林俊宏臺灣大學：生化科學研究所許富翔Hsu, Fu-HsianFu-HsianHsu2007-11-262018-07-062007-11-262018-07-062006http://ntur.lib.ntu.edu.tw//handle/246246/52771UDP-MurNAc-pentapeptide和Lipid I是peptidoglycan生合成的重要的中間物, UDP-MurNAc-pentapeptide的生合成主要是發生在細菌的細胞質內,分別由六個酵素參予反應。 UDP-MurNAc-pentapeptide是酵素MraY的受質,它的功能是將muramyl -pentapeptide接到位在細胞膜上55個碳的undecaprenyl phosphate上面; 也就是所謂的Lipid I。之後MurG 再將一個GlcNAc接到 muramyl基團的羥基上面形成所謂的Lipid II。Lipid II之後再翻轉至細胞膜外側被另外兩個酵素行聚合以及交叉結合的動作,形成peptidoglycan。 為了發展新一代的抗生素,我們建立了一套有效率的方式去得到UMP以及Lipid I。UMP純化來自於一株Gram-negative的菌,稱為Bacillus cereus T, 我們利用過去已知的步驟再經過部分的修飾之後來純化UMP. 並且在UMP上面加上了了一個螢光基團,如FITC和dansyl。另外利用酵素和化學的方式去研究將UDP-MurNAc-pentapeptide和脂肪鍊接在一起,合成Lipid和它的類似物。UDP-MurNAc-pentapeptide (UMP) and Lipid I are the key intermediates for peptidoglycan biosynthesis. The biosynthesis of UDP-MurNAc-pentapeptide takes place in the cytoplasm, where six enzymes are involved. An undecaprenyl chain is then attached to the muramyl pentapeptide to give Lipid I. Additional glycosylation of GlcNAc to the C4-OH of muramyl group produces Lipid II that is subjected to further sugar chain polymerization and peptide chain cross-linked to form peptidoglycans. In order for development of new antibiotics, we aim to establish efficient preparation of UMP and Lipid I. UMP was purified from Bacillus cereus T on the basis of a modified procedure and labeled with a fluorescence group (such a FITC and dansyl group). Chemical and enzymatic methods were then studied to incorporate a long lipid chain for preparation of Lipid I and its analogues.Aknowlegement………………………………………………………….I Table of contents……………………………………………………………II Figure……………………………………………………………………V List of abbreviation…………………………………………………………VI Abstract………………………………………………………………VII Chapter 1. Introduction 1. The emergence of antibiotics resistance 1 1.1 How do antibiotics work? 1 1.2 Drug resistance in bacteria 2 2. The bacterial cell wall as a target4 2.1 The three stages of peptidoglycan biosynthesis 4 2.2 Antibiotics that act on peptidoglycan biosynthesis 6 3. The membrane associated stage of peptidoglycan 8 3.1 MraY 9 3.1.1 Introduction of MraY 9 3.1.2 MraY substrate specificity 10 3.2 MurG 11 3.2.1 Introduction of MurG 11 3.2.2 Structure of E. coli MurG 11 3.2.3 MurG Substrate specificity 13 4. Synthesis Lipid I and Lipid II 13 4.1 Synthesis of Lipid I 14 4.2 Synthesis of Lipid II 15 4.3 Lipid-linked glycosyl pyrophosphate coupling 15 5. specific aims 17 Chapter 2. Materials and Methods 18 Materials and Instruments 18 Methods 19 1. Purification of UDP-MurNAc-pentapeptide 19 1.1 Strain and growth condition 19 1.2 Cells disruption and pre-treatment 19 1.3 Purification by C18 open column 20 1.4 Purification of murein precursors by reversed HPLC 20 1.5 Desalting of the nucleotide precursors 21 2. Gene cloning 21 2.1 Isolation the plasmid DNA 21 2.2 DNA agarose gel electrophoresis 22 2.3 DNA recovery and purification 22 2.4 PCR (polymerase chain reaction) 23 2.5 Restriction enzyme digestion 24 2.6 DNA ligation and DNA transformation 25 3. Protein expression and purification 25 3.1.MurG 25 3.3. partial purification of MraY 26 4. Fluorescence labeled UDP-MurNAc-pentapeptide.27 4.1. Preparation of UDP-MurNAc-Nε-FITC-pentapeptide 27 4.2. Preparation of UDP-MurNAc-Nε-dansyl-pentapeptide 28 5. Preparation of polyprenyl phosphate and other saturated lipid phosphate 28 6. Isolation of cytoplasmic membrane fragments 31 7. Synthesis and purification of Lipid I 31 8. HPLC assay 32 9. Semi-synthesis of Lipid I analogues 32 9.1 1-phospho-MurNAc-Nε-FITC-pentapeptide 32 9.2 Lipid I analogues 32 Chapter 3. Results 34 1. Purification of UDP-MurNAc-pentapeptide 34 2. Protein expression and purification 35 2.1 MraY expression and purification 35 2.2 MurG expression and purification 36 3. Preparation of polyprenyl phosphate and other saturated lipid phosphate 37 4. Fluorescence labeled UDP-MurNAc-pentapeptide 39 5. Synthesis and purification of Lipid I 40 6. HPLC assay of MraY 41 7. Semi-synthesis of Lipid I analogoues 41 Chapter 4. Discussion 44 1. Purification of UDP-MurNAc-pentapeptide 44 2. Enzymatic synthesis of Lipid I analogues 46 3. Semi-synthesis of Lipid I analogues 47 4. Conclusion 48 Reference 50 Figure Appendix: NMR Spectra of Productsen-US細胞壁抗藥性cell wallantibiotics resistanceother