42 results on '"Jesko Koehnke"'
Search Results
2. Cyclic Peptides: From Bioorganic Synthesis to Applications
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Jesko Koehnke, James Naismith, Wilfred A van der Donk, Jesko Koehnke, James Naismith, Wilfred A van der Donk and Jesko Koehnke, James Naismith, Wilfred A van der Donk, Jesko Koehnke, James Naismith, Wilfred A van der Donk
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- 2017
3. Unusual peptide-binding proteins guide pyrroloindoline alkaloid formation in crocagin biosynthesis
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Sebastian Adam, Dazhong Zheng, Andreas Klein, Carsten Volz, William Mullen, Sally L. Shirran, Brian O. Smith, Olga V. Kalinina, Rolf Müller, Jesko Koehnke, University of St Andrews. Arctic Research Centre, University of St Andrews. School of Biology, University of St Andrews. Institute of Behavioural and Neural Sciences, and University of St Andrews. Biomedical Sciences Research Complex
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MCC ,General Chemical Engineering ,DAS ,QD ,General Chemistry ,QD Chemistry - Abstract
Funding: This work was supported by the European Research Council (Consolidator Grant 101002326 to J.K.). Ribosomally synthesized and post-translationally modified peptide natural products have provided many highly unusual scaffolds. This includes the intriguing alkaloids crocagins, which possess a tetracyclic core structure and whose biosynthesis has remained enigmatic. Here we use in vitro experiments to demonstrate that three proteins, CgnB, CgnC and CgnE, are sufficient for the production of the hallmark tetracyclic crocagin core from the precursor peptide CgnA. The crystal structures of the homologues CgnB and CgnE reveal them to be the founding members of a peptide-binding protein family and allow us to rationalize their distinct functions. We further show that the hydrolase CgnD liberates the crocagin core scaffold, which is subsequently N-methylated by CgnL. These insights allow us to propose a biosynthetic scheme for crocagins. Bioinformatic analyses based on these data led to the discovery of related biosynthetic pathways that may provide access to a structurally diverse family of peptide-derived pyrroloindoline alkaloids. Publisher PDF
- Published
- 2023
4. Photorhabdus luminescens lectin A (PllA) : A new probe for detecting α-galactoside-terminating glycoconjugates
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Jesko Koehnke, Eckhard Wolf, Ghamdan Beshr, Asfandyar Sikandar, Dirk Hauck, Alexander Titz, Nikolai Klymiuk, Eva-Maria Jemiller, Stefanie Wagner, and Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS), Universitycampus E8.1, 66123 Saarbrücken, Germany.
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0301 basic medicine ,chemistry.chemical_classification ,030102 biochemistry & molecular biology ,biology ,Glycoconjugate ,Glycobiology ,Protein subunit ,Ligand binding assay ,Lectin ,Glycobiology and Extracellular Matrices ,Cell Biology ,biology.organism_classification ,Biochemistry ,Molecular biology ,In vitro ,Epitope ,03 medical and health sciences ,030104 developmental biology ,chemistry ,Photorhabdus luminescens ,biology.protein ,Molecular Biology - Abstract
Lectins play important roles in infections by pathogenic bacteria, for example, in host colonization, persistence and biofilm formation. The Gram-negative entomopathogenic bacterium Photorhabdus luminescens symbiotically lives in insect-infecting Heterorhabditis nematodes and kills the insect host upon invasion by the nematode. The P. luminescens genome harbors the gene plu2096 coding for a novel lectin that we named PllA. We analyzed the binding properties of purified PllA with a glycan array and a binding assay in solution. Both assays revealed a strict specificity of PllA for alpha-galactoside-terminating glycoconjugates. The crystal structures of apo PllA and complexes with three different ligands revealed the molecular basis for the strict specificity of this lectin. Furthermore, we found that a 90 degree twist in subunit orientation leads to a peculiar quaternary structure compared with that of its ortholog LecA from Pseudomonas aeruginosa. We also investigated the utility of PllA as a probe for detecting alpha-galactosides. The alpha-Gal epitope is present on wild-type pig cells and the main reason for hyperacute organ rejection in pig to primate xenotransplantation. We noted that PllA specifically recognizes this epitope on the glycan array and demonstrated that PllA can be used as a fluorescent probe to detect this epitope on primary porcine cells in vitro. In summary, our biochemical and structural analyses of the P. luminescens lectin PllA have disclosed the structural basis for PllAs high specificity for alpha-galactoside-containing ligands, and we show that PllA can be used to visualize alpha-Gal epitope on porcine tissues.
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- 2023
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5. Caught in the act
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Jesko, Koehnke
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- 2022
6. Enhancing glycan stability via site-selective fluorination: modulating substrate orientation by molecular design
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Ryan Gilmour, Manfred Fobker, Alexander Axer, Michael Schäfers, Anna K. H. Hirsch, Jesko Koehnke, Andreas Faust, Sebastian Adam, Ravindra P. Jumde, and HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany.
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Glycan ,Stereochemistry ,chemistry.chemical_element ,Cleavage (embryo) ,01 natural sciences ,Catalysis ,Stereocenter ,03 medical and health sciences ,Hydrolysis ,chemistry.chemical_compound ,Blood serum ,Monosaccharide ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,biology ,010405 organic chemistry ,Point mutation ,Substrate (chemistry) ,General Chemistry ,Maltose ,Combinatorial chemistry ,0104 chemical sciences ,Enzyme ,Structural biology ,chemistry ,Docking (molecular) ,Fluorine ,biology.protein - Abstract
Single site OH → F substitution at the termini of maltotetraose leads to significantly improved hydrolytic stability towards α-amylase and α-glucosidase relative to the natural compound. To explore the effect of molecular editing, selectively modified oligosaccharides were preparedviaa convergent α-selective strategy. Incubation experiments in purified α-amylase and α-glucosidase, and in human and murine blood serum, provide insight into the influence of fluorine on the hydrolytic stability of these clinically important scaffolds. Enhancements ofca. 1 order of magnitude result from these subtle single point mutations. Modification at the monosaccharide furthest from the probable enzymatic cleavage termini leads to the greatest improvement in stability. In the case of α-amylase, docking studies revealed that retentive C2-fluorination at the reducing end inverts the orientation in which the substrate is bound. A co-crystal structure of human α-amylase revealed maltose units bound at the active-site. In view of the evolving popularity of C(sp3)-F bioisosteres in medicinal chemistry, and the importance of maltodextrins in bacterial imaging, this discovery begins to reconcile the information-rich nature of carbohydrates with their intrinsic hydrolytic vulnerabilities. © The Royal Society of Chemistry 2020. Western Washington University
- Published
- 2021
7. Non-Heme Monooxygenase ThoJ Catalyzes Thioholgamide β-Hydroxylation
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Jesko Koehnke, Maria Lopatniuk, Asfandyar Sikandar, Andriy Luzhetskyy, and HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany.
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0301 basic medicine ,Stereochemistry ,Heterologous ,Peptide ,Hydroxylation ,Peptides, Cyclic ,01 natural sciences ,Biochemistry ,Mixed Function Oxygenases ,Substrate Specificity ,03 medical and health sciences ,chemistry.chemical_compound ,Bacterial Proteins ,Biosynthesis ,Dioxygenase ,Moiety ,Histidine ,Non heme ,chemistry.chemical_classification ,010405 organic chemistry ,General Medicine ,Monooxygenase ,Streptomyces ,0104 chemical sciences ,Thioamides ,030104 developmental biology ,chemistry ,Molecular Medicine ,Protein Processing, Post-Translational - Abstract
Thioviridamide-like compounds, including thioholgamides, are ribosomally synthesized and post-translationally modified peptide natural products with potent anticancer cell activity and an unprecedented structure. Very little is known about their biosynthesis, and we were intrigued by the β-hydroxy-N1, N3-dimethylhistidinium moiety found in these compounds. Here we report the construction of a heterologous host capable of producing thioholgamide with a 15-fold increased yield compared to the wild-type strain. A knockout of thoJ, encoding a predicted nonheme monooxygenase, shows that ThoJ is essential for thioholgamide β-hydroxylation. The crystal structure of ThoJ exhibits a typical mono/dioxygenase fold with conserved key active-site residues. Yet, ThoJ possesses a very large substrate binding pocket that appears suitable to receive a cyclic thioholgamide intermediate for hydroxylation. The improved production of the heterologous host will enable the dissection of the individual biosynthetic steps involved in biosynthesis of this exciting RiPP family.
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- 2020
8. N-Aryl-3-mercaptosuccinimides as Antivirulence Agents Targeting Pseudomonas aeruginosa Elastase and Clostridium Collagenases
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Andreas M. Kany, Samir Yahiaoui, Anna K. H. Hirsch, Esther Schönauer, Fabian K. Berger, Markus Bischoff, Jesko Koehnke, Hans Brandstetter, Rolf Müller, Rolf W. Hartmann, Anastasia Andreas, Alaa Alhayek, Jörg Haupenthal, Jelena Konstantinović, and HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany.
