03 Fakultät Chemie

Permanent URI for this collectionhttps://elib.uni-stuttgart.de/handle/11682/4

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Open Access
Accessing nature's terpenome by squalene-hopene cyclase mediated asymmetric cationic cyclization cascades
(2022) Schneider, Andreas; Hauer, Bernhard (Prof. Dr.)
Die Arbeit befasst sich mit der Erschließung der Squalen-Hopen Zyklase als Biokatalysator für die organische Chemie. Dadurch werden Synthesewege zu hochkomplexen Terpenen stark vereinfacht.
Open Access
Active-site loop variations adjust activity and selectivity of the cumene dioxygenase
(2021) Heinemann, Peter M.; Armbruster, Daniel; Hauer, Bernhard
Active-site loops play essential roles in various catalytically important enzyme properties like activity, selectivity, and substrate scope. However, their high flexibility and diversity makes them challenging to incorporate into rational enzyme engineering strategies. Here, we report the engineering of hot-spots in loops of the cumene dioxygenase from Pseudomonas fluorescens IP01 with high impact on activity, regio- and enantioselectivity. Libraries based on alanine scan, sequence alignments, and deletions along with a novel insertion approach result in up to 16-fold increases in activity and the formation of novel products and enantiomers. CAVER analysis suggests possible increases in the active pocket volume and formation of new active-site tunnels, suggesting additional degrees of freedom of the substrate in the pocket. The combination of identified hot-spots with the Linker In Loop Insertion approach proves to be a valuable addition to future loop engineering approaches for enhanced biocatalysts.
Open Access
Analysis of 3760 hematologic malignancies reveals rare transcriptomic aberrations of driver genes
(2024) Cao, Xueqi; Huber, Sandra; Ahari, Ata Jadid; Traube, Franziska R.; Seifert, Marc; Oakes, Christopher C.; Secheyko, Polina; Vilov, Sergey; Scheller, Ines F.; Wagner, Nils; Yépez, Vicente A.; Blombery, Piers; Haferlach, Torsten; Heinig, Matthias; Wachutka, Leonhard; Hutter, Stephan; Gagneur, Julien
Background. Rare oncogenic driver events, particularly affecting the expression or splicing of driver genes, are suspected to substantially contribute to the large heterogeneity of hematologic malignancies. However, their identification remains challenging. Methods. To address this issue, we generated the largest dataset to date of matched whole genome sequencing and total RNA sequencing of hematologic malignancies from 3760 patients spanning 24 disease entities. Taking advantage of our dataset size, we focused on discovering rare regulatory aberrations. Therefore, we called expression and splicing outliers using an extension of the workflow DROP (Detection of RNA Outliers Pipeline) and AbSplice, a variant effect predictor that identifies genetic variants causing aberrant splicing. We next trained a machine learning model integrating these results to prioritize new candidate disease-specific driver genes. Results. We found a median of seven expression outlier genes, two splicing outlier genes, and two rare splice-affecting variants per sample. Each category showed significant enrichment for already well-characterized driver genes, with odds ratios exceeding three among genes called in more than five samples. On held-out data, our integrative modeling significantly outperformed modeling based solely on genomic data and revealed promising novel candidate driver genes. Remarkably, we found a truncated form of the low density lipoprotein receptor LRP1B transcript to be aberrantly overexpressed in about half of hairy cell leukemia variant (HCL-V) samples and, to a lesser extent, in closely related B-cell neoplasms. This observation, which was confirmed in an independent cohort, suggests LRP1B as a novel marker for a HCL-V subclass and a yet unreported functional role of LRP1B within these rare entities. Conclusions. Altogether, our census of expression and splicing outliers for 24 hematologic malignancy entities and the companion computational workflow constitute unique resources to deepen our understanding of rare oncogenic events in hematologic cancers.
