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Screening of Prenyldiphosphate Mimetics For SCREENING OF PRENYLDIPHOSPHATE MIMETICS FOR THEIR POTENTIAL TO INHIBIT 4-HYDROXYBENZOATE OLIGOPRENYL TRANSFERASE Dissertation To obtain the Doctoral Degree in Natural Sciences (Dr. rer. nat.) Submitted to The Faculty I of Natural Sciences (Biological Sciences) Martin Luther University Halle-Wittenberg (MLU), Germany Institute of Pharmacy by Amina Msonga Born on 16.01.1980 in Nzega, Tanzania Halle (Saale), Germany SCREENING OF PRENYLDIPHOSPHATE MIMETICS FOR THEIR POTENTIAL TO INHIBIT 4-HYDROXYBENZOATE OLIGOPRENYL TRANSFERASE Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) der Naturwissenschaftlichen Fakultät I – Biowissenschaften der Martin-Luther-Universität Halle-Wittenberg Institut für Pharmazie vorgelegt von Frau Amina Msonga geboren am 16.01.1980 in Nzega, Tanzania Gutachter: Prof. Dr. L. A. Wessjohann (Martin-Luther-University, Halle-Wittenberg (& Leibniz-IPB)) Prof. Dr. M. Pietzsch (Martin-Luther-University, Halle-Wittenberg) Prof. Dr. O. Kayser (Technical University, Dortmund) Verteidigt am: 25.01.2017 ii ACKNOWLEDGEMENT Accomplishment of this work has been contributed by direct or indirect support of some people who helped me in one-way or another. Firstly, I would like to express my heartfelt appreciations to my supervisor; Prof. Dr. L. A. Wessjohann for welcoming me at IPB and accepting to supervise me. His kind guidance, continuous support, patience, availability for consultation and for giving me opportunities to attend workshops and a conference are higly appreciated. Furthermore, his valuable advice, suggestions, comments, constructive criticism and encouragement led to successful completion of this study and made it meaningful to the society. Secondly, I would like to thank Prof. Dr. M. Pietzsch for accepting to be my mentor. My sincere acknowledgement goes to the DAAD/MoEVT and IPB for financial support of the study. Many thanks to Dr. Jeanette Keim, Dr. Julia Kufka, Dr. Felix Stehle and Dr. Stephanie Block for laboratory training and valuable scientific discussions on my work. Special thanks to Dr. Martin Dippe, my immediate supervisor in the laboratory, Mrs. Martina Lerbs for ESI-MS measurements, Ms Anja Ehrlich who analyzed (HPLC measurements) my samples tirelessly and Dr. Jürgen Schmidt for LC-MS measurements. Dr. Hans Felix Schreckenbach is specifically thanked for his valuable scientific discussions and ideas on my work and for his efforts to hydrolyze the esters and providing me with the hydrolysis intermediate that I tested. I would also like to thank Ms. Anne-Katrin Bauer, Mr. Benjamin Weigel and Dr. Danilo Meyer for their cooperation in the laboratory and scientific ideas on my work and Mr. Hannes iii Rost, Dr. Dimitar Vasilev and Dr. Serge Fobofou for sharing opinions and ideas in the field of chemistry. I am thankful to group leaders, technical staffs and all colleagues in the Department of Bioorganic Chemistry and Natural Products for any assistance and their contributions to my study through various discussions. I would like to express my gratitude to Mrs. Ines Stein, the secretary of the department for her help on administrative matters and Dr. Frank Broda and Mr Holger Bartz for their help on IT problems and all members of IPB for their cooperation. Thanks also to Prof. Dr. O. Kayser for accepting to review my dissertation. My sincere thanks go to all my close friends for their inspiration, advice and encouragement. I owe my late mother a great debt of gratitude and I am deeply indebted to all my family members and relatives who were close to me for their moral support, prayers and patience for the whole period that I was away from home. Thanks are also due to Mkwawa University College of Education, granting me a study leave that made for a peaceful completion of my study. Last but not least, thanks to God the Almighty, the most exalted, the eternal owner of sovereignty who, against all odds strengthened me throughout to the successful completion of my study. iv ABSTRACT 4-Hydroxybenzoate oligoprenyl transferase (UbiA-prenyltransferase) (EC 2.5.1.39) is an important enzyme in ubiquinone biosynthesis in E. coli. It is a membrane-bound enzyme consisting of 290 amino acids (32.5 kDa). It catalyzes prenylation of 4-hydroxybenzoate (4- HB) at position 3 to form 3-oligoprenyl-4-hydroxybenzoate. In this study some prenyldiphosphate mimetics prepared by Vasilev (2015) at IPB were tested in vitro for their inhibitory potential against UbiA-prenyltransferase catalysed geranylation of 4-HB. The UbiA membrane protein was prepared from E. coli C41 (DE3):pALMU3 which contains the wildtype UbiA construct. The effect of Mg2+ ion on UbiA activity was checked whereby there was no formation of GHB in absence of Mg2+ ion supporting the fact that Mg2+ ion is essential for