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01 natural sciences ,Article ,Microbiology ,03 medical and health sciences ,Clostridium ,Clostridium histolyticum ,Drug Discovery ,medicine ,Cytotoxicity ,030304 developmental biology ,0303 health sciences ,Metalloproteinase ,biology ,Chemistry ,biology.organism_classification ,In vitro ,0104 chemical sciences ,3. Good health ,010404 medicinal & biomolecular chemistry ,Cell culture ,Collagenase ,Molecular Medicine ,Ex vivo ,medicine.drug - Abstract
In light of the global antimicrobial-resistance crisis, there is an urgent need for novel bacterial targets and antibiotics with novel modes of action. It has been shown that Pseudomonas aeruginosa elastase (LasB) and Clostridium histolyticum (Hathewaya histolytica) collagenase (ColH) play a significant role in the infection process and thereby represent promising antivirulence targets. Here, we report novel N-aryl-3-mercaptosuccinimide inhibitors that target both LasB and ColH, displaying potent activities in vitro and high selectivity for the bacterial over human metalloproteases. Additionally, the inhibitors demonstrate no signs of cytotoxicity against selected human cell lines and in a zebrafish embryo toxicity model. Furthermore, the most active ColH inhibitor shows a significant reduction of collagen degradation in an ex vivo pig-skin model.
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- 2020
9. Tutuilamides A–C: Vinyl-Chloride-Containing Cyclodepsipeptides from Marine Cyanobacteria with Potent Elastase Inhibitory Properties
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Anthony J. O’Donoghue, Hendrik Luesch, Brian M. Suzuki, K.M. Canuto, Brendan M. Duggan, Jehad Almaliti, Evgenia Glukhov, Asfandyar Sikandar, C. Liu, Jesko Koehnke, William H. Gerwick, Dan Luo, Lena Keller, C. B. Naman, and HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstraße 7, 38124 Braunschweig, Germany.
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0301 basic medicine ,Lung Neoplasms ,Drug Screening Assays ,01 natural sciences ,Biochemistry ,chemistry.chemical_compound ,Models ,Tandem Mass Spectrometry ,Depsipeptides ,Amino Acids ,Enzyme Inhibitors ,chemistry.chemical_classification ,Cyclic ,Chromatography ,Tumor ,Pancreatic Elastase ,Molecular Structure ,Aminobutyrates ,Elastase ,General Medicine ,Biological Sciences ,Cyclic peptide ,Amino acid ,High Pressure Liquid ,Molecular Medicine ,Two-dimensional nuclear magnetic resonance spectroscopy ,Protein Binding ,Cell Survival ,Stereochemistry ,Antiparasitic ,medicine.drug_class ,Vinyl Chloride ,Antineoplastic Agents ,Cyanobacteria ,Cell Line ,03 medical and health sciences ,Residue (chemistry) ,Hydrolase ,medicine ,Humans ,Piperidones ,Natural product ,010405 organic chemistry ,Organic Chemistry ,Molecular ,Antitumor ,0104 chemical sciences ,030104 developmental biology ,chemistry ,Chemical Sciences ,Peptides - Abstract
Marine cyanobacteria (blue-green algae) have been shown to possess an enormous capacity to produce structurally diverse natural products that exhibit a broad spectrum of potent biological activities, including cytotoxic, antifungal, antiparasitic, antiviral, and antibacterial activities. Using mass-spectrometry-guided fractionation together with molecular networking, cyanobacterial field collections from American Samoa and Palmyra Atoll yielded three new cyclic peptides, tutuilamides A-C. Their structures were established by spectroscopic techniques including 1D and 2D NMR, HR-MS, and chemical derivatization. Structure elucidation was facilitated by employing advanced NMR techniques including nonuniform sampling in combination with the 1,1-ADEQUATE experiment. These cyclic peptides are characterized by the presence of several unusual residues including 3-amino-6-hydroxy-2-piperidone and 2-amino-2-butenoic acid, together with a novel vinyl chloride-containing residue. Tutuilamides A-C show potent elastase inhibitory activity together with moderate potency in H-460 lung cancer cell cytotoxicity assays. The binding mode to elastase was analyzed by X-ray crystallography revealing a reversible binding mode similar to the natural product lyngbyastatin 7. The presence of an additional hydrogen bond with the amino acid backbone of the flexible side chain of tutuilamide A, compared to lyngbyastatin 7, facilitates its stabilization in the elastase binding pocket and possibly explains its enhanced inhibitory potency.
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- 2020
10. The core of the matter
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Jesko Koehnke
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chemistry.chemical_classification ,chemistry ,Peptide ,Substrate recognition ,Cell Biology ,Ribosomal RNA ,Molecular Biology ,Combinatorial chemistry - Abstract
Graspetides are an important class of ribosomal natural products with potent bioactivities. New structural information provides insights into substrate recognition and catalysis, including a rare glimpse into the interactions between a tailoring enzyme and the core of the precursor peptide.
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- 2021
11. Enhancing glycan stability
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Alexander, Axer, Ravindra P, Jumde, Sebastian, Adam, Andreas, Faust, Michael, Schäfers, Manfred, Fobker, Jesko, Koehnke, Anna K H, Hirsch, and Ryan, Gilmour
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Chemistry - Abstract
Single site OH → F substitution at the termini of maltotetraose leads to significantly improved hydrolytic stability towards α-amylase and α-glucosidase relative to the natural compound. To explore the effect of molecular editing, selectively modified oligosaccharides were prepared via a convergent α-selective strategy. Incubation experiments in purified α-amylase and α-glucosidase, and in human and murine blood serum, provide insight into the influence of fluorine on the hydrolytic stability of these clinically important scaffolds. Enhancements of ca. 1 order of magnitude result from these subtle single point mutations. Modification at the monosaccharide furthest from the probable enzymatic cleavage termini leads to the greatest improvement in stability. In the case of α-amylase, docking studies revealed that retentive C2-fluorination at the reducing end inverts the orientation in which the substrate is bound. A co-crystal structure of human α-amylase revealed maltose units bound at the active-site. In view of the evolving popularity of C(sp3)–F bioisosteres in medicinal chemistry, and the importance of maltodextrins in bacterial imaging, this discovery begins to reconcile the information-rich nature of carbohydrates with their intrinsic hydrolytic vulnerabilities., Single site OH → F substitution at the termini of maltotetraose leads to significantly improved hydrolytic stability towards α-amylase and α-glucosidase relative to the natural compound.
- Published
- 2021
12. Leader peptide exchange to produce hybrid, new-to-nature ribosomal natural products
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Laura Franz, Jesko Koehnke, and HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany.
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Signal peptide ,Amino Acid Motifs ,Peptide ,Protein Sorting Signals ,010402 general chemistry ,Mannose-Binding Lectin ,Peptides, Cyclic ,01 natural sciences ,Catalysis ,chemistry.chemical_compound ,Bacterial Proteins ,Sortase ,Materials Chemistry ,chemistry.chemical_classification ,Biological Products ,Peptide modification ,Natural product ,010405 organic chemistry ,Metals and Alloys ,General Chemistry ,Ribosomal RNA ,Aminoacyltransferases ,0104 chemical sciences ,Surfaces, Coatings and Films ,Electronic, Optical and Magnetic Materials ,Cysteine Endopeptidases ,Enzyme ,Biochemistry ,chemistry ,Ceramics and Composites ,Peptides ,Ribosomes - Abstract
Ribosomal natural products contain exquisite post-translational peptide modifications that are installed by a range of pathway-specific enzymes. We present proof of principle for a Sortase A-based approach that enables peptide modification by enzymes from unrelated pathways. This allowed the one-pot synthesis of a new-to-nature, hybrid ribosomal natural product.
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- 2021
13. The structure of CgnJ, a domain of unknown function protein from the crocagin gene cluster
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Andreas Klein, Sebastian Adam, Jesko Koehnke, and Frank Surup
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Protein Conformation ,Biophysics ,Regulator ,Peptide ,Computational biology ,Bacillus subtilis ,Crystallography, X-Ray ,Peptides, Cyclic ,Biochemistry ,Research Communications ,chemistry.chemical_compound ,Biosynthesis ,Structural Biology ,Gene cluster ,Genetics ,chemistry.chemical_classification ,Biological Products ,biology ,computer.file_format ,Condensed Matter Physics ,Protein Data Bank ,biology.organism_classification ,Enzyme ,chemistry ,Multigene Family ,Protein Processing, Post-Translational ,computer ,Function (biology) ,Protein Binding - Abstract
Natural products often contain interesting new chemical entities that are introduced into the structure of a compound by the enzymatic machinery of the producing organism. The recently described crocagins are novel polycyclic peptides which belong to the class of ribosomally synthesized and post-translationally modified peptide natural products. They have been shown to bind to the conserved prokaryotic carbon-storage regulator Ain vitro. In efforts to understand crocagin biosynthesis, the putative biosynthetic genes were expressed and purified. Here, the first crystal structure of a protein from the crocagin-biosynthetic gene cluster, CgnJ, a domain of unknown function protein, is reported. Possible functions of this protein were explored by structural and sequence homology analyses. Even though the sequence homology to proteins in the Protein Data Bank is low, the protein shows significant structural homology to a protein with known function within the competency system ofBacillus subtilis, ComJ, leading to the hypothesis of a similar role of the protein within the producing organism.
