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Biosynthesis of Cobalamin (Vitamin B12) in Salmonella Typhimurium

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Biosynthesis of Cobalamin (Vitamin B12) in Salmonella Typhimurium Biosynthesis of cobalamin (vitamin B^^) in Salmonella typhimurium and Bacillus megaterium de Bary; Characterisation of the anaerobic pathway. By Evelyne Christine Raux A thesis submitted to the University of London for the degree of doctorate (PhD.) in Biochemistry. -k H « i d University College London Department of Molecular Genetics, Institute of Ophthalmology, London. Jan 1999 ProQuest Number: U121800 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest U121800 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Abstract The transformation of uroporphyrinogen HI into cobalamin (vitamin B 1 2 ) requires about 25 enzymes and can be performed by either aerobic or anaerobic pathways. The aerobic route is dependent upon molecular oxygen, and cobalt is inserted after the ring contraction process. The anaerobic route occurs in the absence of oxygen and cobalt is inserted into precorrin- 2 , several steps prior to the ring contraction. A study of the biosynthesis in both S. typhimurium and B. megaterium reveals that two genes, cbiD and cbiG, are essential components of the pathway and constitute genetic hallmarks of the anaerobic pathway. The genes responsible for the cobalt chelation, the S. typhimurium CbiK and the B. megaterium CbiX, were identified within coh opérons and were characterised. Moreover, the activity of the multifunctional iron chelatase/ dehydrogenase enzymes (£. coli CysG and S. cerevisiae MetSp) involved in sirohaem biosynthesis have been investigated for their ability to act as a cobalt chelatase in corrin biosynthesis. Cobalamin can be produced from the S. typhimurium cob operon with any of these chelatases whereas precorrin-2 dehydrogenase activity is required with the B. megaterium cob operon. The X- ray structure of CbiK has been solved at 2.4Â and is highly similar to the structure of the B. subtilis protoporphyrin IX ferrochelatase suggesting a common mechanism. Unlike the P. denitrificans cobalt chelatase complex, which requires three proteins and ATP (similar to the protoporphyrin IX magnesium chelatase), CbiK belongs to the unique protein-ATP independent chelatase family. Conserved amino acids have been characterised as key residues within the CbiK active site. Genomic comparisons of Bi 2 -producing organisms highlight divergences between the methyltransferases, which separate into aerobic and anaerobic pathway subgroups. Further insights into the methyltransferases have been gained from the X-ray structure of the B. megaterium CbiF (solved at 2.4Â resolution). Finally, the molecular structure of cobyric acid produced from the S. typhimurium cob operon in E. coli has been deduced from a number of spectrometric studies, an approach which could be used in the future to characterise other intermediates along the anaerobic cobalamin pathway. From the results obtained in this thesis, it becomes apparent that the terms "aerobic" and "anaerobic" pathways are misleading and should be replaced by "late-cobalt insertion" and "early-cobalt insertion" pathways respectively. Nature's most beautiful cof^ctor, cobalamiiL / The methyl group linked to the cobalt has been removed in this diagram and the loop has been detached from the cobalt (purple), as it occurs when the methyl-cobalamin is bound in the methionine synthase. Atoms in yellow indicate a carbon; in blue, nitrogen; in red, oxygen and the purple atom in the lower part of the figure is a phosphore [Drennan et al. (1994) Science, 266: 1669-1674]. Acknowledgements I started research on vitamin biosynthesis in October 1988, and for over ten years, this molecule has never ceased to fascinate me. Many people have been involved in this work and I would like to thank them here. In France: • I am very grateful to Dr Alain Rambach for having introduced me to the world, trusting me when I worked in Gif-sur-Yvette and to have allowed me to use his Bj2 collection strains afterwards. • I would also like to thank everyone who was working with me in Gif-sur- Yvette between 1988 and 1995. A very special thanks to Dr Claude Thermes, for having taught me the basis of molecular biology and for all those endless discussions about Bj 2 - Thanks to Anne Lanois and Yves D'aubenton for their continuous help and support during those years. In England: • I give my most sincere thanks to my supervisor. Dr Martin Warren. This thesis would never have been realised without his help, guidance and discussions. Thanks also for correcting my English with