Studies of Isoprenoid Biosynthesis

描述(由申请人提供)：该项目将支持对类异戊二烯生物合成途径中的酶的结构和机理的研究，以及开发抑制甲基赤藓糖醇磷酸途径中的酶以合成异戊烯和二甲基烯丙基二磷酸的候选药物的筛选程序。要研究的酶包括甲基赤藓糖醇磷酸途径中的一些蛋白质(甲基赤藓糖醇磷酸合成酶，参与甲基赤藓糖醇吸收的转运体/激酶，将羟基二甲基烯丙基二磷酸转化为异戊烯基二磷酸和二甲基烯丙基二磷酸的酶)，I型和II型异戊烯基二磷酸异构酶，以及催化环丙烷合成反应的酶(角鲨烯合成酶、植酸烯合成酶和菊花二磷酸合成酶)。拟议的工作在国家关注的两个与健康相关的领域产生了影响。高血清胆固醇是导致冠心病的主要决定因素，在美国，冠心病是主要的死亡原因。尽管现在市场上的他汀类药物有效，但一些患者出现了严重的副作用，这些药物对患有最严重的纯合子形式的家族性高胆固醇血症的患者无效。角鲨烯合成酶催化一种分子的法尼基二磷酸与另一种分子的环丙烷化反应是胆固醇生物合成中的第一条途径特异性反应。角鲨烯合成酶直接靶向于他汀类药物途径的甾醇分支，是药物开发的一个有吸引力的靶点。甲基赤藓糖醇磷酸途径是许多细菌中类异戊二烯生物合成的唯一途径，并且与人类类异戊二烯生物合成的甲戊酸途径是正交的。由于类异戊二烯的生物合成是细菌生长所必需的，开发抑制甲基赤藓糖醇磷酸途径中的酶的化合物是具有吸引力的低哺乳动物毒性抗菌化合物。该项目将采用遗传学、分子生物学、酶学、结构和合成工具的组合来实现上述目标。所有被研究的酶的重组版本，包括野生型和突变型，都将在细菌中过度生产。机制方面的工作将集中在酶催化反应的化学机制以及对催化重要的蛋白质结构特征上。结构工作将以合作进行的X射线研究和现场进行的核磁共振研究为特色。鼠伤寒沙门氏菌将被用来构建适合于筛选化合物作为甲基赤藓糖醇磷酸途径酶抑制剂的菌株，包括来自难以处理的病原体的酶。

DESCRIPTION (provided by applicant): This project will support structural and mechanistic studies of enzymes in the isoprenoid biosynthetic pathway and the development of screening procedures for drug candidates that inhibit enzymes in the methylerythritol phosphate pathway to isopentenyl and dimethylallyl diphosphate. Enzymes to be studied include some of the proteins in the methylerythritol phosphate pathway (methylerythritol phosphate synthase, the transporter/kinase involved in uptake of methylerythritol, the enzyme that converts hydroxydimethylallyl diphosphate to isopentenyl diphosphate and dimethylallyl diphosphate), the type I and type II isopentenyl diphosphate isomerases, and the enzymes that catalyze cyclopropanation building reactions (squalene synthase, phytoene synthase, and chrysanthemyl diphosphate synthase). The proposed work has impact in two health-related areas of national concern. High serum cholesterol is a major determinant in coronary heart disease, the leading cause of death in the U.S. Although statins now on the market are effective, some individuals experience severe side effects and the drugs are not effective for individuals with the most severe homozygote form of familial hypercholesterolemia. The cyclopropanation of one molecule of farnesyl diphosphate by another catalyzed by squalene synthase is the first pathway specific reaction in cholesterol biosynthesis. Squalene synthase is an attractive target for drug development as an alternative to the statins by directly targeting the sterol branch of the pathway. The methylerythritol phosphate pathway is the exclusive route for isoprenoid biosynthesis in many bacteria and is orthogonal to the mevalonate pathway for isoprenoid biosynthesis in humans. Since isoprenoid biosynthesis is essential for bacterial growth, development of compounds that inhibit enzymes in the methylerythritol phosphate pathway are attractive drug candidates as antibacterial compounds with low mammalian toxicity. This project will employ a combination of genetic, molecular biological, enzymological, structural, and synthetic tools to achieve the goals stated above. Recombinant versions of all of the enzymes studied, both wild type and mutant, will be overproduced in bacteria. Mechanistic work will focus on the chemical mechanisms of the enzyme catalyzed reactions and those structural features of the protein important for catalysis. Structural work will feature X-ray studies conducted in collaboration and NMR studies performed on site. Salmonella typhimurium will be the vehicle used to construct strains suitable to screen compounds as inhibitors of enzymes in the methylerythritol phosphate pathway, including enzymes from difficult to handle pathogens.