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- 2019
14. The role of protein–protein interactions in the biosynthesis of ribosomally synthesized and post-translationally modified peptides
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Jesko Koehnke and Asfandyar Sikandar
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Peptide Biosynthesis ,Substrate channeling ,Peptide ,010402 general chemistry ,Peptides, Cyclic ,01 natural sciences ,Biochemistry ,Protein–protein interaction ,chemistry.chemical_compound ,Biosynthesis ,Drug Discovery ,Protein Interaction Domains and Motifs ,Genomic organization ,chemistry.chemical_classification ,010405 organic chemistry ,Organic Chemistry ,Biosynthetic enzyme ,0104 chemical sciences ,Enzyme ,chemistry ,Multiprotein Complexes ,Peptides ,Protein Processing, Post-Translational ,Ribosomes - Abstract
Covering: up to 02/2019 This review covers the role of protein–protein complexes in the biosynthesis of selected ribosomally synthesized and post-translationally modified peptide (RiPP) classes. The genomic organization of RiPP systems usually allows the expression of each biosynthetic enzyme as an individual unit, which is in stark contrast to the giant assembly lines found in non-ribosomal peptide and polyketide synthesis systems. Evidence is mounting however that the formation of multi-enzyme complexes is critical for efficient RiPPs biosynthesis and that these complexes may be involved in substrate channeling or conformational sampling. In some pathways, polyfunctional enzymes have evolved, which can be viewed as perpetual protein complexes. We summarize what is currently known on enzyme complexes in RiPP systems for lasso peptides, cyanobactins, linear azolic peptides, thiopeptides, and lanthipeptides.
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- 2019
15. Characterization of the Stereoselective P450 Enzyme BotCYP Enables the
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Sebastian, Adam, Laura, Franz, Mohammed, Milhim, Rita, Bernhardt, Olga V, Kalinina, and Jesko, Koehnke
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Methicillin-Resistant Staphylococcus aureus ,Thiazoles ,Cytochrome P-450 Enzyme System ,Multigene Family ,Stereoisomerism ,Oxidation-Reduction ,Peptides, Cyclic ,Protein Processing, Post-Translational - Abstract
Bottromycins are ribosomally synthesized and post-translationally modified peptide natural product antibiotics that are effective against high-priority human pathogens such as methicillin-resistant
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- 2020
16. Characterization of the Stereoselective P450 Enzyme BotCYP Enables the Biosynthesis of the Bottromycin Core Scaffold
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Sebastian Adam, Mohammed Milhim, Rita Bernhardt, Laura Franz, Jesko Koehnke, Olga V. Kalinina, and HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany.
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Natural product ,Chemistry ,Stereochemistry ,Thiazoline ,Total synthesis ,General Chemistry ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Catalysis ,0104 chemical sciences ,chemistry.chemical_compound ,Colloid and Surface Chemistry ,Biosynthesis ,Gene cluster ,Thiazole ,Oxidative decarboxylation ,Bottromycin - Abstract
Bottromycins are ribosomally synthesized and post-translationally modified peptide natural product antibiotics that are effective against high-priority human pathogens such as methicillin-resistant Staphylococcus aureus. The total synthesis of bottromycins involves at least 17 steps, with a poor overall yield. Here, we report the characterization of the cytochrome P450 enzyme BotCYP from a bottromycin biosynthetic gene cluster. We determined the structure of a close BotCYP homolog and used our data to conduct the first large-scale survey of P450 enzymes associated with RiPP biosynthetic gene clusters. We demonstrate that BotCYP converts a C-terminal thiazoline to a thiazole via an oxidative decarboxylation reaction and provides stereochemical resolution for the pathway. Our data enable the two-pot in vitro production of the bottromycin core scaffold and may allow the rapid generation of bottromycin analogues for compound development.
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- 2020
17. Biosynthesis of Cittilins, Unusual Ribosomally Synthesized and Post-translationally Modified Peptides from
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Joachim J, Hug, Jan, Dastbaz, Sebastian, Adam, Ole, Revermann, Jesko, Koehnke, Daniel, Krug, and Rolf, Müller
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Myxococcus xanthus ,Bacterial Proteins ,Cytochrome P-450 Enzyme System ,Peptides ,Protein Processing, Post-Translational ,Ribosomes ,Biosynthetic Pathways - Abstract
Cittilins are secondary metabolites from myxobacteria comprised of three l-tyrosines and one l-isoleucine forming a bicyclic tetrapeptide scaffold with biaryl and aryl-oxygen-aryl ether bonds. Here we reveal that cittilins belong to the ribosomally synthesized and post-translationally modified peptide (RiPP) family of natural products, for which only the crocagins have been reported from myxobacteria. A 27 amino acid precursor peptide harbors a C-terminal four amino acid core peptide, which is enzymatically modified and finally exported to yield cittilins. The small biosynthetic gene cluster responsible for cittilin biosynthesis also encodes a cytochrome P450 enzyme and a methyltransferase, whereas a gene encoding a prolyl endopeptidase for the cleavage of the precursor peptide is located outside of the cittilin biosynthetic gene cluster. We confirm the roles of the biosynthetic genes responsible for the formation of cittilins using targeted gene inactivation and heterologous expression in
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- 2020
18. Biosynthesis of Cittilins, Unusual Ribosomally Synthesized and Post-translationally Modified Peptides from Myxococcus xanthus
- Author
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Sebastian Adam, Jesko Koehnke, Rolf Müller, Jan Dastbaz, Daniel Krug, Joachim J. Hug, Ole Revermann, and HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany.
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0301 basic medicine ,chemistry.chemical_classification ,biology ,Tetrapeptide ,010405 organic chemistry ,Chemistry ,General Medicine ,biology.organism_classification ,01 natural sciences ,Biochemistry ,0104 chemical sciences ,Amino acid ,03 medical and health sciences ,chemistry.chemical_compound ,030104 developmental biology ,Prolyl endopeptidase ,Myxobacteria ,Biosynthesis ,Gene cluster ,medicine ,Molecular Medicine ,Heterologous expression ,Myxococcus xanthus ,medicine.drug - Abstract
Cittilins are secondary metabolites from myxobacteria comprised of three L-tyrosines and one L-isoleucine forming a bicyclic tetrapeptide scaffold with biaryl and aryl-oxygen-aryl ether bonds. Here we reveal that cittilins belong to the ribosomally synthesized and post-translationally modified peptide (RiPP) family of natural products, for which only the crocagins have been reported from myxobacteria. A 27 amino acid precursor peptide harbors a C-terminal four amino acid core peptide, which is enzymatically modified and finally exported to yield cittilins. The small biosynthetic gene cluster responsible for cittilin biosynthesis also encodes a cytochrome P450 enzyme and a methyltransferase, whereas a gene encoding a prolyl endopeptidase for the cleavage of the precursor peptide is located outside of the cittilin biosynthetic gene cluster. We confirm the roles of the biosynthetic genes responsible for the formation of cittilins using targeted gene inactivation and heterologous expression in Streptomyces. We also report first steps towards the biochemical characterization of the proposed biosynthetic pathway in vitro. An investigation of the cellular uptake properties of cittilin A connected it to a potential biological function as an inhibitor of the prokaryotic carbon storage regulator A (CsrA).Abstract Figure
- Published
- 2020
19. The bottromycin epimerase BotH defines a group of atypical α/β-hydrolase-fold enzymes
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Jesko Koehnke, Sebastian Adam, Asfandyar Sikandar, Andrew W. Truman, Liliya Horbal, Laura Franz, Olga V. Kalinina, Javier Santos-Aberturas, Andriy Luzhetskyy, and HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany.
