ISOPRENOID BIOSYNTHESIS: NON-MEVALONATE PATHWAY STUDIES

医学界面临着越来越多的细菌感染，这些细菌感染对许多目前使用的抗生素具有耐药性。识别细菌中的新分子靶标是一种可以导致开发可能有助于对抗耐药性的抗生素的方法。最近通过生物合成研究在一些细菌、高等植物和藻类中发现的非甲羟戊酸途径（NMP）可能就是这样一个靶点。基于最近沉积的全基因组DNA序列的数据库搜索，已经鉴定出许多致病生物含有与NMP前两个基因高度同源的基因，这表明这些生物可能对靶向该途径的抗生素敏感。与结核病（结核分枝杆菌）、某些溃疡（幽门螺杆菌）、性传播疾病（沙眼衣原体）和疟疾（恶性疟原虫）的病原体有关的生物体也包括在这一清单中。本研究的长期目标是彻底了解NMP酶的作用机制，并确定抑制这些酶作为开发新型抗菌剂的可行性。该项目的第一个具体目标将是充分表征1-脱氧葡糖-5-磷酸还原异构酶（DXR），该酶是该途径中的第二种酶，已被已知的抗菌剂磷霉素抑制。1)从聚囊蓝藻PCC6803和幽门螺杆菌中纯化重组酶并获得其动力学数据。2)利用DXP在C3处进行特异性氘化，考察还原步骤的立体化学性质；3)制备和表征11个DXR位点定向突变体；4)合成替代底物和拟议的抑制剂，并分析它们对其的影响；5)测试体外抑制剂的抗菌活性。第二个目标将是检查来自聚囊藻sp.的1-脱氧醛糖-5-磷酸合成酶，这是参与该途径的第一种酶。这将通过1)克隆、过量生产、纯化和表征重组DXP合成酶来完成，2)用DXP合成酶测试替代底物，3)用DXP测试已知的TPP依赖酶抑制剂，4)设计和测试双底物类似物，为以后开发双底物抑制剂奠定基础。

The medical community is faced with increasing numbers of bacterial infections that are resistant to many of the currently used antibiotics. Identifying new molecular targets in bacteria is an approach that can lead to the development of antibiotics that may help to combat resistance. The non-mevalonate pathway (NMP) to isoprenoids which has recently been identified in some bacteria, higher plants, and algae through biosynthetic studies may prove to be such a target. Based on database searches of recently deposited whole genome DNA sequences, a number of pathogenic organisms have been identified that harbor genes with high homology to the first two genes identified for the NMP, suggesting that these organisms might be susceptible to antibiotics targeted to this pathway. Organisms implicated as causative agents of tuberculosis (Mycobacterium tuberculosis), some ulcers (Helicobacter pylori), sexually transmitted diseases (Chlamydia trachomatis), and malaria (Plasmodium falciparum) are included in this list. The long-term objectives of this research are to thoroughly understand the mechanisms of the enzymes of the NMP and to determine the feasibility of inhibiting these enzymes as a means to develop new antibacterial agents. The first specific aim of this project will be to fully characterize 1- deoxyxylulose-5-phosphate reductoisomerase (DXR), the second enzyme in the pathway and one which has been shown to be inhibited by the known antibacterial agent, fosmidomycin. This aim will be accomplished by 1) Purifying and obtaining kinetic data for the recombinant enzymes from the cyanobacterium Synechocystis sp. PCC6803 and Helicobacter pylori. 2) Examining the stereochemistry of the reduction step by using DXP specifically deuterated at C3 3) Preparing and characterizing eleven site-directed mutants of DXR 4) Synthesizing alternate substrates and proposed inhibitors and assaying for their effects on and 5) Testing in vitro inhibitors for antibacterial activity. The second aim will be to examine 1-deoxyxylulose-5-phosphate synthase from Synechocystis sp., the first enzyme involved in the pathway. This will be accomplished by 1) Cloning, overproducing, purifying, and characterizing the recombinant DXP synthase, 2) Testing alternate substrates with DXP synthase 3) Testing known inhibitors of TPP dependent enzymes with DXP, and 4) Designing and testing bisubstrate analogs to build the foundation for the later development of bisubstrate inhibitors.