such patience... • A very warm thanks to Dr Heidi Schubert for her invaluable and efficient collaboration. • Many friendly thanks for the help and collaboration of Dr Sarah Woodcock and Dr Jenny Roper. • To everyone in the Molecular Genetics department, and especially to Natalie Senior, Sarah Awan, Richard Newbold, Sussie Dalvin and Seraphina Idowu. • A big thanks to Dr Helen Wilcock for her everyday support over the three years. • My warmest thanks to Isabelle, Daniel and Nicole Evain for their advice and support, and to my parents for always having respected my choices, whatever they were. • And finally, the last word is for you, Phil... Remerciements J'ai commencé à étudier la biosynthèse de la vitamin B 1 2 en octobre 1988, et depuis plus de dix ans, cette molécule n'a pas jamais cessé de me fasciner. Beaucoup de personnes ont participé à cet ambitieux projet, j'aimerai aujourd'hui les remercier. En France: • Je suis très reconnaissante à Monsieur Alain Rambach de m'avoir fait découvrir le monde de la B 1 2 , puis de m'avoir fait confiance lorsque je travaillais à Gif-sur-Yvette, ainsi que de m'avoir permis d'utiliser sa collection de souches productrices de B 1 2 , après ma venue à Londres. • Je remercie aussi tous ceux qui travaillaient à Gif-sur-Yvette entre 1988 et 1995. Un merci particulier à Claude Thermes de m'avoir appris les bases de la biologie moléculaire ainsi que pour toutes ces interminables discussions à propos de B 1 2 . Merci également à Anne Lanois et Yves D'aubenton pour leur complicité de chaque jour durant ces années. En Angleterre: • J'adresse mes plus sincères remerciements au Dr Martin Warren. Sans son aide, ses conseils et les multiples discussions que nous avons eues, cette thèse n'aurait jamais vu le jour. Merci aussi d'avoir corrigé mon anglais avec tant de patience... • Je remercie très chaleureusement le Dr Heidi Schubert pour son inestimable collaboration. • Un merci amical aux Dr Sarah Woodcock et Jenny Roper pour l'aide qu'elles m'ont fournie chaque jour et pour leur collaboration, ainsi qu'à tous ceux du département de génétique moléculaire, et plus particulièrement à Natalie Senior, Sarah Awan, Richard Newbold, Sussie Dalvin and Seraphina Idowu. • Un merci spécial au Dr Helen Wilcock pour son soutien de chaque jour pendant plus de 3 ans. • Mes plus chaleureux remerciements vont à Isabelle, Daniel et Nicole Evain pour leur écoute et leurs conseils, ainsi qu'à mes parents pour avoir toujours respecté mes choix, quels qu'ils soient. • Le mot de la fin est pour toi, Phil... Contents. page Title. 1 Abstract. 2 Nature's most beautiful cofactor, cobalamin. 3 Acknowledgements. 4 Remerciements. 5 Contents. 6 List of tables. 14 List of figures. 16 Abbreviations. 2 0 Amino acids and their abbreviations. 2 1 Genetic code. 2 2 Names assigned to cobalamin biosynthetic proteins. 23 Tetrapyrrole numbering system and nomenclature. 24 Meeting / conferences 25 List of publications 26 Chapter 1 : Introduction to tetrapyrroles and cobalamin in particular. 28 1-1- General introduction to Nature's tetrapyrroles. 29 1-1-1- Haem. 30 1-1-2- Chlorophyll and bacteriochlorophyll. 31 1-1-3- Phycobilins and phytochrome. 32 1-1-4- Isobacteriochlorins. 33 1-1-5- Coenzyme F 4 3 0 . 34 1-1-6- Vitamin 34 1-2- Cobalamin dependent enzymes. 35 1-2-1- Reactions catalysed by cobalamin. 37 1-2-2- Vitamin iri human life. 38 1-3- Uroporphyrinogen III biosynthesis. 39 1-4- Sirohaem biosynthesis. 42 1-4-1- In Escherichia coli and Salmonella typhimurium 43 1-4-2- In Saccharomyces cerevisiae. 44 1-5- Vitamin biosynthesis pathway in Pseudomonas deni­ trificans. 44 1-6- Vitamin B^^ biosynthesis in Salmonella typhimurium. 50 1-7- Vitamin B^^ biosynthesis in Bacillus megaterium. 51 1-8- Vitamin B^^ biosynthesis inPropionibacterium shermanii. 53 1-9- Two different pathways for cobalamin biosynthesis. 54 1-10- Purpose of this thesis. 57 Chapter 2: Identification of the genes required for vitamin Bj^ bio­ synthesis via the anaerobic pathway. 58 2-1- Introduction 59 2-2- Use of E. coli as host for the coh opérons and for generating coh phenotypes. 61 2-2-1- E, coli as a host for the S. typhimurium cob operon. 61 2 -2 -2 - Role of CysG in cobalamin synthesis. 62 2-2-3- E. coli as a host for the B. megaterium cobi operon. 64 2-2-4- Production of cobyric acid in E. coli occurs only under anaerobic conditions. 65 2-3- Role of chiD and chiG in cobalamin synthesis. 65 2-3-1- Introduction of separate mutations in cbiD and in cbiG of the cob opérons. 6 6 a- In the S. typhimurium cob operon. 6 6 b- In the B. megaterium cob operon. 6 6 2-3-2- cbiD and cbiG are essential genes for the synthesis of cobalamin.
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