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Models, Molecular ,Protein Folding ,Protein Conformation ,Racemases and Epimerases ,Peptide ,Crystallography, X-Ray ,Peptides, Cyclic ,Substrate Specificity ,Evolution, Molecular ,03 medical and health sciences ,chemistry.chemical_compound ,Protein structure ,Biosynthesis ,Bacterial Proteins ,ddc:570 ,Hydrolase ,Molecular Biology ,Gene ,030304 developmental biology ,Bottromycin ,chemistry.chemical_classification ,0303 health sciences ,Aspartic Acid ,030302 biochemistry & molecular biology ,Cell Biology ,Streptomyces ,Enzyme ,chemistry ,Biochemistry ,Multigene Family ,Protein folding - Abstract
Nature chemical biology 16(9), 1013 - 1018 (2020). doi:10.1038/s41589-020-0569-y, $\small{D}$-amino acids endow peptides with diverse, desirable properties, but the post-translational and site-specific epimerization of $\small{L}$-amino acids into their d-counterparts is rare and chemically challenging. Bottromycins are ribosomally synthesized and post-translationally modified peptides that have overcome this challenge and feature a $\small{D}$-aspartate ($\small{D}$-Asp), which was proposed to arise spontaneously during biosynthesis. We have identified the highly unusual $α/β$-hydrolase (ABH) fold enzyme BotH as a peptide epimerase responsible for the post-translational epimerization of $\small{L}$-Asp to $\small{D}$-Asp during bottromycin biosynthesis. The biochemical characterization of BotH combined with the structures of BotH and the BotH–substrate complex allowed us to propose a mechanism for this reaction. Bioinformatic analyses of BotH homologs show that similar ABH enzymes are found in diverse biosynthetic gene clusters. This places BotH as the founding member of a group of atypical ABH enzymes that may be able to epimerize non-Asp stereocenters across different families of secondary metabolites., Published by Nature Publishing Group, Basingstoke
- Published
- 2019
20. Macroamidine Formation in Bottromycins Is Catalyzed by a Divergent YcaO Enzyme
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Sebastian Adam, Jesko Koehnke, Andrew W. Truman, Laura Franz, and Javier Santos-Aberturas
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Peptide Biosynthesis ,0301 basic medicine ,Macrocyclic Compounds ,Stereochemistry ,Amidines ,Peptides, Cyclic ,Biochemistry ,Catalysis ,03 medical and health sciences ,chemistry.chemical_compound ,Colloid and Surface Chemistry ,Nucleophile ,Molecule ,Amino Acid Sequence ,Peptide sequence ,Bottromycin ,chemistry.chemical_classification ,Molecular Structure ,Chemistry ,Thiazoline ,General Chemistry ,030104 developmental biology ,Enzyme ,Cyclization ,Phosphorylation ,Peptides - Abstract
The YcaO superfamily of proteins catalyzes the phosphorylation of peptide backbone amide bonds, which leads to the formation of azolines and azoles in ribosomally synthesized and post-translationally modified peptides (RiPPs). Bottromycins are RiPPs with potent antimicrobial activity, and their biosynthetic pathway contains two divergent, stand-alone YcaO enzymes, IpoC and PurCD. From an untargeted metabolomics approach, it had been suggested that PurCD acts with a partner protein to form the 12-membered macroamidine unique to bottromycins. Here we report the biochemical characterization of IpoC and PurCD. We demonstrate that IpoC installs a cysteine-derived thiazoline, whereas PurCD alone is sufficient to create the macroamidine structure. Both enzymes are catalytically promiscuous, and we generated 10 different macroamidines. Our data provide important insights into the versatility of YcaO enzymes, their ability to utilize different nucleophiles and provide a framework for the creation of novel bottromycin derivatives with enhanced bioactivity.
- Published
- 2017
21. Thiazoline-specific amidohydrolase PurAH is the gatekeeper of bottromycin biosynthesis
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Asfandyar Sikandar, Laura Franz, Okke Melse, Jesko Koehnke, and Iris Antes
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chemistry.chemical_classification ,Amidohydrolase ,Chemistry ,Stereochemistry ,Thiazoline ,Mutagenesis ,Peptide ,General Chemistry ,010402 general chemistry ,Cleavage (embryo) ,01 natural sciences ,Biochemistry ,Peptides, Cyclic ,Catalysis ,Streptomyces ,0104 chemical sciences ,Amidohydrolases ,chemistry.chemical_compound ,Colloid and Surface Chemistry ,Enzyme ,Biosynthesis ,Bacterial Proteins ,Mutation ,Biocatalysis ,Bottromycin - Abstract
The ribosomally synthesized and post-translationally modified peptide (RiPP) bottromycin A2 possesses potent antimicrobial activity. Its biosynthesis involves the enzymatic formation of a macroamidine, a process previously suggested to require the concerted efforts of a YcaO enzyme (PurCD) and an amidohydrolase (PurAH) in vivo. In vitro, PurCD alone is sufficient to catalyze formation of the macroamidine, but the process is reversible. We set out to probe the role of PurAH in macroamidine formation in vitro. We demonstrate that PurAH is highly selective for macroamidine-containing precursor peptides and cleaves C-terminal of a thiazoline, thus removing the follower peptide. After follower cleavage, macroamidine formation is irreversible, indicating PurAH as the gatekeeper of bottromycin biosynthesis. The structure of PurAH suggests residues involved in catalysis, which were probed through mutagenesis.
- Published
- 2019
22. Adaptation of a Bacterial Multidrug Resistance System Revealed by the Structure and Function of AlbA
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Katarina Cirnski, Asfandyar Sikandar, Mark Brönstrup, Rolf Müller, Giambattista Testolin, Jesko Koehnke, Carsten Volz, and Olga V. Kalinina
- Subjects
0301 basic medicine ,Xanthomonas ,medicine.drug_class ,030106 microbiology ,Antibiotics ,Drug resistance ,Plasma protein binding ,Microbial Sensitivity Tests ,medicine.disease_cause ,Biochemistry ,Catalysis ,Microbiology ,Evolution, Molecular ,03 medical and health sciences ,chemistry.chemical_compound ,Colloid and Surface Chemistry ,Antibiotic resistance ,Bacterial Proteins ,Drug Resistance, Multiple, Bacterial ,medicine ,Organic Chemicals ,Gene ,Mutation ,Natural product ,Chemistry ,Klebsiella oxytoca ,General Chemistry ,Nitro Compounds ,Anti-Bacterial Agents ,Multiple drug resistance ,030104 developmental biology ,Cyclization ,Asparagine ,Carrier Proteins ,Protein Binding - Abstract
To combat the rise of antimicrobial resistance, the discovery of new antibiotics is paramount. Albicidin and cystobactamid are related natural product antibiotics with potent activity against Gram-positive and, crucially, Gram-negative pathogens. AlbA has been reported to neutralize albicidin by binding it with nanomolar affinity. To understand this potential resistance mechanism, we determined structures of AlbA and its complex with albicidin. The structures revealed AlbA to be comprised of two domains, each unexpectedly resembling the multiantibiotic neutralizing protein TipA. Binding of the long albicidin molecule was shared pseudosymmetrically between the two domains. The structure also revealed an unexpected chemical modification of albicidin, which we demonstrate to be promoted by AlbA, and to reduce albicidin potency; we propose a mechanism for this reaction. Overall, our findings suggest that AlbA arose through internal duplication in an ancient TipA-like gene, leading to a new binding scaffold adapted to the sequestration of long-chain antibiotics.
- Published
- 2018
23. Author Correction: The bottromycin epimerase BotH defines a group of atypical α/β-hydrolase-fold enzymes
- Author
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Asfandyar Sikandar, Javier Santos-Aberturas, Andriy Luzhetskyy, Jesko Koehnke, Liliya Horbal, Andrew W. Truman, Olga V. Kalinina, Laura Franz, and Sebastian Adam
- Subjects
chemistry.chemical_classification ,chemistry.chemical_compound ,Enzyme ,Chemistry ,Hydrolase ,Cell Biology ,Molecular Biology ,Molecular biology ,Bottromycin - Published
- 2020
24. The natural product carolacton inhibits folate-dependent C1 metabolism by targeting FolD/MTHFD
- Author
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Irene Wagner-Döbler, Asfandyar Sikandar, Jannik Donner, Jennifer Herrmann, Michael Reck, Chengzhang Fu, Nestor Zaburannyi, Jesko Koehnke, Rolf Müller, and Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124Braunschweig, Germany.
