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(Thdp)-Dependent Enzymes for Carboligation

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Dörte Gocke New and optimised thiamine diphosphate (ThDP)-dependent enzymes for carboligation Creation of a toolbox for chiral 2-hydroxy ketones Thesis for doctoral degree (Dr. rer. nat.) New and optimised thiamine diphosphate (ThDP)- dependent enzymes for carboligation - Creation of a toolbox for chiral 2-hydroxy ketones Inaugural-Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Heinrich-Heine-Universität Düsseldorf vorgelegt von Dörte Gocke aus Dorsten Köln, November 2007 Aus dem Institut für Molekulare Enzymtechnologie der Heinrich-Heine-Universität Düsseldorf Gedruckt mit der Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Heinrich-Heine-Universität Düsseldorf Referent: PD Dr. Martina Pohl Korreferent: Prof. Dr. Dieter Willbold Tag der mündlichen Prüfung: 14. April 2008 "Das Schönste, was wir erleben können, ist das Geheimnisvolle. Es ist das Grundgefühl, das an der Wiege von wahrer Kunst und Wissenschaft steht. Wer es nicht kennt und sich nicht mehr wundern, nicht mehr staunen kann, der ist sozusagen tot und sein Auge erloschen." Albert Einstein, aus ‚Mein Weltbild’ Für Jörn, David und Valentin Content I ABSTRACT Various thiamine diphosphate (ThDP)dependent enzymes catalyse the carboligation of aldehydes to chiral 2hydroxy ketones with high stereoselectivity. Since 2hydroxy ketones are versatile building blocks for organic syntheses, the aim of the thesis was the creation of a ThDPdependent enzyme toolbox in order to access a diversely substituted and enantio complementary 2hydroxy ketone platform. Several goals could be achieved: 1. Development of a quick and reliable high-throughput assay to screen large toolboxes of purified enzymes or whole cell catalysts for the product formation of diversely substituted 2hydroxy ketones. 2. Enlargement of the enzyme toolbox by six wild type enzymes and four variants showing carboligase activity. The platform now encompasses 15 wild type 2keto acid decarboxylases and two benzaldehyde lyases as well as 70. For almost all newly added enzymes a biochemical characterisation of the substrate spectrum for the decarboxylase and carboligase activity including the determination of optimal reaction conditions was conducted. 3. With the new catalysts and the highthroughput assay an effective enlargement of the 2-hydroxy ketone platform was achieved. Branchedchain aliphatic 2hydroxy ketones, substituted (R)phenylacetylcarbinolanalogue products as well as several (S) 2hydroxy ketones are now for the first time biocatalytically available. By combining decarboxylase activity and carboligase activity the ligation of instable aldehydes is also possible. (S)selective CC bond formation was possible due to an understanding of the underlying structure-function relationship of the active site. By gaining two further wild type crystal structures the pool of structure information was broadened. Superimposition of all known structures combined with biochemical information and modelling studies enabled the mapping of catalytically important regions in the active site. It was concluded that the substrate ranges of both, the decarboxylase and the ligase activity of ThDPdependent enzymes as well as the chemo and enantioselectivity of the carboligase reaction is a result of optimal stabilisation of the substrates in the active site. This knowledge paves the way for product prediction by modelling studies and therewith the design of new catalysts with desired ligase activity. Content II KURZFASSUNG Die Carboligation von Aldehyden zu chiralen 2Hydroxyketonen wird von einer Vielzahl Thiamindiphosphat (ThDP)abhängiger Enzyme mit hoher Stereoselektivität katalysiert. Da diese chiralen Verbindungen als Ausgangsstoffe zahlreicher organischer Synthesen dienen, war der Aufbau einer Plattform vielfältig substituierter und enantiokomplementärer 2Hydroxyketone durch Bereitstellung eines Baukastens ThDPabhängiger Enzyme Ziel dieser Doktorarbeit. Folgende Ergebnisse führten zur erfolgreichen Umsetzung der Aufgabenstellung: 1. Entwicklung eines schnellen, reproduzierbaren Hochdurchsatz-Tests zur Untersuchung der Produktbildung in Ganzzelltransformationen sowie einer Vielzahl gereinigter Enzyme. 