UbiA activity. In addition, the effect of EDTA on UbiA activity was also checked. It was found that as concentration of EDTA increases, the amount of GHB formed decreases because EDTA chelates Mg2+ ion from the reaction mixture, thus, rendering the solution with little or no available Mg2+ ion. Among the tested compounds, 11, 26, 4, 28 and 36 showed inhibitory activity at 1 mM. IC50 values were determined for 26, 4, 28 and 36 and were found to be 1.51 ± 0.14 mM, 0.75 ± 0.02 mM, 0.69 ± 0.03 mM and 1.25 ± 0.03 mM, respectively. The IC50 value of 11 was not determined due to limited availability of the substance. Most of the potential inhibitors were FPP analogues. Thus, effect of FPP, also a native substrate on UbiA, versus geranylation was also checked and it showed significant competition at 1 mM; its IC50-like value was found to be 0.88 ± 0.06 mM. Results from this study show that inhibitory potential of the tested compounds depends on chain length, head group and chain linker. v ZUSAMMENFASSUNG 4-Hydroxybenzoat-Oligoprenyl-Transferase (UbiA-Prenyltransferase) (EC 2.5.1.39) ist ein wichtiges Enzym in der Ubichinon-Biosynthese von E. coli. Es ist ein membrangebundenes Enzym, das aus 290 Aminosäuren (32,5 kDa) aufgebaut ist. Es katalysiert die Prenylierung von 4-Hydroxybenzoat (4-HB) an Position 3 zu 3-Oligoprenyl-4-Hydroxybenzoat. In dieser Studie wurden Prenyldiphosphat-Mimetika, die von Vasilev (2015) am IPB hergestellt wurden, in vitro auf ihr inhibitorisches Potential gegen die UbiA-Prenyltransferase- katalysierte Geranylierung von 4-HB getestet. Das UbiA Membranprotein wurde aus E. coli C41 (DE3):pALMU3 isoliert, welcher ein Plasmid enthält, das Wildtyp UbiA kodiert. Die Wirkung von Mg2+-Ionen auf UbiA Aktivität wurde geprüft, wobei keine Bildung von GHB in Abwesenheit von Mg2+-Ionen stattfand. Dies unterstützte die Tatsache, dass Mg2+-Ionen für die UbiA Aktivität wesentlich sind. Darüber hinaus wurde die Wirkung von EDTA auf die UbiA Aktivität geprüft. Es konnte gezeigt werden, dass mit steigender Konzentration von EDTA, die Menge an gebildetem GHB abnimmt, da EDTA Mg2+-Ionen aus dem Reaktionsgemisch chelatiert, wodurch nur wenige oder keine Mg2+-Ionen in der Lösung verfügbar sind. Unter den getesteten Verbindungen zeigten 11, 26, 4, 28 und 36 inhibitorische Aktivität bei 1 mM. IC50 Werte wurden für 26, 4, 28 und 36 ermittelt und betrugen 1,51 ± 0,14 mM, 0,75 ± 0,02 mM, 0,69 ± 0,03 mM beziehungsweise 1,25 ± 0,03 mM. Der IC50 Wert von 11 wurde wegen der begrenzten Verfügbarkeit der Substanz nicht bestimmt. Die meisten der potentiellen Inhibitoren waren FPP Analoga. Daher wurde die Wirkung von FPP, ebenfalls natives Substrat von UbiA, gegen Geranylierung geprüft. FPP zeigte eine signifikante Kompetion bei 1 mM und der gemessene IC50-ähnliche Wert betrug 0,88 ± 0,06 mM. Die Ergebnisse dieser Studie zeigen, dass das inhibitorische Potenzial der getesteten Verbindungen von Kettenlänge, Kopfgruppe und Ketten-Linker abhängt. vi TABLE OF CONTENT ACKNOWLEDGEMENT ...................................................................................................... iii ABSTRACT .............................................................................................................................. v ZUSAMMENFASSUNG ........................................................................................................ vi TABLE OF CONTENT ......................................................................................................... vii LIST OF FIGURES ................................................................................................................ xi LIST OF TABLES ................................................................................................................ xiii LIST OF ABBREVIATIONS ............................................................................................... xiv 1 INTRODUCTION AND LITERATURE REVIEW .......................................................... 1 1.1 General Introduction ................................................................................................. 1 1.2 Literature Review ....................................................................................................... 5 1.2.1 Prenyltransferases .................................................................................................. 5 1.2.1.1 Isoprenyl Diphosphate Synthases ................................................................... 8 1.2.1.2 Protein Prenyltransferases ............................................................................ 11 1.2.1.3 Terpene Synthases ........................................................................................ 12 1.2.1.4 Aromatic Prenyltransferases .......................................................................
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