp motif that is identical to Cut190. Microscopic analyses showed that K. jeonii cells are indeed able to bind on and colonize PET surfaces after a few days of incubation. Homologs of PET27 and PET30 were detected in metagenomes, predominantly aquatic habitats, encompassing a wide range of different global climate zones and suggesting a hitherto unknown influence of this bacterial phylum on man-made polymer degradation.Item Open Access Mechanistic studies on the DNA methyltransferases DNMT3A and DNMT3B(2021) Dukatz, Michael; Jeltsch, Albert (Prof. Dr.)In this work, both regulatory and catalytic mechanisms of de novo methyltransferases were investigated, which include interactions with other proteins and the specific recognition of the substrate sequence. Another part of this work strived to elucidate how enzymatic generation of 3-methylcytosine by DNMT3A can occur.Item Open Access Assembly of a Rieske non-heme iron oxygenase multicomponent system from Phenylobacterium immobile E DSM 1986 enables pyrazon cis-dihydroxylation in E. coli(2021) Hunold, Andreas; Escobedo-Hinojosa, Wendy; Potoudis, Elsa; Resende, Daniela; Farr, Theresa; Syrén, Per-Olof; Hauer, BernhardPhenylobacterium immobile strain E is a soil bacterium with a striking metabolism relying on xenobiotics, such as the herbicide pyrazon, as sole carbon source instead of more bioavailable molecules. Pyrazon is a heterocyclic aromatic compound of environmental concern and its biodegradation pathway has only been reported in P. immobile. The multicomponent pyrazon oxygenase (PPO), a Rieske non-heme iron oxygenase, incorporates molecular oxygen at the 2,3 position of the pyrazon phenyl moiety as first step of degradation, generating a cis-dihydrodiendiol. The aim of this work was to identify the genes encoding for each one of the PPO components and enable their functional assembly in Escherichia coli. P. immobile strain E genome sequencing revealed genes encoding for RO components, such as ferredoxin-, reductase-, α- and β-subunits of an oxygenase. Though, P. immobile E displays three prominent differences with respect to the ROs currently characterized: (1) an operon-like organization for PPO is absent, (2) all the elements are randomly scattered in its DNA, (3) not only one, but 19 different α-subunits are encoded in its genome. Herein, we report the identification of the PPO components involved in pyrazon cis-dihydroxylation in P. immobile, its appropriate assembly, and its functional reconstitution in E. coli. Our results contributes with the essential missing pieces to complete the overall elucidation of the PPO from P. immobile.Item Open Access Modeling of biocatalytic reactions: a workflow for model calibration, selection, and validation using Bayesian statistics(2019) Eisenkolb, Ina; Jensch, Antje; Eisenkolb, Kerstin; Kramer, Andrei; Buchholz, Patrick C. F.; Pleiss, Jürgen; Spiess, Antje; Radde, NicoleWe present a workflow for kinetic modeling of biocatalytic reactions which combines methods from Bayesian learning and uncertainty quantification for model calibration, model selection, evaluation, and model reduction in a consistent statistical frame-work. Our workflow is particularly tailored to sparse data settings in which a considerable variability of the parameters remains after the models have been adapted to available data, a ubiquitous problem in many real-world applications. Our workflow is exemplified on an enzyme-catalyzed two-substrate reaction mechanism describing the symmetric carboligation of 3,5-dimethoxy-benzaldehyde to (R)-3,3',5,5'-tetramethoxybenzoin catalyzed by benzaldehyde lyase from Pseudomonas fluorescens. Results indicate a substrate-dependent inactivation of enzyme, which is in accordance with other recent studies.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 Molecular dynamics simulations of the substrate- and product specificity and mechanism of DNA- and protein lysine methyltransferases(2024) Schnee, Philipp; Jeltsch, Albert (Prof. Dr.)Protein Lysine Methyltransferases (PKMTs) regulate the epigenetic code of cells and their alteration via somatic mutations are often associated with cancer. The aim of this project is to rationalize the product and substrate specificity of this enzyme family by a combination of biochemical experiments and molecular dynamics simulations. Based on this, a detailed view of the underlying mechanism behind the disease associated mutations shall be gained, which may provide new possibilities for personalized cancer therapies.