- Subjects
0301 basic medicine ,General Physics and Astronomy ,Plasma protein binding ,Crystallography, X-Ray ,Formate-Tetrahydrofolate Ligase ,chemistry.chemical_compound ,0302 clinical medicine ,Aminohydrolases ,Neoplasms ,Myxococcales ,lcsh:Science ,chemistry.chemical_classification ,Multidisciplinary ,biology ,Anti-Bacterial Agents ,Mitochondria ,Biochemistry ,030220 oncology & carcinogenesis ,Macrolides ,Protein Binding ,Science ,Antineoplastic Agents ,Microbial Sensitivity Tests ,Article ,General Biochemistry, Genetics and Molecular Biology ,Minor Histocompatibility Antigens ,Inhibitory Concentration 50 ,03 medical and health sciences ,Folic Acid ,Bacterial Proteins ,Multienzyme Complexes ,Oxidoreductase ,Cell Line, Tumor ,Humans ,Enzyme Assays ,Sorangium cellulosum ,Methylenetetrahydrofolate Dehydrogenase (NADP) ,Biological Products ,Natural product ,General Chemistry ,Metabolism ,biology.organism_classification ,Multifunctional Enzymes ,030104 developmental biology ,Enzyme ,chemistry ,Cell culture ,Biofilms ,Cancer cell ,lcsh:Q - Abstract
The natural product carolacton is a macrolide keto-carboxylic acid produced by the myxobacterium Sorangium cellulosum, and was originally described as an antibacterial compound. Here we show that carolacton targets FolD, a key enzyme from the folate-dependent C1 metabolism. We characterize the interaction between bacterial FolD and carolacton biophysically, structurally and biochemically. Carolacton binds FolD with nanomolar affinity, and the crystal structure of the FolD–carolacton complex reveals the mode of binding. We show that the human FolD orthologs, MTHFD1 and MTHFD2, are also inhibited in the low nM range, and that micromolar concentrations of carolacton inhibit the growth of cancer cell lines. As mitochondrial MTHFD2 is known to be upregulated in cancer cells, it may be possible to use carolacton as an inhibitor tool compound to assess MTHFD2 as an anti-cancer target., The mechanisms behind the antibacterial activity of the natural product carolacton are unknown. Here, the authors show that carolacton is a potent inhibitor of FolD/MTHFD enzymes (involved in folate-dependent C1 metabolism in bacteria and humans) and inhibits the growth of cancer cell lines
- Published
- 2017
25. In vitro reconstitution of α-pyrone ring formation in myxopyronin biosynthesis
- Author
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Rolf Müller, E. Prusov, Jan Henning Sahner, Rolf W. Hartmann, Jesko Koehnke, Hilda Sucipto, and Silke C. Wenzel
- Subjects
0303 health sciences ,biology ,010405 organic chemistry ,Stereochemistry ,Thiolase ,General Chemistry ,Condensation reaction ,01 natural sciences ,0104 chemical sciences ,03 medical and health sciences ,chemistry.chemical_compound ,Polyketide ,Myxopyronin ,chemistry ,Biosynthesis ,Biochemistry ,Polyketide synthase ,biology.protein ,Transferase ,Polymerase ,030304 developmental biology - Abstract
Myxopyronins are α-pyrone antibiotics produced by the terrestrial bacterium Myxococcus fulvus Mx f50 and possess antibacterial activity against Gram-positive and Gram-negative pathogens. They target the bacterial RNA polymerase (RNAP) “switch region” as non-competitive inhibitors and display no cross-resistance to the established RNAP inhibitor rifampicin. Recent analysis of the myxopyronin biosynthetic pathway led to the hypothesis that this secondary metabolite is produced from two separate polyketide parts, which are condensed by the stand-alone ketosynthase MxnB. Using in vitro assays we show that MxnB catalyzes a unique condensation reaction forming the α-pyrone ring of myxopyronins from two activated acyl chains in form of their β-keto intermediates. MxnB is able to accept thioester substrates coupled to either N-acetylcysteamine (NAC) or a specific carrier protein (CP). The turnover rate of MxnB for substrates bound to CP was 12-fold higher than for NAC substrates, demonstrating the importance of protein–protein interactions in polyketide synthase (PKS) systems. The crystal structure of MxnB reveals the enzyme to be an unusual member of the ketosynthase group capable of binding and condensing two long alkyl chains bound to carrier proteins. The geometry of the two binding tunnels supports the biochemical data and allows us to propose an order of reaction, which is supported by the identification of novel myxopyronin congeners in the extract of the producer strain. Insights into the mechanism of this unique condensation reaction do not only expand our knowledge regarding the thiolase enzyme family but also opens up opportunities for PKS bioengineering to achieve directed structural modifications.
- Published
- 2015
26. Thioholgamides: Thioamide-Containing Cytotoxic RiPP Natural Products
- Author
-
Asfandyar Sikandar, Jennifer Herrmann, Nestor Zaburannyi, Sebastian Adam, Louise Kjaerulff, Rolf Müller, and Jesko Koehnke
- Subjects
Stereochemistry ,Peptide ,Antineoplastic Agents ,Biology ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Peptides, Cyclic ,Cell Line, Tumor ,Cytotoxic T cell ,Moiety ,Humans ,Cytotoxicity ,Thioamide ,chemistry.chemical_classification ,Biological Products ,010405 organic chemistry ,General Medicine ,HCT116 Cells ,Combinatorial chemistry ,Streptomyces ,0104 chemical sciences ,Amino acid ,Chemical screening ,Biosynthetic Pathways ,Thioamides ,chemistry ,Cell culture ,Multigene Family ,Molecular Medicine ,Protein Processing, Post-Translational - Abstract
Thioviridamide is a structurally unique ribosomally synthesized and post-translationally modified peptide that contains several thioamide bonds and is active against a number of cancer cell lines. In the search for naturally occurring thioviridamide analogs, we employed genome mining that led to the identification of several related gene clusters. Chemical screening followed by cultivation and isolation yielded thioholgamides A and B, two new additions to the thioviridamide family with several amino acid substitutions, a different N-capping moiety, and with one less thioamide bond. Thioholgamides display improved cytotoxicity in the submicromolar range against a range of cell lines and an IC50 of 30 nM for thioholgamide A against HCT-116 cells. Herein, we report the isolation and structural elucidation of thioholgamides A and B, a proposed biosynthetic cluster for their production, and their bioactivities against a larger panel of microorganisms and cancer cell lines.
- Published
- 2017
27. Cyclic Peptides
- Author
-
James H. Naismith, Wilfred A. van der Donk, and Jesko Koehnke
- Subjects
chemistry.chemical_classification ,Chemistry ,Combinatorial chemistry ,Cyclic peptide - Published
- 2017
28. An Efficient Method for the In Vitro Production of Azol(in)e-Based Cyclic Peptides
- Author
-
Louise Thomas, Rosemary I. Adaba, Jesko Koehnke, Nathalie Pieiller, Andrew R. McEwan, Usama W. Hawas, Jioji N. Tabudravu, Andrew F. Bent, Margaret C. M. Smith, Huanting Liu, Marcel Jaspars, Ulrich Schwarz-Linek, Greg Mann, Wael E. Houssen, James H. Naismith, BBSRC, University of St Andrews. School of Chemistry, University of St Andrews. Biomedical Sciences Research Complex, University of St Andrews. School of Biology, and University of St Andrews. EaSTCHEM
- Subjects
Azoles ,F165 ,medicine.medical_treatment ,Molecular Conformation ,Peptide ,Biosynthesis ,010402 general chemistry ,Peptides, Cyclic ,01 natural sciences ,Chemical synthesis ,Catalysis ,chemistry.chemical_compound ,cyanobactins ,medicine ,QD ,ribosomal peptides ,SDG 14 - Life Below Water ,Cyclic peptides ,Patellamides ,health care economics and organizations ,chemistry.chemical_classification ,Cyanobactins ,Protease ,010405 organic chemistry ,Substrate (chemistry) ,cyclic peptides ,General Medicine ,General Chemistry ,QD Chemistry ,Combinatorial chemistry ,Communications ,Cyclic peptide ,0104 chemical sciences ,Amino acid ,patellamides ,Enzyme ,chemistry ,Phosphorus-Oxygen Lyases ,biosynthesis ,Oxidoreductases ,Ribosomal peptides ,Peptide Hydrolases - Abstract
This paper was funded by: Funded Access, Leverhulme Trust. Grant Number: RPG-2012-504, TSB. Grant Number: 131181, ERC. Grant Number: 339367, and BBSRC. Grant Number: BB/K015508/1 Heterocycle-containing cyclic peptides are promising scaffolds for the pharmaceutical industry but their chemical synthesis is very challenging. A new universal method has been devised to prepare these compounds by using a set of engineered marine-derived enzymes and substrates obtained from a family of ribosomally produced and post-translationally modified peptides called the cyanobactins. The substrate precursor peptide is engineered to have a non-native protease cleavage site that can be rapidly cleaved. The other enzymes used are heterocyclases that convert Cys or Cys/Ser/Thr into their corresponding azolines. A macrocycle is formed using a macrocyclase enzyme, followed by oxidation of the azolines to azoles with a specific oxidase. The work is exemplified by the production of 17 macrocycles containing 6-9 residues representing 11 out of the 20 canonical amino acids. Publisher PDF
- Published
- 2014
29. The Cyanobactin Heterocyclase Enzyme: A Processive Adenylase That Operates with a Defined Order of Reaction
- Author
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Tomas Lebl, Sally L. Shirran, Jesko Koehnke, Andrew F. Bent, D. Zollman, Xiaofeng Zhu, Wael E. Houssen, Marcel Jaspars, James H. Naismith, Ulrich Schwarz-Linek, Greg Mann, Richard Scharff, Kieran G. Smith, Catherine H. Botting, BBSRC, University of St Andrews. School of Chemistry, University of St Andrews. School of Biology, University of St Andrews. Biomedical Sciences Research Complex, and University of St Andrews. EaSTCHEM
- Subjects
Computer science ,Cyanobacteria ,010402 general chemistry ,01 natural sciences ,Adenylation ,Catalysis ,Bacterial protein ,Bacterial Proteins ,cyanobactins ,ribosomal peptide pathways ,QD ,Ribosomal peptide pathways ,License ,Cyanobactins ,Information retrieval ,010405 organic chemistry ,General Chemistry ,Creative commons ,General Medicine ,Adenylyl Cyclases ,QD Chemistry ,Heterocyclase ,heterocyclase ,0104 chemical sciences - Abstract
Counting backwards: The cyanobactin class of heterocyclases, exemplified by TruD, possess an almost unique combination of processivity, specificity, chemical versatility, and promiscuity. TruD is shown by biochemical assay to be an adenylase, and processes cysteines in a defined order. The entire substrate leader can be removed and TruD will process a single specific cysteine residue; however the role of leader is to permit processivity through a balance of recognition. ATP/AMP=adenosine tri/monophosphate; PPi=pyrophosphate.
