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Investigation of the Antibacterial Activity and the Biosynthesis Gene Cluster of the Peptide Antibiotic Feglymycin

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Investigation of the Antibacterial Activity and the Biosynthesis Gene Cluster of the Peptide Antibiotic Feglymycin Investigation of the antibacterial activity and the biosynthesis gene cluster of the peptide antibiotic feglymycin vorgelegt von Diplom-Biochemikerin Saskia Rausch aus Berlin Von der Fakultät II – Mathematik und Naturwissenschaften der Technischen Universität Berlin zur Erlangung des akademischen Grades Doktor der Naturwissenschaften - Dr. rer. nat. – genehmigte Dissertation Promotionsausschuss: Vorsitzende: Prof. Dr. Marga Lensen 1. Berichter/Gutachter: Prof. Dr. Roderich Süssmuth 2. Berichter/Gutachter: Prof. Dr. Dirk Schwarzer Tag der wissenschaftlichen Aussprache: 09.12.2011 Berlin 2012 D 83 Zusammenfassung: Untersuchung zur antibakteriellen Wirkung und zum Biosynthese-Genclusters des Peptidantibiotikum Feglymycin Feglymycin ist ein aus Streptomyces sp. DSM 11171 isoliertes, lineares 13mer-Peptid, das zu einem hohen Anteil aus den nicht-proteinogenen Aminosäuren Hpg (4-Hydroxyphenylglycine) und Dpg (3,5- Dihydroxyphenylglycine) besteht. Zudem besitzt es eine interessante, alternierende Abfolge von D- und L- Aminosäuren und strukturelle Ähnlichkeiten mit den Glycopeptiden der Vancomycin-Gruppe von Antibiotika und den Glycodepsipeptid-Antibiotika Ramoplanin und Enduracidin. Außerdem besitzt Feglymycin eine interessante Bioaktivität. Es wirkt in vivo antibakteriell gegen MRSA-Stämme (multi- resistente Staphylococcus aureus Stämme) und inhibitiert in vitro die Replikation von HIV-Viren im Zellkulturtest. Aufgrund seiner molekularen Masse und strukturellen Ähnlichkeit mit bekannten Zellwandbiosynthese-Inhibitoren wie z.B. Vancomycin und Ramoplanin, wurde auch Feglymycin als Zellwandbiosynthese-Inhibitor getestet. In diesen Tests zeigte Feglymycin keinen Effekt auf die membran-gebundenen zweite und die dritte Stufe der Peptidoglycanbiosynthese. Jedoch deuteten die Experimente auf eine Inhibition der früheren Biosyntheseschritte hin. Ziel dieser Arbeit war es, die antibakterielle Wirkung von Feglymycin auf die bakterielle Zellwandbiosynthese im Detail zu untersuchen und das biologische Target zu identifizieren. Zusätzlich wurde das Feglymycin Biosynthese-Gencluster untersucht. In LC-MS „one-pot assays“ wurde die Wirkung von Feglymycin auf die isolierten E. coli-Enzyme MurA-F getestet. Hierbei konnte reproduzierbar gezeigt werden, das Feglymycin die Enzyme MurA (Enopyruvyl-UDP-GlcNAc Synthase) und MurC (UDP-N-Acetyl-muramyl-L-alanin Ligase) inhibiert. In spektrophotometischen Assays mit den E. coli-Enzymen MurA und MurC konnte ein Ki-Wert von 0.33 +/- 0.04 µM für das MurC Enzym und ein Ki-Wert von 3.4 +/- 1.1 µM für das MurA Enzym bestimmt werden. Weitere Untersuchungen zeigten, dass Feglmycin auch die MurA (IC50 = 3.5 +/- 1.3 µM) und MurC (IC50 = 1.0 +/- 0.6 µM) Enzyme des gram-positiven Bakteriums Staphylococcus aureus inhibiert. Feglymycin zeigte dabei eine nicht-kompetitive Inhibition gegenüber der Bindung der Substrate des MurA Enzyms PEP (Phosphoenolpyruvat) und UDP-GlcNAc (UDP-N-Acetylglucosamin) und der Substrate des MurC Enzyms UDP-MurNAc (UDP-N-Acetylmuramat), ATP (Adenosintriphosphat) and L-Alanin. Feglmycin ist daher der erste Naturstoff der das MurC Enzym inhibiert. Zudem zeigt Feglymycin einen nicht- kompetitive Inhibitionstyp. Circulardichromismus (CD) Experimente mit den isolierten E. coli-Enzymen MurA und MurC und Feglymycin deuten einen möglichen allosterischen Effekt des Inhibitors auf die Enzyme an. Zusätzlich wurde die Feglmycinproduktion durch den Stamm Streptomyces sp. DSM 11171 und die Feglymycin-Detektion mittels LC-MS optimiert. Durch die Sequezierung des Genoms von Streptomyces sp. DSM 11171 konnte das Feglymycin Biosynthese-Gencluster identifiziert werden. Bei der Annotation des Genclusters zeigte sich, dass es sich bei Feglymycin um ein nicht-ribosomal synthetisiertes Peptid (NRPS) handelt dessen Biosynthese der Biosynthese der Glycopeptidantibiotika der Vancomycin-Gruppe von Antibiotika ähnelt. Zudem konnten im Streptomyces sp. DSM 11171 Genom weitere NRPS und Polyketidsynthase (PKS) Gencluster identifiziert und annotiert werden. Abstract: Investigation of the antibacterial activity and the biosynthesis gene cluster of the peptide antibiotic feglymycin Feglymycin is a linear 13-mer peptide produced by Streptomyces sp. DSM 11171 containing largely the non-proteinogenic Hpg (4-hydroxyphenylglycine) and the non-proteinogenic Dpg (3,5- dihydroxyphenylglycine) amino acids and an interesting alternation of D and L amino acids. It shows structural homogies to the glycopeptides of the vancomycin group of antibiotics and the glycodepsipeptide antibiotics ramoplanin and enduracidin. Feglymycin additionally shows an interesting biological activity. It possesses