Open Access
Analysis of the substrate specificity of the SMYD2 protein lysine methyltransferase and discovery of novel non-histone substrates
(2019) Weirich, Sara; Schuhmacher, Maren Kirstin; Kudithipudi, Srikanth; Lungu, Cristiana; Ferguson, Andrew D.; Jeltsch, Albert
The SMYD2 protein lysine methyltransferase methylates various histone and non-histone proteins and is overexpressed in several cancers. Using peptide arrays, we investigated the substrate specificity of the enzyme, revealing a recognition of leucine (or weaker phenylalanine) at the -1 peptide site and disfavor of acidic residues at the +1 to +3 sites. Using this motif, novel SMYD2 peptide substrates were identified, leading to the discovery of 32 novel peptide substrates with a validated target site. Among them, 19 were previously reported to be methylated at the target lysine in human cells, strongly suggesting that SMYD2 is the protein lysine methyltransferase responsible for this activity. Methylation of some of the novel peptide substrates was tested at the protein level, leading to the identification of 14 novel protein substrates of SMYD2, six of which were more strongly methylated than p53, the best SMYD2 substrate described so far. The novel SMYD2 substrate proteins are involved in diverse biological processes such as chromatin regulation, transcription, and intracellular signaling. The results of our study provide a fundament for future investigations into the role of this important enzyme in normal development and cancer.
Open Access
Analyzing bioaccessibility of polyphenols in six commercial and six traditional apples (Malus domestica Borkh.) during in vitro and ex vivo oral digestion
(2023) Kaeswurm, Julia Anna Helene; Sempio, Rebecca; Manca, Federica; Burandt, Melanie Regina; Buchweitz, Maria
Scope: Apples are an important polyphenol (PP) source. To compare the health benefits of traditional and commercial varieties, the phenolic contents and profiles as well as their release from the matrix (bioaccessibility) during oral digestion are determined. Furthermore, based on these data the proposed beneficial effect of PP on the variety specific allergenicity is discussed. Methods and results: Phenolics are quantified by HPLC‐DAD. Total phenolic contents (TPC) are in the range of 111-645 and 343-1950 mg 100 g−1 dry weight for flesh and peel, respectively. Matrix release during oral digestion is investigated ex vivo, with centrifuged and non‐centrifuged human saliva and in vitro with simulated saliva fluid (SSF). The overall bioaccessibility is similar in all digestion media, ranging between 40-80% and 39-65% of the TPC in flesh and peel, respectively. Analyzing the correlation among Mal‐d 1‐allergen‐content, unoxidized PP, and the allergenic potential for the samples reveals a negligible effect of phenolics. Conclusion: Due to higher phenolic contents in combination with a similar release, increased PP concentrations in the oral phase and an improved uptake of PP from traditional varieties are assumed. However, the proposed beneficial effect of phenolics on allergenicity cannot be confirmed.
Open Access
Application of dual reading domains as novel reagents in chromatin biology reveals a new H3K9me3 and H3K36me2/3 bivalent chromatin state
(2017) Mauser, Rebekka; Kungulovski, Goran; Keup, Corinna; Reinhardt, Richard; Jeltsch, Albert
Histone post-translational modifications (PTMs) play central roles in chromatin-templated processes. Combinations of two or more histone PTMs form unique interfaces for readout and recruitment of chromatin-interacting complexes, but the genome-wide mapping of co-existing histone PTMs remains an experimentally difficult task. We introduce here a novel type of affinity reagents consisting of two fused recombinant histone modification interacting domains (HiMID) for direct detection of doubly modified chromatin. To develop the method, we fused the MPP8 Chromodomain and DNMT3A PWWP domain which have a binding specificity for H3K9me3 and H3K36me2/3, respectively. We validate the novel reagent biochemically and in ChIP applications and show its specific interaction with H3K9me3-H3K36me2/3 doubly modified chromatin. Modification specificity was confirmed using mutant double-HiMIDs with inactivated methyllysine binding pockets. Using this novel tool, we mapped co-existing H3K9me3-H3K36me2/3 marks in human cells by chromatin interaction domain precipitation (CIDOP). CIDOP-seq data were validated by qPCR, sequential CIDOP/ChIP and by comparison with CIDOP- and ChIP-seq data obtained with single modification readers and antibodies. The genome-wide distribution of H3K9me3-H3K36me2/3 indicates that it represents a novel bivalent chromatin state, which is enriched in weakly transcribed chromatin segments and at ZNF274 and SetDB1 binding sites.