- Published
- 2013
30. An Enzymatic Route to Selenazolines
- Author
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Sally L. Shirran, Iain A. Smellie, Catherine H. Botting, Marcel Jaspars, Matthew Fuszard, Jesko Koehnke, Wael E. Houssen, James H. Naismith, Andrew F. Bent, Falk Morawitz, Margaret C. M. Smith, BBSRC, University of St Andrews. School of Chemistry, University of St Andrews. School of Biology, University of St Andrews. EaSTCHEM, and University of St Andrews. Biomedical Sciences Research Complex
- Subjects
Threonine ,Macrocyclization ,Biosynthesis ,010402 general chemistry ,Peptides, Cyclic ,01 natural sciences ,Biochemistry ,Iodoacetamide ,Selenium ,chemistry.chemical_compound ,Multienzyme Complexes ,Serine ,Organic chemistry ,QD ,Amino Acid Sequence ,Cysteine ,Patellamides ,Oxazoles ,Molecular Biology ,chemistry.chemical_classification ,Selenocysteine ,010405 organic chemistry ,Organic Chemistry ,Selenazolines ,selenazolines ,Line ,QD Chemistry ,Multienzyme complexes ,Communications ,3. Good health ,0104 chemical sciences ,patellamides ,Thiazoles ,Enzyme ,chemistry ,Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ,Molecular Medicine ,biosynthesis ,Derivatives - Abstract
Supported by a Wellcome Trust Capital Award (086036) Ringing the changes: Selenazolines have applications in medicinal chemistry, but their synthesis is challenging. We report a new convenient and less toxic route to these heterocycles that starts from commercially available selenocysteine. The new route depends on a heterocyclase enzyme that creates oxazolines and thiazolines from serines/threonines and cysteines. Publisher PDF
- Published
- 2013
31. The Discovery of New Cyanobactins fromCyanothecePCC 7425 Defines a New Signature for Processing of Patellamides
- Author
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Jeremie Vendome, Jesko Koehnke, Margaret C. M. Smith, Marcel Jaspars, Wael E. Houssen, James H. Naismith, Andrea Raab, and D. Zollman
- Subjects
Models, Molecular ,food.ingredient ,Cyanothece ,medicine.medical_treatment ,Molecular Sequence Data ,Peptide ,Biology ,Peptides, Cyclic ,Biochemistry ,food ,Hydrolase ,medicine ,Amino Acid Sequence ,Molecular Biology ,chemistry.chemical_classification ,Serine protease ,Protease ,Organic Chemistry ,Ribosomal RNA ,Cyclic peptide ,Protein Structure, Tertiary ,Enzyme ,chemistry ,biology.protein ,Molecular Medicine ,Serine Proteases ,Protein Processing, Post-Translational - Abstract
Cyanobactins, including patellamides, are a group of cyanobacterial post-translationally modified ribosomal cyclic peptides. The final product should in theory be predictable from the sequence of the precursor peptide and the associated tailoring enzymes. Understanding the mechanism and recognition requirements of these enzymes will allow their rational engineering. We have identified three new cyanobactins from a Cyanothece PCC 7425 culture subjected to a heat shock. One of these compounds revealed a novel signature signal for ThcA, the subtilisin-like serine protease that is homologous to the patellamide protease PatA. The crystal structure of the latter and modelling studies allow rationalisation of the recognition determinants for both enzymes, consistent with the ribosomal biosynthetic origin of the new compounds.
- Published
- 2012
32. Structure and Substrate Recognition of the Bottromycin Maturation Enzyme BotP
- Author
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Greg Mann, Rolf Müller, Sebastian Adam, Liujie Huo, Nicholas J. Westwood, Jeremie Vendome, Jesko Koehnke, Brunello Nardone, European Research Council, University of St Andrews. School of Chemistry, University of St Andrews. EaSTCHEM, and University of St Andrews. Biomedical Sciences Research Complex
- Subjects
0301 basic medicine ,Models, Molecular ,Stereochemistry ,NDAS ,Molecular Conformation ,Sequence (biology) ,Peptide ,Biology ,Biosynthesis ,Crystallography, X-Ray ,Biochemistry ,Peptides, Cyclic ,Substrate Specificity ,03 medical and health sciences ,chemistry.chemical_compound ,Leucyl Aminopeptidase ,Hydrolase ,Leucyl-amino peptidase ,QD ,Molecular Biology ,Bottromycin ,chemistry.chemical_classification ,Methionine ,BotP ,Organic Chemistry ,Substrate (chemistry) ,QD Chemistry ,RiPPs ,030104 developmental biology ,Enzyme ,chemistry ,Molecular Medicine - Abstract
JK would like to thank the University of St Andrews, which is supported by a Wellcome Trust Capital Award (086036) and the Deutsche Forschungsgemeinschaft for an Emmy Noether fellowship (KO4116/3-1). BN would like to thank the European Research Council (339367). The bottromycins are a family of highly modified peptide natural products displaying potent antimicrobial activity against Gram-positive bacteria including methicillin-resistant Staphyloccoccus aureus. Bottromycins have recently been shown to be ribosomally synthesized and post-translationally modified peptides (RiPPs). Unique amongst RiPPs the precursor peptide BotA contains a C-terminal "follower" sequence, rather than the canonical N- terminal "leader" sequence. We report the structural and biochemical characterization of BotP, a leucyl-aminopeptidase like enzyme from the bottromycin pathway. We demonstrate that BotP is responsible for the removal of the N-terminal methionine from the precursor peptide. Determining the crystal structures of apo BotP and of BotP in complex with Mn2+ allowed us to model a BotP/substrate complex and to rationalize substrate recognition. Our data represent the first step towards targeted compound modification to unlock the full antibiotic potential of bottromycin. Postprint
- Published
- 2016
33. Splice Form Dependence of β-Neurexin/Neuroligin Binding Interactions
- Author
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Lawrence Shapiro, Fabiana Bahna, Goran Ahlsen, Phinikoula S. Katsamba, Jeremie Vendome, Jesko Koehnke, Xiangshu Jin, and Barry Honig
- Subjects
Models, Molecular ,Glycosylation ,PROTEINS ,Cell Adhesion Molecules, Neuronal ,Neuregulin-1 ,Neuroscience(all) ,Neurexin ,Neuroligin ,Plasma protein binding ,Biology ,Crystallography, X-Ray ,Transfection ,MOLNEURO ,Biophysical Phenomena ,03 medical and health sciences ,0302 clinical medicine ,Humans ,Immunoprecipitation ,Protein Isoforms ,NLS ,splice ,Binding site ,Cell Line, Transformed ,030304 developmental biology ,0303 health sciences ,Binding Sites ,General Neuroscience ,Alternative splicing ,Surface Plasmon Resonance ,Molecular biology ,Protein Structure, Tertiary ,Cell biology ,Alternative Splicing ,Mutation ,RNA splicing ,CELLBIO ,Calcium ,Ultracentrifugation ,030217 neurology & neurosurgery ,Protein Binding - Abstract
Alternatively spliced beta-neurexins (beta-NRXs) and neuroligins (NLs) are thought to have distinct extracellular binding affinities, potentially providing a beta-NRX/NL synaptic recognition code. We utilized surface plasmon resonance to measure binding affinities between all combinations of alternatively spliced beta-NRX 1-3 and NL 1-3 ectodomains. Binding was observed for all beta-NRX/NL pairs. The presence of the NL1 B splice insertion lowers beta-NRX binding affinity by approximately 2-fold, while beta-NRX splice insertion 4 has small effects that do not synergize with NL splicing. New structures of glycosylated beta-NRXs 1 and 2 containing splice insertion 4 reveal that the insertion forms a new beta strand that replaces the beta10 strand, leaving the NL binding site intact. This helps to explain the limited effect of splice insert 4 on NRX/NL binding affinities. These results provide new structural insights and quantitative binding information to help determine whether and how splice isoform choice plays a role in beta-NRX/NL-mediated synaptic recognition.
