ISOPRENOID BIOSYNTHESIS: NON-MEVALONATE PATHWAY STUDIES

医学界面临着越来越多的细菌感染，这些细菌感染对目前使用的许多抗生素都有抗药性。在细菌中识别新的分子靶点是一种可能导致开发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类异戊二烯的非甲氧戊酸途径(NMP)可能就是这样一个靶标。基于对最近保存的全基因组DNA序列的数据库搜索，一些病原体被鉴定出含有与NMP的前两个基因高度同源性的基因，这表明这些生物可能对针对这一途径的抗生素敏感。与结核病(结核分枝杆菌)、一些溃疡(幽门螺杆菌)、性传播疾病(沙眼衣原体)和疟疾(恶性疟原虫)有关的微生物被包括在这份清单中。这项研究的长期目标是彻底了解NMP酶的机制，并确定抑制这些酶作为开发新抗菌剂的手段的可行性。该项目的第一个具体目标将是充分表征1-脱氧果糖-5-磷酸还原异构酶(DXR)，这是该途径中的第二种酶，已被已知的抗菌剂磷霉素所抑制。这一目标将通过以下方法实现：1)从蓝藻聚胞藻中纯化重组酶并获得其动力学数据。PCC6803和幽门螺杆菌。2)用DXP在C3上特异性的氚代来研究还原步骤的立体化学；3)制备和鉴定11个DXR的定点突变体；4)合成替代底物和建议的抑制剂，并分析它们对抑制菌作用的影响；5)测试体外抗菌活性。第二个目标是检测聚球藻的1-脱氧硫糖-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、高产、纯化和鉴定重组DXP合成酶，2)测试DXP合成酶的替代底物，3)用DXP测试已知的TPP依赖酶的抑制剂，以及4)设计和测试双底物类似物，为以后双底物抑制剂的开发奠定基础。

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.