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, Bernhard
Phenylobacterium 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.
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, Juergen; 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.
Open Access
Biocatalytic allylic oxidation with bacterial P450 monooxygenases
(2023) Bogazkaya, Anna Maria; Hauer, Bernhard (Prof. Dr.)
Diese Forschungsarbeit konzentriert sich auf die Identifizierung und Charakterisierung geeigneter Cytochrom P450 Monooxygenasen (CYPs) in Streptoyceten. Durch eine Dünnschichtchromatographie (DC)-Methode wurden zwei vielversprechende Streptomyces-Stämme identifiziert. Aus diesen Stämmen wurden die Enzyme CYP105A1, CYP105B1, CYP105D5 und CYP170A1 kloniert und in E. coli zur weiteren Untersuchung exprimiert. Da die natürlichen Redoxpartner dieser CYPs unbekannt sind, wurde ein heterologes Redoxsystem aus Pseudomonas putida verwendet. Diese Arbeit liefert wichtige Einblicke für die industrielle Anwendung dieser Enzyme in der biokatalytischen Oxidation. Interessanterweise zeigten die Studienergebnisse, dass drei der ausgewählten CYPs (CYP105B1, CYP105D5 und CYP170A1) zyklische Modell Substrate wie α- oder β-Ionon auf eine regioselektive Weise zu Allylalkoholen hydroxylierten. Diese Reaktion erzeugt spezifisch Allylalkohole, was eine hohe Regioselektivität zeigt - das heißt, die Enzyme zielten gezielt auf bestimmte Teile der Moleküle ab, um sie zu oxidieren. Andererseits führte die Oxidation von azyklischen Substraten mit diesen Enzymen zu einer Mischung von regioisomeren Epoxiden. Dies zeigt eine Vielseitigkeit der Enzyme, aber auch eine Variation in der Produktverteilung, abhängig von der Struktur des Ausgangssubstrats. Diese Ergebnisse tragen dazu bei, das Verständnis für die Funktionsweise und die potenzielle Anwendung von CYPs in der Synthese von spezifischen und wertvollen Produkten zu erweitern. Sie unterstreichen das Potenzial von CYPs als leistungsstarke Werkzeuge in der biotechnologischen und pharmazeutischen Industrie, wo selektive und effiziente Oxidationsmethoden dringend benötigt werden. Zur Vereinfachung der Anwendung dieser Ganzzellkatalysatoren in industriellen Prozessen wurde ein innovativer Ansatz zur Immobilisierung dieser Zellen in einer Latex SF091-Schicht erarbeitet, dank der Zusammenarbeit mit Prof. Michael Flickinger von der NC State University, USA. Nach erfolgreicher Immobilisierung der Zellen zeigten die Reaktionen eine bemerkenswerte Produktselektivität: Bei der Oxidation von Nerol mit CYP154E1 wurden 97% 8-Hydroxynerol und nur 3% 2,3Epoxynerol erzeugt.
Open Access
Biochemical analysis of DNA- and protein methyltransferases using recombinant designer nucleosomes
(2022) Bröhm, Alexander; Jeltsch, Albert (Prof. Dr.)
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.
Open Access
Biochemical characterization of protein lysine methyltransferases-regulation, specificity and effect of somatic cancer mutants
(2023) Khella, Mina S.; Jeltsch, Albert (Prof. Dr.)
Open Access
Biochemical investigations of multivalent chromatin reading domains
(2024) Choudalakis, Michel; Jeltsch, Albert (Prof. Dr.)
