03 Fakultät Chemie
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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, 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.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 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 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 EpiCRISPR targeted methylation of Arx gene initiates transient switch of mouse pancreatic alpha to insulin-producing cells(2023) Đorđević, Marija; Stepper, Peter; Feuerstein-Akgoz, Clarissa; Gerhauser, Clarissa; Paunović, Verica; Tolić, Anja; Rajić, Jovana; Dinić, Svetlana; Uskoković, Aleksandra; Grdović, Nevena; Mihailović, Mirjana; Jurkowska, Renata Z.; Jurkowski, Tomasz P.; Jovanović, Jelena Arambašić; Vidaković, MelitaBeta cell dysfunction by loss of beta cell identity, dedifferentiation, and the presence of polyhormonal cells are main characteristics of diabetes. The straightforward strategy for curing diabetes implies reestablishment of pancreatic beta cell function by beta cell replacement therapy. Aristaless-related homeobox (Arx) gene encodes protein which plays an important role in the development of pancreatic alpha cells and is a main target for changing alpha cell identity. In this study we used CRISPR/dCas9-based epigenetic tools for targeted hypermethylation of Arx gene promoter and its subsequent suppression in mouse pancreatic αTC1-6 cell line. Bisulfite sequencing and methylation profiling revealed that the dCas9-Dnmt3a3L-KRAB single chain fusion constructs (EpiCRISPR) was the most efficient. Epigenetic silencing of Arx expression was accompanied by an increase in transcription of the insulin gene (Ins2) mRNA on 5th and 7th post-transfection day, quantified by both RT-qPCR and RNA-seq. Insulin production and secretion was determined by immunocytochemistry and ELISA assay, respectively. Eventually, we were able to induce switch of approximately 1% of transiently transfected cells which were able to produce 35% more insulin than Mock transfected alpha cells. In conclusion, we successfully triggered a direct, transient switch of pancreatic alpha to insulin-producing cells opening a future research on promising therapeutic avenue for diabetes management.Item Open Access Harnessing the structure and dynamics of the squalene‐hopene cyclase for (-)‐ambroxide production(2023) Schneider, Andreas; Curado, Christian; Lystbaek, Thomas B.; Osuna, Sílvia; Hauer, BernhardTerpene cyclases offer enormous synthetic potential, given their unique ability to forge complex hydrocarbon scaffolds from achiral precursors within a single cationic rearrangement cascade. Harnessing their synthetic power, however, has proved to be challenging owing to their generally low catalytic performance. In this study, we unveiled the catalytic potential of the squalene‐hopene cyclase (SHC) by harnessing its structure and dynamics. First, we synergistically tailored the active site and entrance tunnel of the enzyme to generate a 397‐fold improved (-)‐ambroxide synthase. Our computational investigations explain how the introduced mutations work in concert to improve substrate acquisition, flow, and chaperoning. Kinetics, however, showed terpene‐induced inactivation of the membrane‐bound SHC to be the major turnover limitation in vivo. Merging this insight with the improved and stereoselective catalysis of the enzyme, we applied a feeding strategy to exceed 10 5 total turnovers. We believe that our results may bridge the gap for broader application of SHCs in synthetic chemistry.Item Open Access Biochemical analysis of DNA- and protein methyltransferases using recombinant designer nucleosomes(2022) Bröhm, Alexander; Jeltsch, Albert (Prof. Dr.)Item Open Access Mechanistic insights into the allosteric regulation of the Clr4 protein lysine methyltransferase by autoinhibition and automethylation(2020) Khella, Mina S.; Bröhm, Alexander; Weirich, Sara; Jeltsch, AlbertClr4 is a histone H3 lysine 9 methyltransferase in Schizosaccharomyces pombe that is essential for heterochromatin formation. Previous biochemical and structural studies have shown that Clr4 is in an autoinhibited state in which an autoregulatory loop (ARL) blocks the active site. Automethylation of lysine residues in the ARL relieves autoinhibition. To investigate the mechanism of Clr4 regulation by autoinhibition and automethylation, we exchanged residues in the ARL by site-directed mutagenesis leading to stimulation or inhibition of automethylation and corresponding changes in Clr4 catalytic activity. Furthermore, we demonstrate that Clr4 prefers monomethylated (H3K9me1) over unmodified (H3K9me0) histone peptide substrates, similar to related human enzymes and, accordingly, H3K9me1 is more efficient in overcoming autoinhibition. Due to enzyme activation by automethylation, we observed a sigmoidal dependence of Clr4 activity on the AdoMet concentration, with stimulation at high AdoMet levels. In contrast, an automethylation-deficient mutant showed a hyperbolic Michaelis–Menten type relationship. These data suggest that automethylation of the ARL could act as a sensor for AdoMet levels in cells and regulate the generation and maintenance of heterochromatin accordingly. This process could connect epigenome modifications with the metabolic state of cells. As other human protein lysine methyltransferases (for example, PRC2) also use automethylation/autoinhibition mechanisms, our results may provide a model to describe their regulation as well.Item Open Access Structure, activity and function of the NSD3 protein lysine methyltransferase(2021) Rathert, PhilippNSD3 is one of six H3K36-specific lysine methyltransferases in metazoans, and the methylation of H3K36 is associated with active transcription. NSD3 is a member of the nuclear receptor-binding SET domain (NSD) family of histone methyltransferases together with NSD1 and NSD2, which generate mono- and dimethylated lysine on histone H3. NSD3 is mutated and hyperactive in some human cancers, but the biochemical mechanisms underlying such dysregulation are barely understood. In this review, the current knowledge of NSD3 is systematically reviewed. Finally, the molecular and functional characteristics of NSD3 in different tumor types according to the current research are summarized.Item Open Access Refined read‐out : the hUHRF1 Tandem‐Tudor domain prefers binding to histone H3 tails containing K4me1 in the context of H3K9me2/3(2023) Choudalakis, Michel; Kungulovski, Goran; Mauser, Rebekka; Bashtrykov, Pavel; Jeltsch, AlbertUHRF1 is an essential chromatin protein required for DNA methylation maintenance, mammalian development, and gene regulation. We investigated the Tandem-Tudor domain (TTD) of human UHRF1 that is known to bind H3K9me2/3 histones and is a major driver of UHRF1 localization in cells. We verified binding to H3K9me2/3 but unexpectedly discovered stronger binding to H3 peptides and mononucleosomes containing K9me2/3 with additional K4me1. We investigated the combined binding of TTD to H3K4me1-K9me2/3 versus H3K9me2/3 alone, engineered mutants with specific and differential changes of binding, and discovered a novel read-out mechanism for H3K4me1 in an H3K9me2/3 context that is based on the interaction of R207 with the H3K4me1 methyl group and on counting the H-bond capacity of H3K4. Individual TTD mutants showed up to a 10,000-fold preference for the double-modified peptides, suggesting that after a conformational change, WT TTD could exhibit similar effects. The frequent appearance of H3K4me1-K9me2 regions in human chromatin demonstrated in our TTD chromatin pull-down and ChIP-western blot data suggests that it has specific biological roles. Chromatin pull-down of TTD from HepG2 cells and full-length murine UHRF1 ChIP-seq data correlate with H3K4me1 profiles indicating that the H3K4me1-K9me2/3 interaction of TTD influences chromatin binding of full-length UHRF1. We demonstrate the H3K4me1-K9me2/3 specific binding of UHRF1-TTD to enhancers and promoters of cell-type-specific genes at the flanks of cell-type-specific transcription factor binding sites, and provided evidence supporting an H3K4me1-K9me2/3 dependent and TTD mediated downregulation of these genes by UHRF1. All these findings illustrate the important physiological function of UHRF1-TTD binding to H3K4me1-K9me2/3 double marks in a cellular context.