2. Vergrößerung des Enzymbaukastens durch Hinzufügen von sechs Wildtypenzymen und vier Varianten mit Carboligaseaktivität. Die biochemische Charakterisierung des Substratspektrums der Decarboxylase sowie Carboligaseaktivität inklusive Bestim mung optimaler Reaktionsbedingungen wurde für fast alle neuen Enzyme durchgeführt. 3. Durch die neugewonnenen Biokatalysatoren und den Einsatz des HochdurchsatzTests konnte die Plattform an diversen 2-Hydroxyketonen wesentlich erweitert werden. Verzweigtkettige aliphatische 2Hydroxyketone, substituierte (R)Phenylacetyl carbinolanaloge Produkte sowie verschiedene (S)2Hydroxyketone sind nun erstmalig enzymatisch zugänglich. Durch die Kombination der Decarboxylase und Ligaseaktivität ist eine Ligation instabiler Aldehyde mit ThDPabhängigen Decarboxylasen möglich. Die (S)selektive Carboligation wurde erst durch die Aufklärung der Struktur- Funktionsbeziehungen im aktiven Zentrum ermöglicht. Zusätzlich zu den bereits bekannten Strukturen konnten zwei weitere WildtypKristallstrukturen gelöst werden. Durch Übereinanderlagerungen aller Strukturen, Modellierungen und Berücksichtigung entstehender Produkte konnten katalytisch wichtige Regionen im aktiven Zentrum kartiert werden. Das Substratspektrum der Decarboxylase als auch Carboligaseaktivität der Enzyme sowie ihre Chemo und Enantioselektivität ist ein Ergebnis optimaler Substrat stabilisierung im aktiven Zentrum. Diese Erkenntnis ermöglicht den Zugang zur Vorhersage gebildeter Produkte durch Modellierungen und somit zur Entwicklung von Katalysatoren mit gewünschter Ligaseaktivität. Content III LIST OF PUBLICATIONS 1. Gocke, D., Wendorff, M. & Pohl, M. A colorimetric assay for the detection of 2 hydroxy ketones. submitted to Analytical Biochemistry 2. Domínguez de María, P., Pohl, M., Gocke, D., Gröger, H., Trauthwein, H., Walter, L. & Müller, M. (2007). Asymmetric synthesis of aliphatic 2hydroxy ketones by enzymatic carboligation of aldehydes. European Journal of Organic Chemistry, 29402944 3. Spaepen, S., Versées, W., Gocke, D., Pohl, M., Steyaert, J. & Vanderleyden, J. (2007). Characterization of phenylpyruvate decarboxylase, involved in auxin production of Azospirillum brasilense. Journal of Bacteriology, 189 (21), 7626 7633 4. Gocke, D., Berthold, C. L., Graf, T., Brosi, H., FrindiWosch, I., Knoll, M., Stillger, T., Walter, L., Müller, M., Pleiss, J., Schneider, G. & Pohl, M. Pyruvate decarboxylase from Acetobacter pasteurianus: biochemical and structural characterisation. submitted to Biochimica et Biophysica Acta – Proteins and Proteomics 5. Gocke, D., Nguyen, C. L., Pohl, M., Stillger, T., Walter, L. & Müller, M. (2007). Branchedchain keto acid decarboxylase from Lactococcus lactis (KdcA), a valuable thiamine diphosphatedependent enzyme for asymmetric CC bond formation. Advanced Synthesis & Catalysis, 349, 14251435 6. Berthold, C. L., Gocke, D., Wood, M. D., Leeper, F. J., Pohl, M. & Schneider, G. (2007). Crystal structure of the branchedchain keto acid decarboxylase (KdcA) from Lactococcus lactis provides insights into the structural basis for the chemo and enantioselective carboligation reaction. Acta Crystallographica, Section D: Biological Crystallography, 63, 12171224 7. Gocke, D., Walter, L., Gauchenova, E., Kolter, G., Knoll, M., Berthold, C. L., Schneider, G., Pleiss, J., Müller, M. & Pohl, M. (2008). Rational protein design of ThDPdependent enzymes: engineering stereoselectivity. ChemBioChem, 9, 406 412 Content IV LIST OF POSTER PRESENTATIONS 1. Gocke, D., Stillger, T., Knoll, M., Berthold, C., Schneider, G., Pleiss, J., Müller, M., Pohl, M. (2007). New (S)2hydroxy ketones by engineering the active site of benzoylformate decarboxylase. 