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 The triple variant K170D/N174L/D239A compensates the destabilizing effect of variant K170D/N174L in β-hydroxyacid dehydrogenase (βHAD) from Arabidopsis thaliana(2020) Schelle, Luca S.; Stockinger, Peter; Pleiss, Jürgen; Nestl, Bettina M.Chiral amines are essential building blocks in biologically active compounds, fine chemicals, agrochemicals and pharmaceuticals. In the last ten years, various enzymes were identified as new biocatalysts for chiral amine synthesis. Promising enzymes for the synthesis of primary, secondary, and tertiary amines are NADPH-dependent imine reductases (IREDs). Bioinformatics analysis revealed that IREDs are closely related to β-hydroxyacid dehydrogenases (βHADs). In recent work, we engineered the βHAD from Arabidopsis thaliana (βHAD_At) into imine-reducing enzymes by a single amino acid exchange. The exchange of the proton-donor described lysine (K170) in βHAD_At by aspartic acid, the most common amino acid at this position in R-selective IREDs, led to a 12-fold increase in activity for the model substrate 2-methylpyrroline. At the same time, the activity for the natural substrate glyoxylic acid is reduced 885-fold, resulting in a total of 8200-fold change in catalytic activity through the exchange of an amino acid. At the same time, highly decreased soluble expression has been observed by exchanging asparagine at position 174 (N174) with leucine. We thus hypothesized, that the aspartic acid residue (D239) in near proximity to N174 will stabilize the underlying α-helix. Consequently, replacement of D239 with alanine should result in soluble expression of variants containing the N174 mutations. We generated variants K170D/D239A, as well as, and tested them on imine reduction of test substrates 2-methylpyrroline, 3,4-dihydroisoquinoline and 6-phenyl-2,3,4,5-tetrahydropyridine. Due to loss of essential cofactor and precipitation of purified proteins during purification procedure, activities of variants were determined using cell lysates. Notably, variants N174L/D239A and K170D/N174L/D239A demonstrated soluble expression and imine-reducing activities of up to 98 mU per mg of variant.Item Open Access Oleathydratase katalysierte stereoselektive Hydratisierungsreaktionen kurzkettiger Alkene(2025) Härterich, Natalie; Hauer, Bernhard (Prof. Dr.)Item Open Access Distinct specificities of the HEMK2 protein methyltransferase in methylation of glutamine and lysine residues(2024) Weirich, Sara; Ulu, Gizem T.; Chandrasekaran, Thyagarajan T.; Kehl, Jana; Schmid, Jasmin; Dorscht, Franziska; Kublanovsky, Margarita; Levy, Dan; Jeltsch, AlbertThe HEMK2 protein methyltransferase has been described as glutamine methyltransferase catalyzing ERF1-Q185me1 and lysine methyltransferase catalyzing H4K12me1. Methylation of two distinct target residues is unique for this class of enzymes. To understand the specific catalytic adaptations of HEMK2 allowing it to master this chemically challenging task, we conducted a detailed investigation of the substrate sequence specificities of HEMK2 for Q- and K-methylation. Our data show that HEMK2 prefers methylation of Q over K at peptide and protein level. Moreover, the ERF1 sequence is strongly preferred as substrate over the H4K12 sequence. With peptide SPOT array methylation experiments, we show that Q-methylation preferentially occurs in a G-Q-X3-R context, while K-methylation prefers S/T at the first position of the motif. Based on this, we identified novel HEMK2 K-methylation peptide substrates with sequences taken from human proteins which are methylated with high activity. Since H4K12 methylation by HEMK2 was very low, other protein lysine methyltransferases were examined for their ability to methylate the H4K12 site. We show that SETD6 has a high H4K12me1 methylation activity (about 1000-times stronger than HEMK2) and this enzyme is mainly responsible for H4K12me1 in DU145 prostate cancer cells.