ISOPRENOID BIOSYNTHESIS: NON-MEVALONATE PATHWAY STUDIES

医学界面临着越来越多的细菌感染，这些细菌对许多目前使用的抗生素具有耐药性。 在细菌中识别新的分子靶点是一种方法，可以导致抗生素的开发，这可能有助于对抗耐药性。 最近通过生物合成研究在一些细菌、高等植物和藻类中鉴定出的非甲羟戊酸途径（NMP）可能是这样的靶点。 基于对最近保藏的全基因组DNA序列的数据库检索，已经鉴定出许多致病生物体具有与针对NMP鉴定的前两个基因具有高度同源性的基因，这表明这些生物体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）的病原体有关的微生物也包括在此列表中。本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是充分表征1-脱氧木酮糖-5-磷酸还原异构酶（DXR），这是该途径中的第二种酶，并且已被证明可被已知的抗菌剂磷霉素抑制。 这一目的将通过1）纯化和获得来自蓝细菌集胞藻属PCC 6803和幽门螺杆菌的重组酶的动力学数据来实现。 2)3）制备和表征DXR的11种定点突变体4）合成替代底物和所提出的抑制剂并测定它们的作用和5）测试体外抑制剂的抗菌活性。第二个目的是检测来自集胞藻的1-脱氧木酮糖-5-磷酸合酶，第一个参与这个过程的酶。 这将通过1）克隆、过量生产、纯化和表征重组DXP合酶，2）用DXP合酶测试替代底物，3）用DXP测试TPP依赖性酶的已知抑制剂，和4）设计和测试双底物类似物以建立双底物抑制剂的后期开发的基础来实现。

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.