Studies of Isoprenoid Biosynthesis

描述（由申请人提供）：该项目将支持类异戊二烯生物合成途径中酶的结构和机制研究，以及抑制甲基赤藓糖醇磷酸途径中酶生成异戊烯基和二磷酸二甲基烯丙基的候选药物筛选程序的开发。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，参与甲基赤藓糖醇摄取的转运体/激酶，将二磷酸羟二甲基烯丙基转化为二磷酸异戊烯基和二磷酸二甲基烯丙基的酶），I型和II型二磷酸异戊烯基异构酶，以及催化环丙烷生成反应的酶(角鲨烯合成酶，植物烯合成酶，和菊花二磷酸合成酶)。拟议的工作对国家关注的两个与卫生有关的领域产生了影响。高血清胆固醇是冠心病的主要决定因素，而冠心病是美国人死亡的主要原因。尽管他汀类药物目前在市场上是有效的，但有些人会出现严重的副作用，而且这些药物对家族性高胆固醇血症最严重的纯合子形式的患者无效。在角鲨烯合成酶的催化下，一个二磷酸法尼酯分子与另一个二磷酸法尼酯分子的环丙化反应是胆固醇生物合成中的第一途径特异性反应。角鲨烯合成酶作为他汀类药物的替代品，通过直接靶向该途径的固醇分支而成为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合成类异戊二烯的唯一途径，与人类合成类异戊二烯的甲羟戊酸途径是正交的。由于类异戊二烯的生物合成对细菌生长至关重要，因此开发抑制甲基赤藓糖醇磷酸途径酶的化合物是具有低哺乳动物毒性的有吸引力的抗菌化合物候选药物。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。所研究的所有酶的重组版本，无论是野生型还是突变型，都会在细菌中过量产生。机制方面的工作将集中在酶催化反应的化学机制和对催化作用重要的蛋白质的结构特征上。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。鼠伤寒沙门氏菌将成为构建适合筛选甲基赤藓糖醇磷酸途径中酶抑制剂的菌株的载体，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.