以酵素法合成Lipid II及其衍生物與發現具雙功能之十一異戊烯磷酸酯去磷酸酶

細菌廣布於世界各個角落，最重要的特徵是有一個肽聚醣構造的細胞壁。自從抗藥性細菌的出現，對細菌細胞壁生合成的研究以促進新型抗生素的研發已是刻不容緩的事。本論文將分為兩個章節，第一章在於建立細菌細胞壁轉醣酶受質lipid II的酵素合成方法，而lipid II是細胞壁轉醣酶的唯一受質。第二章節在於運用生化方法研究十一異戊烯醇加磷酸酶的功能，該酶與細胞壁酯質的生成代謝有密切關聯。肽聚醣是一層網狀構造包覆細菌用以保護細菌免受環境傷害，他的生物合成牽涉到許多步驟，根據合成位置可概略分為細胞內、膜上以及周質間三個部分。其中最後一個步驟也就是把lipid II聚合上新生成的肽聚醣是由細胞壁轉醣酶負責，由於該酶位於細胞膜外，也是細胞壁合成的必需品又在不同細菌間皆有相似的酶存在，所以是個研發細菌抗生素的重要標的。我們開發出酵素合成的方法來製造目前非常不易取得的lipid II，該方法運用十一異戊烯醇加磷酸酶、MraY、MurG以及輔因子的再生系統來克服化學生產上的瓶頸，並且可以將此方法運用在合成其他不同酯類的衍生物上。另外我們意外發現十一異戊烯醇加磷酸酶在有ADP存在下同時具有去磷酸酶的功能，這兩個功能對彼此皆不可缺少，他們能將十一異戊烯醇在加減磷酸根上做相互轉化是細胞壁合成上膜部分的重要步驟。Bacteria are widespread in environment and are characterized to have a unique peptidoglycan cell wall structure. The emergence of antibiotic resistance has promoted the study of peptidoglycan biosynthesis and the development of new antibiotics with new mode of action. This thesis is separated to two chapters: one is the enzymatic synthesis of lipid II, which is the only substrate for peptidoglycan transglycosylases; another is the biochemistry study of an enzyme undecaprenol kinase (UK), which is involved in the lipid metabolism of cell wall synthesis. Peptidoglycan is a network-like structure surrounding bacteria for protection. Its biosynthesis involves multiple steps, and is generally separated to cytosolic, membrane and periplasmic parts based on the location. Transglycosylase responding to the last step for polymerizing lipid II on nascent peptidoglycan is an attractive antibiotic target because of the periplasmic location, the essentiality for bacterial survival and the conservation among different bacteria. To produce the limited substrate lipid II, an enzymatic synthesis method was developed that UK, MraY and MurG were used coupling with cofactor regenerations. This method was also applied to the synthesis of lipid II analogues with a wide range of lipids. Interesting, an ADP-dependent phosphatase activity of UK was discovered. The bifunctional activities of kinase and phosphatase were indispensable to each other and provided reciprocal conversion of lipids which were involved in the membrane part of peptidoglycan synthesis.Table of Contents i List of Figures iii List of Tables iv Abstract 1 摘要 2 Chapter 1. Enzymatic synthesis of lipid II and analogues 3 1.1 ABSTRACT 3 1.2 INTRODUCTION 3 1.2.1 Structure of bacterial cell walls 3 1.2.2 The biosynthesis process of peptidoglycan 5 1.2.3 The antibiotics targets in cell walls polymerization 10 1.2.4 The current methods for lipid II synthesis 12 1.3 RESULTS AND DISSCUSSIONS 13 1.3.1 Enzymatic synthesis of UDP-MurNAc pentapeptide 14 1.3.2 Preparation of membrane associated enzymes for Lipid II synthesis 14 1.3.3 Enzymatic synthesis of lipid II analogues 16 1.3.4 Transglycosylation of PBPs using lipid II and analogues 18 1.4 METHODS 19 1.4.1 Materials 19 1.4.2 General methods 20 1.4.3 Plasmid constructions 20 1.4.4 Protein purification 21 1.4.5 Synthesis of UDP-MurNAc pentapeptide 21 1.4.6 Preparation of NBD-labeled UDP-MurNAc pentapeptide 22 1.4.7 Synthesis of polyprenyl phosphates 23 1.4.8 Synthesis of Lipid II and analogues 25 1.4.9 Enzymatic activity assays 28 Chapter 2. The undecaprenyl phosphate phosphatase activity of undecaprenol kinase regulates lipid pool on bacteria 45 2.1 ABSTRACT 45 2.2 INTRODUCTION 46 2.2.1 Undecaprenyl phosphate as a membrane glycan carrier for cell wall synthesis 46 2.2.2 The presence of undecaprenol in Gram-positive bacteria 46 2.3 RESULTS 48 2.3.1 The dephosphorylation of undecaprenyl phosphate by DgkA 48 2.3.2 The substrate specificity of dephosphorylation activities of UK 49 2.3.3 ADP dependency of C55P dephosphorylation 49 2.3.4 The release of inorganic phosphate 50 2.3.5 The effects of ATP analogues or inhibitors on C55OH phosphorylation and C55P dephosphorylation 51 2.3.6 The activities for DgkA mutants 52 2.3.7 The in vivo activity of UK 53 2.4 DISCUSSIONS 54 2.5 METHODS 57 2.5.1 Materials 57 2.5.2 Protein expression and purification 58 2.5.3 Activities measured by thin layer chromatography (TLC) 58 2.5.4 Activities measured by high-performance liquid chromatography (HPLC) 59 2.5.5 Determination of free inorganic phosphates 60 2.5.6 Determination of ATP concentrations 60 2.5.7 Membrane lipids extraction 61 References 81 Appendix 9