In eukaryotes, the negatively charged nuclear DNA wraps around cationic histone proteins to form nucleosomes and compact the genetic information. Histones carry several post-translational modifications (PTMs) that appear in combinatorial patterns. These marks are interpreted by non-covalent interactions with proteins containing histone modification interacting domains (HiMIDs), also known as “reader” domains. Thirty years ago, it was proposed that the histone marks act as signals in the regulation of transcription and other chromatin functions. With time, this concept has been refined to suggest that combinatorial patterns of marks represent context-specific signals, termed a 'histone code'. It functions as one of the epigenetic regulatory mechanisms, which control reversible and heritable changes in cellular phenotype. Intermolecular models demonstrate thermodynamic benefits from multivalent engagement of nucleosomes, suggesting their widespread occurrence. However, so far only few multivalent readers are known and dissecting their function has been very challenging. This thesis focuses on HiMIDs with complex roles that simultaneously interact with two histone PTMs or two different substrates. Introducing the theoretical foundation, I discuss the thermodynamic and biological basis of how multivalent interactions can guide effector protein complexes, targeting their functions to distinct regions and chromatin states. Then, I present data from the characterisation of the readers DNMT3A-PWWP, DDX19A, and UHRF1-TTD in the context of multivalent engagement of histone PTMs and biomolecules. Starting with DNMT3A-PWWP, I quantified the binding of the wild-type (WT) and a mutant domain to histone H3K36me2/3 peptides, showing negligible differences, while my colleagues showed that the mutant has drastically reduced binding to DNA and nucleosomal substrates. I, then, studied the R-loop helicase DDX19A to demonstrate a very strong binding to H3K27me3 peptides in the nanomolar range, complementing the findings of a complex functional study. The latter showed that interaction with H3K27me3 is necessary for robust DDX19A-mediated R-loop resolution, and LSD1-target gene silencing. With UHRF1-TTD, I discovered and quantified its preferential binding to H3K4me1-K9me2/3 peptides vs H3K9me2/3 alone and engineered mutants with specific and differential binding changes leading to the discovery of a novel Kme1 read-out mechanism, based on the interaction of R207 methylene groups with the H3K4me1 methyl group and on counting the H-bond capacity of H3K4. High-throughput sequencing (HTS) data revealed strong TTD binding at chromatin sites with H3K4me1 peaks and broad H3K9me2/3 signal, which are enriched on enhancers and promoters of cell-type specific genes at the flanks of cell-type specific transcription factor binding sites. Data from the full-length protein in mouse and human cells evidenced the physiological role of the H3K4me1-K9me2/3 double marks in TTD-mediated UHRF1 recruitment. To further illustrate this point, I investigated UHRF1-dependent silencing of repeat elements (RE). To this end, I developed RepEnTools, improving the previously available programmes for RE enrichment analysis in chromatin pulldown studies by leveraging new tools, with carefully chosen and validated settings, enhancing accessibility, and adding some key functions. RepEnTools analyses showed that chromatin binding of hUHRF1-TTD and full-length mUHRF1 was strongly enriched on different REs promoters with the H3K4me1-K9me3 double mark where UHRF1 represses their expression. The data suggest a novel functional role for the H3K4me1-K9me3 signal of the histone code that is both sequence independent and conserved in two distinct mammals. Taken together, the work presented here is consistent with and supports the histone code theory, best illustrated by UHRF1-TTD which binds a specific double mark that has a biological meaning going beyond the meaning of the individual marks. In this thesis, I presented various mechanisms that influence epigenomic regulation, including chromatin 3D-architecture, accessibility, transcription factor recruitment, and chromatin marks. Especially in the context of UHRF1-TTD functions, I discussed how DNA, RNA, histones, and covalent modifications thereof interweave to produce the signalling network necessary throughout the lifetime of the mammalian cell, during differentiation, development and every other phase of life. Thus, within the three-dimensional scaffold of chromatin structures these biomolecules and their modifications collectively form the context-specific network of effectors and maintainers of the epigenomic modifications. The ways in which they influence transcription and translation are only now becoming unravelled. Hence, the recent data suggest the existence of not just a histone code, but a 3D-chromatin modification code, which dictates how biomolecules and their modifications collectively implement epigenomic regulation by interactions along the chromatin and through 3D space. As shown in these projects, readers commonly use the mechanism of multivalent interactions to interpret such contextual signals and guide epigenomic effectors to their targets. The tools and workflows that were developed and applied in this work can be employed to reveal more instances of refined read-out among HiMIDs. Additionally, I leveraged my experience with fluorescence spectroscopy and made contributions to another two published studies. The first study demonstrated that the DNMT3A-ADD Zn-finger domain, which is a known H3K4me0 reader, also binds to a domain from the MECP2 protein. The association was quantified, and the specificity demonstrated with a binding deficient triple mutant. This interaction offers complex additional regulation options to DNMT3A and MECP2, in interplay with the histone code. The second study focused on SETD2, a H3K36me3 depositing enzyme, and the mechanism of its preference for a designed “super substrate” peptide. By elegantly combining computational simulations and experimental data, the study demonstrated that an H3 peptide substrate predominantly exists in an extended conformation in solution, while the super substrate forms a hairpin conformation. Upon binding to the enzyme, the hairpin is opened and the super substrate adopts a similar conformation as the canonical substrate. These results highlighted the dynamic nature of solubilised peptides' conformations, their impact on protein-protein interactions, and the significance of dynamic conformational changes in interactions.
