Investigating the biosynthesis of heam d1 in pseudomonas aeruginosa: a cofactor for dissimilatory nitrite reductase. Parmar-Bhundia, Vina The copyright of this thesis rests with the author and no quotation from it or information derived from it may be published without the prior written consent of the author For additional information about this publication click this link. https://qmro.qmul.ac.uk/jspui/handle/123456789/509 Information about this research object was correct at the time of download; we occasionally make corrections to records, please therefore check the published record when citing. For more information contact [email protected] INVESTIGATING THE BIOSYNTHESIS OF HAEM d1 IN Pseudomonas aeruginosa; A COFACTOR FOR DISSIMILATORY NITRITE REDUCTASE Vina Parmar-Bhundia B.Sc (Hons), MRes. A thesis submitted to the UNIVERSITY OF LONDON for the degree of DOCTOR OF PHILOSOPHY Vina Parmar Abstract: page i ABSTRACT Haem d1 is a modified tetrapyrrole unique to the periplasmic enzyme nitrite reductase - where it acts in catalysing the reduction of nitrite (NO2 ) to nitric oxide (NO), as part of denitrification. As with all modified tetrapyrroles, haem d1 shares a common biosynthetic pathway starting from 5-aminolaevulinic acid (ALA), up to the formation of uroporphyrinogen III (UIII). UIII is the branch point from which the pathway diverges to form the various metallo-prosthetic groups including vitamin B12. The precise mechanism of transformation from UIII to haem d1 is unknown. Examination of both structures shows a requirement of methylation at C2 and C7; decarboxylation of acetate side chains at C12 and C18; loss of propionic side chains at C3 and C8 with subsequent oxidation at C3 and C8; dehydrogenation of C17 propionate side chain gives the acrylate substituent and ferrochelation. Of particular interest is the addition of oxygen to the macrocycle under anaerobic conditions. Only one other intermediate, compound 800, has been isolated thus far but it is unknown how it is part of the pathway. Genetic studies have implicated seven nir genes, called nirF, nirD, nirL, nirG, nirH, nirJ and nirE, are required for haem d1 biogenesis. Here, experiments and data show for the first time that it proceeds from UIII to precorrin-2 using the enzyme NirE. This study is the first to experimentally show the production of precorrin-2 as part of the pathway using anaerobic enzyme assays. This thesis illustrates the intense work that has focused on cloning the genes individually and as multigene constructs in an attempt to characterise the proteins overproduced. Heterologous expression in Escherichia coli has been successful as well as the development of a homologous expression system in Pseudomonas aeruginosa. The data represented shows the various aspects entailed in the optimisation of overproduction and the stabilisation of the Nir proteins. It also documents the first concerted attempt to take the operon and engineer strains to make haem d1 both in vivo and in vitro, using the Link and Lock method to clone the nir genes consecutively into a plasmid. This thesis therefore provides a foundation for understanding the molecular biology and biochemistry of haem d1 synthesis for the future. Vina Parmar Contents: page ii CONTENTS Abstract i Contents ii List of Figures viii List of Tables xii Abbreviations xiii Acknowledgments xvi CHAPTER 1: AN INTRODUCTION TO TETRAPYRROLES, THEIR SYNTHESIS IN PSEUDOMONADS AND THE ENIGMA THAT IS HAEM d1. 1. Introduction 2 1.1. Modified tetrapyrroles 2 1.1.1. Chlorophyll and Bacteriochlorophyll 5 1.1.2. Haem 7 1.1.3. Sirohaem 9 1.1.4. Coenzyme F430 12 1.1.5. Vitamin B12 13 1.2. Haem d1 15 1.2.1. Haem d1 in cytochrome cd1 17 1.2.2. Denitrification 20 1.2.3. The denitrification enzyme system 21 1.3. The biosynthesis of Haem d1 23 1.3.1. Synthesis of 5-aminolaevulinic acid (ALA) 23 1.3.2. Synthesis of the first macrocyclic intermediate, uroporphyrinogen III (UIII) from 5-aminolaevulinic acid (ALA) 25 1.3.3. The methylation of UIII and the formation of PC-2 27 1.3.3.1. CobA in the vitamin B12 pathway 28 1.3.3.2. CysGA in E. coli and S. enterica in the sirohaem pathway 29 1.3.3.3. SirA in Bacilli in the sirohaem pathway 31 1.3.3.4. Met1p in Saccharomyces cerevisiae in the sirohaem Vina Parmar Contents: page iii pathway 31 1.3.3.5. CorA in Methanobacterium ivanovii 31 1.3.3.6. UPM1 from A. thaliana and ZmSUMT1 from maize 32 1.3.4. The biosynthesis of haem d1 33 1.3.4.1. NirE as a SUMT 35 1.3.4.2. The enigma of haem d1 biosynthesis 37 1.4. Ps. aeruginosa as a candidate species 39 1.5. Purpose of this thesis 40 2. CHAPTER 2: MATERIALS AND METHODS. 