antibiotic activity against MRSA (multi-resistant Staphylococcus aureus) strains in vivo and inhibits syncytium formation in HIV infection in vitro. Due to its molecular mass and structural analogies to known inhibitors of the cell-wall biosynthesis, i.e. vancomycin and ramoplanin, feglymycin was tested as cell-wall biosynthesis inhibitor. In these tests feglymycin showed no effect on the membrane-bound second and third step of the peptidoglycan biosynthesis but the experiments indicated an inhibition of earlier biosynthetic steps. Aim of this work was to investigate the antibacterial activity of feglymycin on the bacterial cell- wall biosynthesis in more detail and to identify the biological target. Additionally the feglymycin biosynthesis gene cluster was investigated. Feglymycin was tested in a LC-MS one-pot assay against the isolated enzymes MurA-F from E. coli. Dereplication revealed that feglymycin specifically inhibits the enzymes MurA (enolpyruvyl-UDP-GlcNAc synthase) and MurC (UDP-N-acetyl-muramyl-L-alanine ligase). In in vitro assays with the enzymes MurA and MurC from gram-negative E. coli, a Ki value of 0.33 +/- 0.04 µM was determined for the MurC enzyme and a Ki value of 3.4 +/- 1.1 µM for the MurA enzyme. Further investigations showed that feglymycin also inhibits the MurA (IC50 = 3.5 +/- 1.3 µM) and MurC (IC50 = 1.0 +/- 0.6 µM) enzyme from gram-positive Staphylococcus aureus. The inhibition mode of feglymycin was found to be non-competitive with the binding of PEP (phosphoenolpyruvate) and UDP-GlcNAc (UDP-N-acetylglucosamine) in case of the MurA enzyme and non-competitive with binding of UDP-MurNAc (UDP-N-acetylmuramic acid), ATP (adenosine-triphosphate) and L-alanine in case of the MurC enzyme. Feglymycin is therefore the first natural compound found to inhibit the MurC enzyme showing a non-competitive inhibition type. Circular dichroism (CD) experiments with the isolated enzymes MurA and MurC from E. coli and feglymycin indicated a possible allosteric effect of feglymycin. Furthermore the feglymycin production by Streptomyces sp. DSM 11171 and feglymycin detection by LC-MS were optimized. Sequencing of the genome of Streptomyces sp. DSM 11171 allowed the idenfitication of the feglymycin biosynthesis gene cluster. Annotation of the gene cluster showed that feglymycin is a non-ribosomal synthesized peptide (NRPS) closely related to the glycopeptides of the vancomycin group of antibiotics. Additionally further NRPS and polyketide synthase (PKS) gene clusters were identified in the Streptomyces sp. DSM 11171 genome and annotated. Teile der vorliegenden Arbeit wurden bereits veröffentlicht: Publikationen: “Feglymycin is an inhibitor of the enzymes MurA and MurC of the peptidoglycan biosynthesis pathway” Saskia Rausch, Anne Hänchen, Alexander Denisiuk, Marius Löhken, Tanja Schneider, Roderich D. Süssmuth ChemBioChem, 2011, 8, 1171-1173. Poster: “Feglymycin inhibits the enzymes MurA and MurC of the peptidoglycan biosynthesis pathway” Saskia Rausch, Anne Hänchen, Alexander Denisiuk, Marius Löken and Roderich Süßmuth 10th German Peptide Symposium, FU-Berlin, 7.03-10.03.2011 “Feglymycin inhibits the enzymes MurA and MurC of the peptidoglycan biosynthesis pathway” Saskia Rausch, Anne Hänchen, Alexander Denisiuk, Marius Löken and Roderich Süßmuth Bayer PhD student Course, Köln, 10.-14.07.2011 Vorträge: “Antimicrobial activity of the peptide antibiotic Feglymycin” Saskia Rausch, Anne Hänchen, Alexander Denisiuk, Marius Löken and Roderich Süßmuth International workshop, DGMS „New Developments of Lysosomal Storage Disease Diagnostics & Therapeutics, Protein – Carbohydrate Interactions, Konstanz, 3-5.10.2011 “It is the search for the truth, not possession of the truth which is the way of philosophy. Its questions are more relevant than its answers, and every answer becomes a new question.” Karl Theodor Jaspers, Way to Wisdom: An Introduction to Philosophy “It’s a bacteria-eat-man world out there, filled with a nearly endless variety of germs that see us as spawning grounds.” W. Wayt Gibbs, Nanobodies Die vorliegende Arbeit wurde unter der Leitung von Herrn Prof. Dr. Roderich Süßmuth In der Zeit vom September 2008 bis August 2011 am Institut für Chemie der Fakultät II der Technischen Universität Berlin angefertigt. Danksagungen: Mein besondere Dank gilt meinen Doktorvater Prof. Roderich Süssmuth für meine Aufnahme in den Arbeitskreis Süßmuth, das interessante Promotionsthema und die exzellente Betreuung und Förderung wärend meiner Promotionszeit. Ich bin dankbar für das mir von ihm entgegengebrachte Vertrauen und das freundschaftliche Verhältnis, dass das Arbeiten auch in besonders stressigen Zeiten noch angenehm machte. Ich bin ebenfalls dankbar für die vielen Möglichkeiten die mir geboten wurden so viele verschiedene Methoden zu erlernen und so vielfältige und spannende Projekte zu bearbeiten. Insbesondere möchte ich mich auch für die intensive
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