Open Access
Charakterisierung der Substratspezifität von Protein-Methyltransferasen
(2023) Schnee, Philipp; Weirich, Sara; Jeltsch, Albert
The regulation of cellular activities is a key hallmark in the development of complex life forms. Among other factors, it is facilitated by protein lysine methyltransferases (PKMTs), which modify proteins in a highly specific manner and regulate their biological activities. Here, we describe methods to decipher the PKMT-substrate specificity by biochemical experiments and molecular dynamics simulations. This led to the discovery of novel PKMT substrates and rational design of even better non-natural substrates, which represent a promising starting point for the design of novel PKMT inhibitors.
Open Access
Chromatin-dependent allosteric regulation of DNMT3A activity by MeCP2
(2018) Rajavelu, Arumugam; Lungu, Cristiana; Emperle, Max; Dukatz, Michael; Bröhm, Alexander; Broche, Julian; Hanelt, Ines; Parsa, Edris; Schiffers, Sarah; Karnik, Rahul; Meissner, Alexander; Carell, Thomas; Rathert, Philipp; Jurkowska, Renata Z.; Jeltsch, Albert
Despite their central importance in mammalian development, the mechanisms that regulate the DNA methylation machinery and thereby the generation of genomic methylation patterns are still poorly understood. Here, we identify the 5mC-binding protein MeCP2 as a direct and strong interactor of DNA methyltransferase 3 (DNMT3) proteins. We mapped the interaction interface to the transcriptional repression domain of MeCP2 and the ADD domain of DNMT3A and find that binding of MeCP2 strongly inhibits the activity of DNMT3A in vitro. This effect was reinforced by cellular studies where a global reduction of DNA methylation levels was observed after overexpression of MeCP2 in human cells. By engineering conformationally locked DNMT3A variants as novel tools to study the allosteric regulation of this enzyme, we show that MeCP2 stabilizes the closed, autoinhibitory conformation of DNMT3A. Interestingly, the interaction with MeCP2 and its resulting inhibition were relieved by the binding of K4 unmodified histone H3 N-terminal tail to the DNMT3A-ADD domain. Taken together, our data indicate that the localization and activity of DNMT3A are under the combined control of MeCP2 and H3 tail modifications where, depending on the modification status of the H3 tail at the binding sites, MeCP2 can act as either a repressor or activator of DNA methylation.
Open Access
Controlling monoterpene isomerization by guiding challenging carbocation rearrangement reactions in engineered squalene‐hopene cyclases
(2024) Ludwig, Julian; Curado‐Carballada, Christian; Hammer, Stephan C.; Schneider, Andreas; Diether, Svenja; Kress, Nico; Ruiz‐Barragán, Sergi; Osuna, Sílvia; Hauer, Bernhard
The interconversion of monoterpenes is facilitated by a complex network of carbocation rearrangement pathways. Controlling these isomerization pathways is challenging when using common Brønsted and Lewis acid catalysts, which often produce product mixtures that are difficult to separate. In contrast, natural monoterpene cyclases exhibit high control over the carbocation rearrangement reactions but are reliant on phosphorylated substrates. In this study, we present engineered squalene‐hopene cyclases from Alicyclobacillus acidocaldarius (AacSHC) that catalyze the challenging isomerization of monoterpenes with unprecedented precision. Starting from a promiscuous isomerization of (+)‐β‐pinene, we first demonstrate noticeable shifts in the product distribution solely by introducing single point mutations. Furthermore, we showcase the tuneable cation steering by enhancing (+)‐borneol selectivity from 1 % to >90 % (>99 % de) aided by iterative saturation mutagenesis. Our combined experimental and computational data suggest that the reorganization of key aromatic residues leads to the restructuring of the water network that facilitates the selective termination of the secondary isobornyl cation. This work expands our mechanistic understanding of carbocation rearrangements and sets the stage for target‐oriented skeletal reorganization of broadly abundant terpenes.
