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Durham E-Theses Durham E-Theses Fluorometabolite biosynthesis in streptomyces cattleya Moss, Steven J. How to cite: Moss, Steven J. (1999) Fluorometabolite biosynthesis in streptomyces cattleya, Durham theses, Durham University. Available at Durham E-Theses Online: http://etheses.dur.ac.uk/4603/ Use policy The full-text may be used and/or reproduced, and given to third parties in any format or medium, without prior permission or charge, for personal research or study, educational, or not-for-prot purposes provided that: • a full bibliographic reference is made to the original source • a link is made to the metadata record in Durham E-Theses • the full-text is not changed in any way The full-text must not be sold in any format or medium without the formal permission of the copyright holders. Please consult the full Durham E-Theses policy for further details. Academic Support Oce, Durham University, University Oce, Old Elvet, Durham DH1 3HP e-mail: [email protected] Tel: +44 0191 334 6107 http://etheses.dur.ac.uk Fluorometabolite Biosynthesis in Streptomyces cattleya Steven J Moss Nature has evolved the ability to form a C-F bond, as exemplified by the bacterium Streptomyces cattleya, which elaborates fluoroacetate (FAc) and 4-fluorothreonine (4- FT). The mechanism of this bond formation are unknown. This thesis probes the biosynthesis of fluoroacetate and 4-fluorothreonine and in doing so explores the C-F bond forming process. Feeding stable isotope enriched primary metabolites to S. cattleya, followed by 19F NMR and GCMS analysis of the resultant fluorometabolites, highlights the role of the glycolytic pathway in delivering a substrate for fluorination. 3-Fluoro-l- hydroxypropan-2-one was synthesised and feeding studies eliminate this as the initial product of fluorination. Fluoroacetaldehyde was identified as a common fluorinated intermediate in the biosynthesis of both FAc and 4-FT. Whole cell studies demonstrate the rapid oxidation of fluoroacetaldehyde to FAc. 4-FT is produced in low quantities by S. cattleya incubated with fluoroacetaldehyde. The synthesis and feeding of [1- H]- fluoroacetaldehyde provide evidence that the resultant 4-FT is biosynthesised from fluoroacetaldehyde. The biotransformation from fluoroacetaldehyde to FAc was shown in cell free studies to be mediated by an aldehyde dehydrogenase, requiring NAD+ as a co-factor. The substrate specificity of fluoroacetaldehyde dehydrogenase was probed by spectrophotometrically monitoring the production of NADH in the presence of different aldehydes. Further cell free experiments probed the biosynthetic origins of fluoroacetaldehyde. Glycolaldehyde phosphate and various phosphorylated glycolytic intermediates were incubated with cell free extracts of S. cattleya and a plethora of co-factors. In the absence of observing fluorination activity, it was shown that the cell free extract acts to dephosphorylate the substrates. The putative role of glycolaldehyde phosphate was explored by feeding isotopically labelled glycolaldehydes to whole cells of the bacterium. The results were not consistent with direct conversion from glycolaldehyde phosphate to fluoroacetaldehyde. Steven James Moss PhD Thesis 1999 University of Durham Department of Chemistry The copyright of tliis thesis rests with (lie author. No quotation from it should be published without the written consent of the author and information derived from it should be acknowledged. I T JAN 2000 Declaration The work contained in this thesis was carried out by the author in the Department of Chemistry, University of Durham between October 1996 and July 1999. The work was conducted in collaboration with the Queen's University of Belfast. GC-MS analyses, on samples submitted by the author, were performed by Dr Jack Hamilton at the Queen's University of Belfast. Copyright The copyright of this thesis rests with the author. No quotation from it .should be published without their prior written consent and information derived from it should be acknowledged iii Acknowledgements I wish to thank my supervisor, Professor David O'Hagan, not only for the continued support and enthusiasm that he has dedicated to the project, but also for the opportunities that he has given me. Professor David Harper and Dr Cormac Murphy, at the Queen's University of Belfast, provided both technical assistance in developing the resting cell methodology in Durham and hospitality during my stay in Belfast. Sincere thanks go to Dr Jack Hamilton for the GCMS analysis that makes this project possible and for his candid discussions of the results. I acknowledge British Nuclear Fuels Ltd and the BBSRC who provided CASE funding for this work and I thank Dr Peter Binks, Dr Roy Bowden, Mr Martin Green and Dr Harry Eccles, all from BNFL, for the