- Published
- 2010
34. Ubc9 fusion–directed SUMOylation (UFDS): a method to analyze function of protein SUMOylation
- Author
-
Bernhard Korn, Astrid Jakobs, Rainer Niedenthal, Matthias Gaestel, Fabian Himstedt, Martin Funk, and Jesko Koehnke
- Subjects
Protein sumoylation ,Recombinant Fusion Proteins ,SUMO-1 Protein ,HEK 293 cells ,SUMO protein ,SUMO enzymes ,Cell Biology ,Biology ,Kidney ,Protein Engineering ,Subcellular localization ,Biochemistry ,Cell Line ,Protein–protein interaction ,Cell biology ,STAT1 Transcription Factor ,Ubiquitin-Conjugating Enzymes ,Humans ,Phosphorylation ,Tumor Suppressor Protein p53 ,Molecular Biology ,Function (biology) ,Biotechnology - Abstract
Although small ubiquitin-like modifier (SUMO) is conjugated to proteins involved in diverse cellular processes, the functional analysis of SUMOylated proteins is often hampered by low levels of specific SUMOylated proteins in the cell. Here we describe a SUMO-conjugating enzyme (Ubc9) fusion-directed SUMOylation (UFDS) system, which allows efficient and selective in vivo SUMOylation of proteins. Although SUMOylation of overexpressed p53 and STAT1 was difficult to detect in HEK293 cells, up to 40% of p53 and STAT1 were conjugated with endogenous SUMO when fused to Ubc9. We verified the specificity of UFDS using SUMOylation-site mutants and showed that the method is not dependent on SUMO ligases. Using UFDS we demonstrated that SUMOylation of STAT1 inhibits its phosphorylation at Tyr701 and discovered p53 multi-SUMOylation in vivo. We propose that UFDS will be useful for the analysis of function of SUMOylation in protein interactions, subcellular localization as well as enzymatic activity.
- Published
- 2007
35. Structural analysis of leader peptide binding enables leader-free cyanobactin processing
- Author
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Marcel Jaspars, Andrew F. Bent, Sally L. Shirran, Tomas Lebl, Jesko Koehnke, Catherine H. Botting, Wael E. Houssen, James H. Naismith, Greg Mann, Hannes Ludewig, BBSRC, University of St Andrews. School of Chemistry, University of St Andrews. School of Biology, University of St Andrews. EaSTCHEM, and University of St Andrews. Biomedical Sciences Research Complex
- Subjects
Models, Molecular ,Signal peptide ,Molecular Sequence Data ,Protein domain ,NDAS ,Gene Expression ,Context (language use) ,Peptide ,Protein Sorting Signals ,Cyanobacteria ,Protein Engineering ,010402 general chemistry ,Peptides, Cyclic ,01 natural sciences ,Article ,Protein Structure, Secondary ,Substrate Specificity ,03 medical and health sciences ,Adenosine Triphosphate ,Bacterial Proteins ,Hydrolase ,QD ,Amino Acid Sequence ,Cysteine ,Binding site ,Molecular Biology ,Peptide sequence ,R2C ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,Binding Sites ,biology ,Active site ,Stereoisomerism ,Cell Biology ,QD Chemistry ,Recombinant Proteins ,Protein Structure, Tertiary ,0104 chemical sciences ,Thiazoles ,Biochemistry ,chemistry ,Cyclization ,Biocatalysis ,biology.protein ,BDC ,Protein Processing, Post-Translational - Abstract
This work was supported by grants from the ERC 339367 (J.H.N. and M.J.) and BBSRC BB/K015508/1 (J.H.N and M.J.). We acknowledge use of the Diamond (beamlines I02 and I24) and ESRF (beamline ID29) synchrotrons. JHN is Royal Society Wolfson Merit Award Holder and a 1000 talent scholar of the Chinese Government at Sichuan University. Regioselective modification of amino acids within the context of a peptide is common to a number of biosynthetic pathways, and many of the resulting products have potential as therapeutics. The ATP-dependent enzyme LynD heterocyclizes multiple cysteine residues to thiazolines within a peptide substrate. The enzyme requires the substrate to have a conserved N-terminal leader for full activity. Catalysis is almost insensitive to immediately flanking residues in the substrate, suggesting that recognition occurs distant from the active site. Nucleotide and peptide substrate co-complex structures of LynD reveal that the substrate leader peptide binds to and extends the β-sheet of a conserved domain of LynD, whereas catalysis is accomplished in another conserved domain. The spatial segregation of catalysis from recognition combines seemingly contradictory properties of regioselectivity and promiscuity, and it appears to be a conserved strategy in other peptide-modifying enzymes. A variant of LynD that efficiently processes substrates without a leader peptide has been engineered. Postprint
- Published
- 2015
36. The structure of the cyanobactin domain of unknown function from PatG in the patellamide gene cluster
- Author
-
Marcel Jaspars, Jesko Koehnke, Rachael Graham, Wael E. Houssen, Andrew F. Bent, Greg Mann, James H. Naismith, Uli Schwarz-Linek, BBSRC, University of St Andrews. School of Chemistry, University of St Andrews. School of Biology, University of St Andrews. Biomedical Sciences Research Complex, and University of St Andrews. EaSTCHEM
- Subjects
Protein Conformation ,Molecular Sequence Data ,Protein domain ,Biophysics ,Computational biology ,Biology ,Peptides, Cyclic ,Biochemistry ,chemistry.chemical_compound ,Protein structure ,Biosynthesis ,Structural Biology ,cyanobactins ,Gene cluster ,Hydrolase ,Genetics ,Structural Communications ,QD ,Amino Acid Sequence ,PatG ,Nuclear Magnetic Resonance, Biomolecular ,chemistry.chemical_classification ,Sequence Homology, Amino Acid ,Condensed Matter Physics ,QD Chemistry ,Cyclic peptide ,patellamides ,RiPPs ,chemistry ,Cyclization ,Domain of unknown function ,Function (biology) - Abstract
The highly conserved domain of unknown function in the cyanobactin superfamily has a novel fold. The protein does not appear to bind the most plausible substrates, leaving questions as to its role., Patellamides are members of the cyanobactin family of ribosomally synthesized and post-translationally modified cyclic peptide natural products, many of which, including some patellamides, are biologically active. A detailed mechanistic understanding of the biosynthetic pathway would enable the construction of a biotechnological ‘toolkit’ to make novel analogues of patellamides that are not found in nature. All but two of the protein domains involved in patellamide biosynthesis have been characterized. The two domains of unknown function (DUFs) are homologous to each other and are found at the C-termini of the multi-domain proteins PatA and PatG. The domain sequence is found in all cyanobactin-biosynthetic pathways characterized to date, implying a functional role in cyanobactin biosynthesis. Here, the crystal structure of the PatG DUF domain is reported and its binding interactions with plausible substrates are investigated.
- Published
- 2014
37. The structural biology of patellamide biosynthesis
- Author
-
Marcel Jaspars, Wael E. Houssen, Jesko Koehnke, Andrew F. Bent, James H. Naismith, Greg Mann, BBSRC, University of St Andrews. School of Chemistry, University of St Andrews. EaSTCHEM, and University of St Andrews. Biomedical Sciences Research Complex
- Subjects
Protein Conformation ,Stereochemistry ,Molecular Sequence Data ,Peptide ,Biology ,Peptides, Cyclic ,01 natural sciences ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,Protein structure ,Biosynthesis ,Heterocyclic Compounds ,Structural Biology ,Dimethylallyltranstransferase ,QD ,Amino Acid Sequence ,Amino Acids ,Molecular Biology ,Peptide sequence ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,Bacteria ,010405 organic chemistry ,QD Chemistry ,Biosynthetic Pathways ,0104 chemical sciences ,Amino acid ,Enzyme ,chemistry ,Biochemistry ,Structural biology - Abstract
This work was supported by grants from the ERC339367 (JHN and MJ) and BBSRCBB/K015508/1 (JHN and MJ). The biosynthetic pathways for patellamide and related natural products have recently been studied by structural biology. These pathways produce molecules that have a complex framework and exhibit a diverse array of activity due to the variability of the amino acids that are found in them. As these molecules are difficult to synthesize chemically, exploitation of their properties has been modest. The patellamide pathway involves amino acid heterocyclization, peptide cleavage, peptide macrocyclization, heterocycle oxidation and epimerization; closely related products are also prenylated. Enzyme activities have been identified for all these transformations except epimerization, which may be spontaneous. This review highlights the recent structural and mechanistic work on amino acid heterocyclization, peptide cleavage and peptide macrocyclization. This work should help in using the enzymes to produce novel analogs of the natural products enabling an exploitation of their properties. Publisher PDF
- Published
- 2014
38. Structure of PatF from Prochloron didemni
- Author
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Jesko Koehnke, Margaret C. M. Smith, Wael E. Houssen, James H. Naismith, Andrew F. Bent, Marcel Jaspars, BBSRC, University of St Andrews. School of Chemistry, University of St Andrews. EaSTCHEM, and University of St Andrews. Biomedical Sciences Research Complex
- Subjects
System ,natural products ,Astrophysics::High Energy Astrophysical Phenomena ,Molecular Sequence Data ,Biophysics ,Prochloron ,Physics::Optics ,Plasma protein binding ,Lissoclinum-patella ,Biochemistry ,Peptides, Cyclic ,Homology (biology) ,Protein Structure, Secondary ,Prenylation ,Structural Biology ,patellamide ,cyanobactins ,Genetics ,Transferase ,Structural Communications ,QD ,Amino Acid Sequence ,Peptide sequence ,Gene ,Macromolecular crystallography ,biology ,Active site ,Refinement ,Condensed Matter Physics ,biology.organism_classification ,QD Chemistry ,Quality ,Protein Structure, Tertiary ,prenyltransferases ,biology.protein ,Crystallization ,Protein Binding - Abstract
The X-ray crystal structure of PatF from P. didemni was solved by the single-wavelength anomalous diffraction method to a resolution of 2.13 Å., Patellamides are macrocyclic peptides with potent biological effects and are a subset of the cyanobactins. Cyanobactins are natural products that are produced by a series of enzymatic transformations and a common modification is the addition of a prenyl group. Puzzlingly, the pathway for patellamides in Prochloron didemni contains a gene, patF, with homology to prenylases, but patellamides are not themselves prenylated. The structure of the protein PatF was cloned, expressed, purified and determined. Prenylase activity could not be demonstrated for the protein, and examination of the structure revealed changes in side-chain identity at the active site. It is suggested that these changes have inactivated the protein. Attempts to mutate these residues led to unfolded protein.