Open Access
Deep enzymology studies on the mammalian DNA methyltransferases and methylcytosine dioxygenases
(2022) Adam, Sabrina; Jeltsch, Albert (Prof. Dr.)
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.
Open Access
Development of an epigenetic tetracycline sensor system based on DNA methylation
(2020) Ullrich, Timo; Weirich, Sara; Jeltsch, Albert
Bacterial live cell sensors are potentially powerful tools for the detection of environmental toxins. In this work, we have established and validated a flow cytometry readout for an existing bacterial arabinose sensor system with DNA methylation based memory function (Maier et al., 2017, Nat. Comm., 8:15336). Flow cytometry readout is convenient and enables a multiparameter analysis providing information about single-cell variability, which is beneficial for further development of sensor systems of this type in the future. We then designed a tetracycline sensor system, because of the importance of antibiotics pollution in the light of multi-resistant pathogens. To this end, a tetracycline trigger plasmid was constructed by replacing the araC repressor gene and the ara operator of the arabinose trigger plasmid with the tetR gene coding for the tetracycline repressor and the tet operon. After combination with the memory plasmid, the tetracycline sensor system was shown to be functional in E. coli allowing to detect and memorize the presence of tetracycline. Due to a positive feedback between the trigger and memory systems, the combined whole-cell biosensor showed a very high sensitivity for tetracycline with a detection threshold at 0.1 ng/ml tetracycline, which may be a general property of sensors of this type. Moreover, acute presence of tetracycline and past exposure can be detected by this sensor using the dual readout of two reporter fluorophores.
Open Access
Development of super-specific epigenome editing by targeted allele-specific DNA methylation
(2023) Rajaram, Nivethika; Kouroukli, Alexandra G.; Bens, Susanne; Bashtrykov, Pavel; Jeltsch, Albert
Background. Epigenome editing refers to the targeted reprogramming of genomic loci using an EpiEditor which may consist of an sgRNA/dCas9 complex that recruits DNMT3A/3L to the target locus. Methylation of the locus can lead to a modulation of gene expression. Allele-specific DNA methylation (ASM) refers to the targeted methylation delivery only to one allele of a locus. In the context of diseases caused by a dominant mutation, the selective DNA methylation of the mutant allele could be used to repress its expression but retain the functionality of the normal gene. Results. To set up allele-specific targeted DNA methylation, target regions were selected from hypomethylated CGIs bearing a heterozygous SNP in their promoters in the HEK293 cell line. We aimed at delivering maximum DNA methylation with highest allelic specificity in the targeted regions. Placing SNPs in the PAM or seed regions of the sgRNA, we designed 24 different sgRNAs targeting single alleles in 14 different gene loci. We achieved efficient ASM in multiple cases, such as ISG15, MSH6, GPD1L, MRPL52, PDE8A, NARF, DAP3, and GSPT1, which in best cases led to five to tenfold stronger average DNA methylation at the on-target allele and absolute differences in the DNA methylation gain at on- and off-target alleles of > 50%. In general, loci with the allele discriminatory SNP positioned in the PAM region showed higher success rate of ASM and better specificity. Highest DNA methylation was observed on day 3 after transfection followed by a gradual decline. In selected cases, ASM was stable up to 11 days in HEK293 cells and it led up to a 3.6-fold change in allelic expression ratios. Conclusions. We successfully delivered ASM at multiple genomic loci with high specificity, efficiency and stability. This form of super-specific epigenome editing could find applications in the treatment of diseases caused by dominant mutations, because it allows silencing of the mutant allele without repression of the expression of the normal allele thereby minimizing potential side-effects of the treatment.