Studies of Isoprenoid Biosynthesis

描述（由申请人提供）：本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，以及抑制甲基异戊烯基磷酸途径中酶生成异戊烯基和二甲基烯丙基二磷酸的候选药物筛选程序的开发。要研究的酶包括一些在磷酸甲酯途径中的蛋白质（甲基戊烯基二磷酸合酶，参与甲基戊烯基二磷酸摄取的转运蛋白/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，以及催化环丙烷化构建反应的酶（角鲨烯合酶、八氢番茄红素合酶和胆甾醇二磷酸合酶）。拟议的工作在全国关注的两个与健康有关的领域产生影响。高血清胆固醇是冠心病的主要决定因素，冠心病是美国死亡的主要原因。虽然现在市场上的他汀类药物有效，但有些人会出现严重的副作用，而且这些药物对最严重的纯合子型家族性高胆固醇血症患者无效。由角鲨烯合酶催化的一分子法呢基二磷酸与另一分子法呢基二磷酸的环丙烷化是胆固醇生物合成中的第一个途径特异性反应。角鲨烯合酶是一个有吸引力的药物开发的目标，作为替代他汀类药物，直接靶向途径的甾醇分支。磷酸甲脂途径是许多细菌中类异戊二烯生物合成的唯一途径，并且与人类中类异戊二烯生物合成的甲羟戊酸途径正交。由于类异戊二烯生物合成是细菌生长所必需的，因此开发抑制甲基异戊二醇磷酸途径中的酶的化合物作为具有低哺乳动物毒性的抗菌化合物是有吸引力的候选药物。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。所有研究的酶的重组版本，无论是野生型还是突变型，都将在细菌中过量生产。机械工作将集中在酶催化反应的化学机制和那些重要的催化蛋白质的结构特征。结构工作将包括合作进行的X射线研究和现场进行的NMR研究。鼠伤寒沙门氏菌将是用于构建菌株的载体，这些菌株适用于筛选化合物作为磷酸甲酯途径中酶的抑制剂，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.