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The Malonate Decarboxylase Enzyme System of Malonomonas Rubra Identification, Purification and Biochemical Characterization of Components

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The Malonate Decarboxylase Enzyme System of Malonomonas Rubra Identification, Purification and Biochemical Characterization of Components Research Collection Doctoral Thesis The malonate decarboxylase enzyme system of Malonomonas rubra identification, purification and biochemical characterization of components Author(s): Hilbi, Hubert Franz Pius Publication Date: 1994 Permanent Link: https://doi.org/10.3929/ethz-a-000971872 Rights / License: In Copyright - Non-Commercial Use Permitted This page was generated automatically upon download from the ETH Zurich Research Collection. For more information please consult the Terms of use. ETH Library Diss. ETH No. 10766 The malonate decarboxylase enzyme System of Malonomonas rubra: Identification, purification and biochemical characterization of components A dissertation submitted to the SWISS FEDERAL INSTITUTE OF TECHNOLOGY ZÜRICH for the degree of DOCTOR OF NATURAL SCIENCES presented by HUBERT FRANZ PIUS HILBI Dipl. Natw. ETH bomMay30,1%5 Citizen of Zug (ZG) and Flums (SG) accepled on the recommendation of Prof. Dr. P. Dimroth, examiner Prof. Dr. T. Leisinger, coexaminer Zürich 1994 Dank Ich möchte Herrn Prof. Dr. P. Dimroth für die fachliche Betreuung dieser Doktorarbeit danken und insbesondere für die Ideen, die in kritischen Momenten verhindert haben, dass das Projekt abgestürzt ist. Meinem "Diplomvater" Herrn Prof. Dr. T. Leisinger danke ich für die wohlwollende Übernahme des Koreferats. Uschi & Claudia, die jeweils die fürchterlichen Stunden vor dem ersten Kaffee mit mir teilten, sei an dieser Stelle für ihr Verständnis gedankt, und gleichzeitig entschuldige ich mich beim Feuerlöscher LFV D19 für den unbeherrschten Tritt, der ihm fast seinen Halt gekostet hätte. Den übrigen D-Stock-Bewohnerlnnen danke ich für hebevoll ausgewählte Geburtstagsgeschenke, Apdros und Fachdiskussionen; für letztere bedanke ich mich auch auf Institutsebene. Corinne danke ich für die Tage in den Cinque Terre & das Salz auf unserer Haut, Christian für die Idee nach Indien zu fahren & ähnlich schräge Einfälle, Karin für lange Jahre, Urs für nächtliche Gespräche, Marianne für den Weg zwischen Pigalle & Züribar und Martina für gemeinsame Kultur. Meinen Eltern möchte ich herzlich für die grosszügige Unterstützung dieser Ausbildung danken. Table of Contents ZUSAMMENFASSUNG SUMMARY Chafter I: Introduction S 1.1. Physiology of anaerobic, chemotrophic bacteria 5 1.1. Bioenergetic callenges of chemotrophic anaerobes 5 1.2. Complete anaerobic oxidation of organic matter to CO2 9 1.3. The role of sodium in bacterial physiology 11 1.4. Primary sodium pumps 13 1.5. Endergonic Systems depending on ApNa+ 16 I. 2. Conservation of decarboxylation energy 17 2.1. Fermentation of malonate and other saturated dicarboxylates 17 2.2. Soluble decarboxylases and substrate/product antiport 19 2.3. Decarboxylation-linked primary sodium pumps 21 1.3. Aimsofthework 27 1.4. References 29 Chafter II: Malonate decarboxylase of MaUmomonas rubra, a novel type of biotin-containing acetyl enzyme 37 Eur. J. Biochem. 207:117-123 n. 1. Summary 38 II. 2. Introduction 38 n. 3. Materials and Methods 40 n. 4. Results 43 II. 5. Discussion 52 U. 6. References 57 Chafter UI: The malonate decarboxylase enzyme System of Malonomonas rubra: evidence for the cytoplasmic location of the biotin-containing component 59 Arch. Microbiol. 160:126-131 DI. 1. Summary 60 in. 2. Introduction 60 HI. 3. Experimental Procedures 62 HI. 4. Results 65 HI. 5. Discussion 71 DI. 6. References 73 Chafter IV: and Purification characterization of a cytoplasmic enzyme component of the Na+-activated malonate decarboxylase System of Malonomonas rubra: acetyl-S-acyl carrier protein: malonate acyl carrier protein-SH transferase 75 Arch. Microbiol. (in press) IV. 1. Summary 76 IV. 2. Introduction 76 IV. 3. Materials and Methods 79 IV. 4. Results 82 IV. 5. Discussion 93 IV. 6. References 97 Appendix I: N-terminal sequence of the transferase 100 Appendix II: Preliminary experiments on the purification of the biotin protein of malonate decarboxylase and other protein components involved in malonate decarboxylation 101 Summary 102 Introduction 103 Materials and Methods 104 Results 108 Discussion 114 References 117 Chafter V: General Discussion 119 V.l. The malonate decarboxylase enzyme System of M. rubra 119 1.1. Activation of malonate 119 1.2. Purification of a protein thiol transferase and relationship of malonate decarboxylase to citrate lyase 122 1.3. Relationship of malonate decarboxylase to the Na+- transport decarboxylases 125 V. 2. Anaplerotic needs for fermentaüve growth on malonate 127 2.1. Synthesis of Cj-compounds 127 2.2. Fatty acid synthesis 128 2.3. Redox reactions 129 V. 3. Outlook 130 V. 4. References 131 Curriculum Vitae 133 