8th International Symposium on Biocatalysis and Biotransformations, Oviedo (Spain) 2. Gocke, D., Nguyen, C. L., Stillger, T., Müller, M., Pohl, M. (2007). A new enzyme for asymmetric carboligation. 8th International Symposium on Biocatalysis and Biotransformations, Oviedo (Spain) 3. Kolter, G., Gocke, D., Knoll, M., Pleiss, J., Müller, M., Pohl, M. (2007). A new variant of the ThDPdependent enzyme benzoylformate decarboxylase. 8th International Symposium on Biocatalysis and Biotransformations, Oviedo (Spain) 4. Gocke, D., Stillger, T., Müller, M., Pohl, M. (2006). Carboligation of branched chain aliphatic aldehydes with thiamine diphosphate (ThDP)dependent enzymes. 3rd International Congress on Biocatalysis, HamburgHarburg 5. Gocke, D., Graf, T., Wendorff, M., Eggert, T., Müller, M., Pohl, M. (2005). Isolation, cloning and characterisation of the novel thiamine diphosphatedependent pyruvate decarboxylase from Acetobacter pasteurianus. 9th International SFB- Symposium, Aachen 6. Wendorff, M., Dresen, C., Gocke, D., Henning, H., Jaeger, K.E., Kühl, S., Lütz, S., Müller, M., Pohl, M., Eggert, T. (2005). Benzoylformate decarboxylase from Pseudomonas putida: improvement of substrate specificity by directed evolution. 9th International SFB-Symposium, Aachen 7. Dresen, C., Cosp, A., Walter, L., Gocke, D., Wendorff, M., Pohl, M., Müller, M. (2005). ,Unsaturated aldehydes as substrates for asymmetric CC bond forming reactions with thiamine diphosphate (ThDP)dependent biocatalysts. 9th Inter- national SFB-Symposium, Aachen Content V ACKNOWLEDGEMENT I would like to express my sincere gratitude to my supervisor PD Dr. Martina Pohl for her continuous
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  • THE YEAST MEDIATED SYNTHESIS of the L-EPHEDRINE PRECURSOR, L-PHENYLACETYLCARBINOL, in an ORGANIC SOL VENT

    THE YEAST MEDIATED SYNTHESIS of the L-EPHEDRINE PRECURSOR, L-PHENYLACETYLCARBINOL, in an ORGANIC SOL VENT

    THE YEAST MEDIATED SYNTHESIS OF THE l-EPHEDRINE PRECURSOR, l-PHENYLACETYLCARBINOL, IN AN ORGANIC SOL VENT A Thesis submitted for the Degree of DOCTOR OF PPHLOSOPHY By MARGARET MARY KOSTRABY School of Life Sciences and Technology Victoria University of Technology, Footscray Park Campus, Ballarat Road, Footscray, Victoria, 3011 AUSTRALIA ^25'0^'|5g FTS THESIS 615.19 KOS 30001005559408 Kostraby, Margaret Mary The yeast mediated synthesis of the 1-ephedrine precursor, STATEMENT BY THE CAIVDIDATE This is to certify that the research contained in this thesis is the sole work of the candidate. The work has not been submitted for any other academic award. M. M. KOSTRABY 11 ACKNOWLEDGEMENTS I wish to thank my supervisors; Dr. Andrew Smallridge for his advice and guidance with the project and Assoc. Prof. Maurie Trewhella for his assistance and advice. Thanks are extended to my fellow students; Nick Athanasiou, Caroline Medson and Rad Bak. I wish to thank Polychip Pharmaceuticals Fty. Ltd., a wholly owned controlled entity of Circadian Technologies Ltd., who funded the project and Mauri Integrated Ingredients, Footscray, who supplied the yeast used in this study. Thanks are also extended to Victoria University of Technology for the Scholarship which supported me through these studies. Ill SPECIAL MENTIONS To Mum and Dad, I am most grateful for all your assistance especially with looking after the children in the early days of my PhD studies. To my children, Tristan and Kieran, a pair who are absolutely wonderful, kept their mum going through difficult times and were supportive and well behaved when they had to spend many a day in at the University with me whilst I studied.