2.1. Materials 42 2.1.1. Chemicals (source) 42 2.1.2. Bacterial Strains 42 2.1.3. Plasmids 42 2.1.4. Media and Solutions for bacterial work 54 2.1.5. Solutions for DNA work 57 2.1.6. Solutions for Protein work 58 2.1.6.1. Solutions for metal chelate chromatography 58 2.1.6.2. Solutions for SDS-PAGE and Native gels 60 2.1.6.3. Solutions for Western Blotting 62 2.1.6.4. Solutions for Gel Filtration Chromotography (FPLC) 64 2.2. Molecular Biology Methods 65 2.2.1. Isolation of genomic DNA from Ps. aeruginosa 65 2.2.2. Isolation of plasmid DNA 65 2.2.3. Restriction of DNA 66 2.2.4. Electrophoresis of DNA 66 2.2.4.1. Agarose gel 66 2.2.4.2. Visualisation and UV 66 2.2.4.3. Hyperladder (Bioline) 67 2.2.5. Isolation and purification of a DNA fragment from an Agarose gel 67 2.2.6. Ligation of DNA fragments 67 2.2.7. Competent cells and Transformation 68 Vina Parmar Contents: page iv 2.2.7.1. Preparation of E. coli Competent Cells 68 2.2.7.2. Transformation of E. coli Competent Cells 68 2.2.7.3. Preparation of Ps. aeruginosa Competent cells 68 2.2.7.4. Transformation of Ps. aeruginosa Competent cells 69 2.2.7.5. Preparation of S. enterica AR3612 Competent cells 69 2.2.7.6. Transformation of S. enterica AR3612 Competent cells 70 2.2.7.7. S. enterica cysG complementation 70 2.2.7.8. Glycerol stocks of strains 70 2.2.8. Polymerase Chain Reaction 71 2.2.8.1. PCR of nir genes 71 2.2.8.1.1. NdeI and BamHI Primers 71 2.2.8.1.2. NdeI or NheI, RBS and XbaI and BamHI Primers 72 2.2.8.2. Primers for PCR 73 2.2.9. T-Vector cloning of PCR products 74 2.2.10. Subcloning into pET14b: NdeI – BamHI for Protein Expression in pET14b 75 2.2.11. Subcloning into pUCP Nde/Nco for expression in Ps. aeruginosa76 2.2.12. Link and Lock construction of operon 78 2.2.13. Restriction analysis of nir genes in haem d1 operon 81 2.3. Biochemical Methods 82 2.3.1. Protein assay (Bradford Assay) 82 2.3.2. A280 protein concentration estimation 82 2.3.3. Standard cloning, growth and purification of histagged proteins 83 2.3.3.1. Protein overproduction in E.coli 83 2.3.3.2. Protein overproduction in Ps. aeruginosa 83 2.3.3.3. Sonication of bacteria 84 2.3.3.4. Purification by Metal chelate chromatography 84 2.3.3.5. Histag cleavage 85 2.3.3.6. Buffer Exchange / Desalting 85 2.3.3.7. Gel filtration / Fast protein liquid chromatography 85 2.3.4. Poly-Acrylamide Gel Electrophoresis (PAGE) of proteins 86 2.3.4.1. SDS-PAGE of proteins 86 2.3.4.2. Native-PAGE of proteins 86 Vina Parmar Contents: page v 2.3.5. Western blotting of histagged proteins 87 2.3.5.1. SDS-PAGE for Western blotting of proteins 87 2.3.5.2. Western blotting 87 2.3.5.3. Probing for Histagged proteins 88 2.4. Isolation and production of tetrapyrroles 89 2.4.1. UltraViolet-Visible Spectroscopy 89 2.4.2. Production of precorrin-2 and sirohydrochlorin in situ from cell extract 89 2.4.3. Production of Precorrin-2 and sirohydrochlorin from purified protein 90 2.4.4. Production of haem d1 intermediates 91 2.4.4.1. In vivo accumulation of intermediates 91 2.4.4.2. In vitro accumulation of intermediates 91 2.4.5. In vitro Sirohydrochlorin coupled SUMT assay 92 2.4.6. Reverse Phase chromatography Separation of Pigments 93 2.4.7. Ion exchange chromatography separation of pigments – DEAE Sephacyl 93 2.5. Crystallography - Hanging drop method 94 3. CHAPTER 3: CHARACTERISATION OF NirE. 3.1. Introduction 96 RESULTS AND DISCUSSION 102 3.2. Sequence identity of NirE to various SUMTs 102 3.3. The overproduction of NirE 107 3.3.1. Cloning and expression of nirE in E.coli 107 3.3.2. Cloning and expression of nirE in Ps. aeruginosa 107 3.3.3. Purification of recombinant NirE from E. coli and Ps. aeruginosa 108 3.3.4. Analysis of recombinant NirE from E. coli and Ps. aeruginosa 3.3.5. The accumulation of porphyrinoid material 108 Vina Parmar Contents: page vi 3.4. Complementation of Salmonella enterica cysG metH mutant strains by Ps. aeruginosa NirE 115 3.5. Biochemical complementation of NirE 118 3.5.1. Production of PC-2 and SHC in situ using NirE 118 3.5.2. SHC coupled NirE assay 123 3.6. Conclusion 129 4. CHAPTER 4: OPTIMISATION OF PRODUCTION AND STABILISATION OF PROTEINS INVOLVED IN HAEM d1 SYNTHESIS.