interest that they have shown in the project. High field I9F NMR analysis was performed by Mr Ian McKeag and I thank him and the technical support staff in the Department of Chemistry. The O'Hagan group, past and present, made the lab a vibrant and interesting place to work. I specifically would like to acknowledge Dr Jens Nieschalk and Dr Jens Fuchser, for the help and advice they imparted during this project. Finally, I would like to thank my parents for encouraging me throughout my PhD studies and education before that. iv List of Contents Table of Contents v List of Figures x List of Tables xvi List of Abbreviations xix Chapter 1: Introduction 1.1 Secondary metabolism 1 1.1.1 Primary and secondary metabolism 1 1.1.2 Fatty acid biosynthesis and P-oxidation 4 1.1.3 Microbial secondary metabolism 5 1.2 Elucidation of metabolic pathways 7 1.2.1 Biological methods 7 1.2.1.1 Mutant studies 7 1.2.1.2 Inhibition of protein biosynthesis 8 1.2.1.3 Cell free studies 8 g 1.2.2 Chemical methods 1.2.2.1 Radioisotopes 9 1.2.2.2 Stable isotopes 10 1.2.2.3 Single label feeding experiment studies 10 1.2.2.4 Double label feeding studies: 'bond labelling' 10 1.3 Halogenated secondary metabolites 12 1.3.1 Distribution of halogenated metabolites 12 1.3.2 Mechanisms for biohalogenation 13 1.4 Fluorinated secondary metabolites 15 1.4.1 Fluorine bioavailability 15 1.4.2 The Nature oftheC-F bond 15 1.4.3 Fluorinated natural products in plants 17 1.4.3.1 Fluoroacetate 17 1.4.3.2 oo-Fluorinated fatty acids 18 1.4.3.3 Fluoroacetone 19 1.4.4 Fluoroacetate metabolism: (2R, 3i?)-2-fluorocitrate 20 1.4.5 Resistance to fluoroacetate poisoning 23 1.4.6 Fluorometabolites from microorganisms 25 1.4.6.1 Nucleocidin 25 v 1.4.6.2 4-Fluorothreonine and fluoroacetate 26 1.5 Biological fluorination - proposed mechanisms 27 1.5.1 Addition to a carbon-carbon multiple bond 27 1.5.1.1 Pyridoxal phosphate assisted addition of fluoride 27 1.5.1.2 Ethylene and ethylene oxide as putative precursors 30 1.5.1.3 Nucleophilic attack on a halonium ion 30 1.5.2 Fluorination via nucleophilic substitution 32 1.6 Streptomyces cattleya and the fluorometabolites 33 1.6.1 The Streptomycetes 33 1.6.2 Streptomyces cattleya 33 1.6.3 Fluoroacetate and 4-fluorothreonine 35 1.6.4 The biosynthetic relationship between fluoroacetate and 38 4-fluorothreonine 1.6.5 Incorporation of stable isotope enriched glycolate, glycine 39 and pyruvate 1.6.6 The stereochemical processing of glycerol 41 1.6.7 Overview 43 Chapter 2: Fluorometabolite biosynthesis and primary metabolism 2.1 Maintenance and growth of Streptomyces cattleya 45 2.1.1 Preparation of a starter culture of Streptomyces cattleya 46 2.1.2 Preparation of batch cultures of Streptomyces cattleya 46 2.1.3 Time course study on fluorometabolite biosynthesis 47 2.1.3.1 Results: pH analysis 47 2.1.3.2 Results: fluoride analysis 48 2.1.3.3 Discussion 49 2.2 Studying fluorometabolite biosynthesis 50 2.2.1 Preparation of resting cells of Streptomyces cattleya 50 2.2.1.1 Resting cell experiments: controls 51 2.2.2 Studying fluoroacetate and 4-fluorothreonine with l9F NMR 51 2 2.2.2.1 Feeding H20 54 2.2.2.2 Incorporation of [2-l3C]-glycine into the fluorometabolites: 56 19F NMR analysis 2.2.3 GCMS analysis of fluoroacetate and 4-fluorothreonine 57 2.2.3.1 GCMS analysis of fluoroacetate 57 2.2.3.2 GCMS analysis of 4-fluorothreonine 58 2.3 Resting cell incorporation studies 60 2 2.3.1 [6,6- H2]-Glucose 60 vi 2.3.1.1 Background 60 2.3.1.2 Results and discussion 62 2.3.2 Isotopically labelled acetates 66 2.3.2.1 Background 66 2.3.2.2 Results and discussion 66 2.3.3 [2-2H]-Glycerol and [2-2H]-glycerate 71 2.3.3.1 Background 71 2.3.3.2 Results and discussion 72 2.4 Synthesis and feeding of 3-fluoro-l-hydroxypropanone 75 2.4.1 Background 75 2.4.2 3-Fluoro-l-hydroxypropanone 77 2.4.3 Synthesis of 3-fluoro-l-hydroxypropanone 78 2.4.4 Biotransformation of 3-fluoro-l-hydroxypropanone 81 2.5 Conclusions 87 Chapter 3: A role for fluoroacetaldehyde 3.1 Fluoroacetaldehyde 89 3.1.1 Synthesis of fluoroacetaldehyde 89 3.2 Whole cell studies on fluoroacetaldehyde 92 3.2.1 Preliminary studies 92 3.2.1.1 Introduction 92 3.2.1.2 Results and discussion 92 3.2.2 Oxygen requirements of fluoroacetaldehyde metabolism 95 3.2.2.1 Fluoroacetaldehyde metabolism under nitrogen 95 3.2.2.2 Fluoroacetaldehyde metabolism under 1802 96 3.2.2.3 Conclusions 97 3.3 Fluoroacetaldehyde in fluorometabolite biosynthesis 98 3.3.1 Fluoroacetaldehyde metabolism by other Streptomyces spp. 98 3.3.1.1 Growth of Streptomyces cinnamonensis 9 8 3.3.1.2 Feeding fluoroacetaldehyde to resting cells of Streptomyces 99 cinnamonensis 3.3.2 Fluoroacetaldehyde: incubation with alanine and serine 100 3.3.2.1 Experimental considerations 100 3.3.2.2 Results and discussion 101 3.3.3 The synthesis and feeding
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