Defining the O-antigen biosynthetic pathways in zoonotic Coxiella burnetii: studies of dTDP-sugar biosynthesis and LPS extraction

本项目将研究人类和反刍动物病原体伯纳蒂克希菌对罕见糖病毒鼻和二氢羟基链球菌的生物合成。实验方法将涵盖三个主要目的：1)确定ndp -病毒鼻蛋白和ndp -二羟基链球菌的合成途径2)利用纯化酶在体外验证这些途径以确定每种提出的活性3)探测这些途径中不寻常酶的结构-功能关系目的1将主要使用质谱（MS）。Coxiella将在规定的条件下在Dstl培养，提取ms核苷酸糖的细胞代谢物将使用石墨化碳固相萃取分离，然后使用离子交换色谱法分离。然后用LC-MS检测；或水解以释放糖，然后将其硅化以进行GC-MS分析，特别强调在假定的途径中寻找中间体（Flores-Ramirez et al. 2012 Proteome Sci. 10,67）。这些实验将通过使用同位素标记生长基质的研究加以补充，以验证所提出的途径。对于目标2，相关基因将通过大肠杆菌密码子优化合成，并克隆到一组提供一系列溶解度标签的载体中（载体从牛津蛋白质生产设施获得）。这些将在小范围内表达，并为高水平表达和纯化提出最佳结构。纯化可能通过镍亲和层析和尺寸排除层析进行，然后去除标签，最后纯化去除标签和裂解酶。核苷酸结合将使用差示扫描荧光法或色氨酸荧光法来确定，并通过更精确的方法（例如等温滴定量热法，微尺度热泳法）来确定解离常数。每种酶的反应将使用适当的酶分析进行评估，使用连续的，停止的，偶联的分析，或这些的混合物，以获得最佳结果。可能的反应途径的核苷酸-糖前体很容易在商业上获得。为了验证反应结果，将使用HPLC对稳定的产物进行纯化，并用质谱法进行确认；对于不太稳定的中间体，将使用偶联反应，与之前一样再次验证产物。目的3将使用目的2中纯化的蛋白质。结晶将使用我们的道格拉斯结晶机器人进行，该机器人带有一套四个标准结晶屏幕。结晶将与相关核苷酸或核苷酸糖配合进行；并在适当情况下与其他辅因子、底物/底物类似物或产物一起使用。在实验阶段，将在适当情况下对蛋白质进行硒代蛋氨酸标记；或者加入重原子（最好是通过卤化物浸泡，然后使用常规的重原子化合物）。里程碑：12个月：使用GC-MS鉴定代谢中间体，并使用同位素标记的底物确认代谢途径；克隆酶（预计在Dstl的时间将在第一年度过）。24个月：病毒酶生物合成的酶验证；初步晶体学。36个月：二氢羟基链糖生物合成的酶学验证；酶-底物复合物的晶体学。48个月：期末实验，完成论文。

This project will examine the biosynthesis of the unusual sugars virenose and dihydrohydroxystreptose by the human and ruminant pathogen Coxiella burnetii. The experimental approaches will cover three main aims:1) To determine the pathway to synthesis for NDP-virenose and NDP-dihydroxystreptose2) To validate these pathways in vitro using purified enzymes to establish each proposed activity3) To probe the structure-function relationship of unusual enzymes in these pathwaysAim 1 will principally use mass spectrometry (MS). Coxiella will be grown at Dstl in defined conditions, and the cellular metabolites extracted for MS. Nucleotide sugars will be isolated using solid phase extraction with graphitised carbon, followed by ion-exchange chromatography. These will then be examined by LC-MS; or hydrolysed to release the sugars, which will then be silylated for analysis by GC-MS, with particular emphasis on searching for intermediates in putative pathways (Flores-Ramirez et al. 2012 Proteome Sci. 10, 67). These experiments will be supplemented by studies using isotopically labelled growth substrates to verify the proposed pathways.For aim 2, the relevant genes will be synthesised with codon optimisation for E. coli, and cloned into a set of vectors offering a range of solubility tags (vectors obtained from the Oxford Protein Production Facility). These will be expressed in small scale, and optimal constructs taken forward for high level expression and purification. Purification is likely to proceed through nickel affinity chromatography and size exclusion chromatography, followed by removal of the tags and a final purification to remove the tag and cleavage enzyme. Nucleotide binding will be determined using differential scanning fluorimetry or tryptophan fluorescence, and validated by more accurate methods (e.g. isothermal titration calorimetry, microscale thermophoresis) to determine dissociation constants. The reaction of each enzyme will be assessed using appropriate enzymatic assays, using either continuous, stopped, coupled assays, or a mixture of these, to obtain best results. Nucleotide-sugar precursors for the likely reaction paths are readily commercially available. To verify reaction outcomes, the products will be purified where stable using HPLC and confirmed with mass spectrometry; for less stable intermediates, coupled reactions will be used, with the products again verified as before.Aim 3 will use protein purified for the work in aim 2. Crystallisation will be performed using our Douglas crystallisation robot, with a set of four standard crystallisation screens. Crystallisation will be performed in complex with relevant nucleotides or nucleotide sugars; and with other cofactors, substrates/substrate analogues or products where appropriate. For experimental phasing, proteins will be grown selenomethionine labelling where appropriate; or heavy atoms will be added (preferably through halide soaking, following which conventional heavy atom compounds will be used).Milestones:12 months: identify metabolic intermediates using GC-MS and use isotopically labelled substrates to confirm metabolic pathways; clone enzymes (expect that time at Dstl will be spent in year 1).24 months: enzymatic verification of virenose biosynthesis; initial crystallography.36 months: enzymatic verification of dihydrohydroxystreptose biosynthesis; crystallography of enzyme-substrate complexes.48 months: final experiments, completion of thesis.