List of Publications 134 1 Zusammenfassung Malonomonas rubra, ein mikroaerotolerantes, strikt Na+-abhängiges Faulschlamm- Bakterium, wächst anaerob auf Malonat als einziger Kohlenstoff- und Energiequelle. Malonat Decarboxylase, das Schlüsselenzym dieser Fermentation, setzt dabei das Substrat quantitativ zu Acetat und C02 um. Die Energieausbeute dieser Reaktion beträgt nur -17.4 kj pro Mol, und daher muss die ATP-Synthese über chemi- osmotische Prozesse erfolgen. Die Decarboxylierung von Malonat stellt ein chemisches Problem dar, weil die C-C-Bindung für die Spaltung aktiviert werden muss. Ziel der vorliegenden Arbeit war die Aufklärung der Malonat-Aktivierung und die biochemische Charakterisierung der Malonat-Decarboxylase. Zellfreier Extrakt von M. rubra decarboxyliert Malonat mit einer beträchtlichen Aktivität von 2.7 U/mg Protein. Zugabe von ATP und Acetyl-CoA zeigt keine Wirkung in frisch präpariertem Extrakt Da Malonyl-CoA zehnmal langsamer als freies Malonat umgesetzt wird, ist letzteres offensichtlich das Substrat. Indizien für einen Radikal-Mechanismus sind keine gefunden worden. Stattdessen wird Malonat Decarboxylase durch eine Acetylierung aktiviert, ein Mechanismus, der für Citrat Lyase bekannt war. Aktives Enzym wird vollständig gehemmt durch desacetylierende Reagentien wie Hydroxylamin, Mercaptoethanol oder Thiocyanat. Diese Inhibition ist sowohl enzymatisch (mittels einer spezifischen Ligase und ATP/Acetat) als auch chemisch (mit Acetanhydrid) reversibel. Dithioerythritol erhöht die reaktivierbare Aktivität, was auf die Beteiligung eines Thiols schliessen Iässt. Im Verlauf der Katalyse wird der Enzym-gebundene Acetyl-Rest durch einen Malonyl-Rest ersetzt. Das aktivierte Substrat der Malonat Decarboxylase ist daher ein Protein-gebundener Thioester der Malonsäure, nämlich Malonyl-Thio-Acyl-Carrier-Protein (Malonyl-S- ACP). Acetyl-S-Acyl Carrier Protein: Malonat Acyl Carrier Protein-SH Transferase katalysiert die eigentliche Aktivierung von Malonat Diese Transferase setzt als nichtphysiologische Substrate auch die entsprechenden Coenzym A (CoA)-Derivate um. Mit Malonyl-CoA und Acetat als Alternativ-Substraten ist eine lösliche CoA Transferase gereinigt worden, deren Menge 4 % des Proteins im Extrakt beträgt und die an der Malonat Decarboxylierung beteiligt ist. Das monomere Enzym besitzt ein apparentes Molekulargewicht von 67'000 und ein pH-Optimum von 5.5. Die Km- Werte für die CoA Substrate sind 1.9 mM (Malonyl-CoA) und 6.9 mM (Acetyl-CoA) und damit etwa zwei Grössenordnungen grösser als diejenigen von physiologischen CoA Transferasen. Der katalytische Mechanismus läuft nicht über ein kovalentes Transferase-CoA Intermediat, das für physiologische CoA Transferasen nachgewiesen 2 ist Der Umsatz von CoA Derivativen ohne Beteiligung eines kovalenten Enzym-CoA Intermediates ist ebenfalls von der Citrate Lyase bekannt Das Malonat Decarboxylase System enthält neben cytoplasmatischen auch Membran-gebundene Komponenten. Ausserdem ist Biotin als Cofaktor beteiligt und die Umsetzung von Malonat wird im Extrakt spezifisch durch Na+ (Km = 0.8 mM) oder Li+ (Km = 3.3 mM) stimuliert Diese Eigenschaften sind typisch für Na+- Transport Decarboxylasen, die die Decarboxylierungs-Energie zum Aufbau eines elektrochemischen Na+-Gradienten nutzen. Beim Wachstum von M. rubra auf Malonat wird ein einziges Biotin Protein von ungewöhnlicher Grösse (120 kD) exprimiert Aufgrund biochemischer Analysen (Inhibition durch Avidin, Western Blots) als auch mittels Elektronen-Mikroskopie ist dieses Protein im Cytoplasma lokalisiert worden. Das Malonat Decarboxylase Enzym System von Malonomonas rubra ist bezüglich der Substrat-Aktivierung (Acetylierung, ACP-Thiol Transfer) mit der Citrat Lyase verwandt, und es gleicht den Na+-Transport Decarboxylasen bezüglich der Substrat- Decarboxylierung (Carboxyltransfer auf Biotin, Decarboxylierung des C02-Biotin) und der Energiekonservierung. 3 Summary Malonomonas rubra, a microaerotolerant, strictly Na+-dependent bacterium isolated from anoxic Sediments grows anaerobically on malonate as sole source of carbon and energy. Malonate decarboxylase, the key enzyme of this fermentation pathway, decarboxylates the Substrate quantitatively to acetate and CO2. This reaction yields only -17.4 kJ per mol and consequently, the ATP synthesis mandatorily involves chemiosmotic processes. The clecarboxylation of malonate imposes a chemical problem, since the C-C-bond has to be activated for cleavage. Aim of the work presented here was the elucidation of malonate activation and the biochemical characterization of malonate decarboxylase. Cell free extracts of M. rubra decarboxylate malonate with the considerable activity of 2.7 U/mg protein. Addition of ATP and acetyl-CoA has no effect on freshly prepared extracts. Since malonyl-CoA is decarboxylated ten times slower than free malonate, the latter is apparently the Substrate. No evidences for a radical mechanism have been found.
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