- Published
- 2013
39. The mechanism of patellamide macrocyclization revealed by the characterization of the PatG macrocyclase domain
- Author
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Jeremie Vendome, Andrew F. Bent, Catherine H. Botting, Jesko Koehnke, Marcel Jaspars, Sally L. Shirran, D. Zollman, Ada F. Nneoyiegbe, Wael E. Houssen, Laurent Trembleau, James H. Naismith, Margaret C. M. Smith, and Falk Morawitz
- Subjects
Models, Molecular ,Stereochemistry ,Protein Conformation ,Prochloron ,Peptide ,Peptides, Cyclic ,Article ,Substrate Specificity ,Protein structure ,Bacterial Proteins ,Structural Biology ,Hydrolase ,Peptide bond ,Animals ,Amino Acid Sequence ,Subtilisins ,Urochordata ,Symbiosis ,Molecular Biology ,Peptide sequence ,chemistry.chemical_classification ,biology ,Subtilisin ,biology.organism_classification ,Recombinant Proteins ,Amino acid ,Protein Structure, Tertiary ,chemistry ,Oligopeptides - Abstract
Peptide macrocycles are found in many biologically active natural products. Their versatility, resistance to proteolysis and ability to traverse membranes has made them desirable molecules. Although technologies exist to synthesize such compounds, the full extent of diversity found among natural macrocycles has yet to be achieved synthetically. Cyanobactins are ribosomal peptide macrocycles encompassing an extraordinarily diverse range of ring sizes, amino acids and chemical modifications. We report the structure, biochemical characterization and initial engineering of the PatG macrocyclase domain of Prochloron sp. from the patellamide pathway that catalyzes the macrocyclization of linear peptides. The enzyme contains insertions in the subtilisin fold to allow it to recognize a three-residue signature, bind substrate in a preorganized and unusual conformation, shield an acyl-enzyme intermediate from water and catalyze peptide bond formation. The ability to macrocyclize a broad range of nonactivated substrates has wide biotechnology applications.
- Published
- 2012
40. Crystal structures of β-neurexin 1 and β-neurexin 2 ectodomains and dynamics of splice insertion sequence 4
- Author
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Phinikoula S. Katsamba, Peter Scheiffele, Nikola Trbovic, Jesko Koehnke, Lawrence Shapiro, Goran Ahlsen, Barry Honig, Xiangshu Jin, Arthur G. Palmer, and Julia Brasch
- Subjects
Models, Molecular ,Materials science ,PROTEINS ,Cell Adhesion Molecules, Neuronal ,Molecular Sequence Data ,Neurexin ,Neuroligin ,Nerve Tissue Proteins ,Plasma protein binding ,Crystallography, X-Ray ,Models, Biological ,Article ,03 medical and health sciences ,0302 clinical medicine ,Structural Biology ,Animals ,Protein Isoforms ,splice ,Amino Acid Sequence ,Insertion sequence ,Peptide sequence ,Molecular Biology ,030304 developmental biology ,0303 health sciences ,Sequence Homology, Amino Acid ,Alternative splicing ,Membrane Proteins ,Recombinant Proteins ,Protein Structure, Tertiary ,Rats ,Crystallography ,SIGNALING ,RNA splicing ,Biophysics ,Calcium ,RNA Splice Sites ,030217 neurology & neurosurgery ,Protein Binding - Abstract
Presynaptic neurexins (NRXs) bind to postsynaptic neuroligins (NLs) to form Ca2+-dependent protein complexes that bridge neural synapses. NRXs bind NLs through their membrane-proximal LNS domain, which contains a single site of alternative splicing (splice site 4) that gives rise to two isoforms: +4, which includes a 30-residue splice insertion, and Δ which lacks this insertion. Here we present crystal structures of the neuroligin-binding Δ isoforms of the LNS domains from β-NRX1 and β-NRX2, crystallized in the presence of Ca2+ ions. The Ca2+-binding site in the 3.0Å β-NRX2 structure is disordered. In contrast, the 1.7Å β-NRX1 structure reveals a single Ca2+ ion, with one of its coordinating ligands donated by a glutamic acid from an adjacent β-NRX1 molecule, ∼12Å from the splice insertion site. NMR studies of β-NRX1 Δ and β-NRX1+4 show that the splice-inserted sequence forms an unstructured loop in isolation, and remains at least partially disordered in a β-NRX1+4 / NL complex. These results suggest a likely mode of Ca2+-dependent interaction with NLs, and raise the possibility that β-NRX insertion sequence 4 may function in roles independent of neuroligin binding.
- Published
- 2008
41. Crystal structure of the extracellular cholinesterase-like domain from neuroligin-2
- Author
-
Barry Honig, Jesko Koehnke, Elaine C. Budreck, Shoshana Posy, Peter Scheiffele, Lawrence Shapiro, and Xiangshu Jin
- Subjects
Gene isoform ,Models, Molecular ,Protein Conformation ,Cell Adhesion Molecules, Neuronal ,Neurexin ,Nerve Tissue Proteins ,Biology ,Crystallography, X-Ray ,Cell Line ,Mice ,Protein structure ,Animals ,Cholinesterases ,Humans ,splice ,Multidisciplinary ,Binding Sites ,Alternative splicing ,Membrane Proteins ,Biological Sciences ,Transmembrane protein ,Cell biology ,Alternative Splicing ,Membrane protein ,Ectodomain ,Biochemistry ,Dimerization - Abstract
Neuroligins (NLs) are catalytically inactive members of a family of cholinesterase-like transmembrane proteins that mediate cell adhesion at neuronal synapses. Postsynaptic neuroligins engage in Ca 2+ -dependent transsynaptic interactions via their extracellular cholinesterase domain with presynaptic neurexins (NRXs). These interactions may be regulated by two short splice insertions (termed A and B) in the NL cholinesterase domain. Here, we present the 3.3-Å crystal structure of the ectodomain from NL2 containing splice insertion A (NL2A). The overall structure of NL2A resembles that of cholinesterases, but several structural features are unique to the NL proteins. First, structural elements surrounding the esterase active-site region differ significantly between active esterases and NL2A. On the opposite surface of the NL2A molecule, the positions of the A and B splice insertions identify a candidate NRX interaction site of the NL protein. Finally, sequence comparisons of NL isoforms allow for mapping the location of residues of previously identified mutations in NL3 and NL4 found in patients with autism spectrum disorders. Overall, the NL2 structure promises to provide a valuable model for dissecting NL isoform- and synapse-specific functions.
- Published
- 2008
42. Cover Picture: The Discovery of New Cyanobactins fromCyanothecePCC 7425 Defines a New Signature for Processing of Patellamides (ChemBioChem 18/2012)
- Author
-
Marcel Jaspars, Wael E. Houssen, James H. Naismith, D. Zollman, Margaret C. M. Smith, Andrea Raab, Jeremie Vendome, and Jesko Koehnke
- Subjects
food.ingredient ,food ,Biochemistry ,Cyanothece ,Organic Chemistry ,Molecular Medicine ,Cover (algebra) ,Computational biology ,Biology ,Molecular Biology ,Signature (logic) - Published
- 2012
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