Mechanistic studies of enzymes in isoprenoid biosynthesis

描述（由申请人提供）：所有类异戊二烯是由二磷酸异戊烯基（IPP）及其异构体二磷酸二甲基烯基（DMAPP）构成的。磷酸脱氧纤维素（DXP）途径在原核生物IPP和DMAPP的生物合成中发挥着重要作用，而动物中该途径的缺乏使DXP途径酶成为开发广谱抗生素的理想候选酶。DXP途径也是除草剂开发的目标，因为它的植物突变体不能合成正常生长所需的足够数量的类胡萝卜素和叶绿素。低天然丰度的类异戊二烯也刺激了通过生物工程生产的兴趣。由于基于生物工程的类异戊二烯生产的限制因素是IPP和DMAPP的供应不足，对DXP途径的机理研究将指导基于生物工程的类异戊二烯生产宿主菌株的构建。该项目将研究DXP途径限速步骤之一的反应机理，该反应是由含有IspH蛋白的铁硫簇催化的还原性脱水反应。在初步研究中取得的几项重大成就是拟议工作的基础。在初步研究中，与文献报道相比，IspH活性提高了97倍。此外，使用底物类似物，研究了几种IspH机制选项，并将其缩小到与所有当前数据一致的模型。[57Fe]标记的IspH被大量分离（一天纯化约500 mg）。初始EPR和Mvssbauer表征表明，IspH蛋白具有高度的铁硫簇负载和均匀性。根据这些成就，该小组已获得进行拟议研究所需的所有必要材料和规程。具体来说：在目标1中，酶和底物为基础的中间体将被捕获并使用生物有机和生物物理方法的组合进行表征。一些证据表明，IspH以蛋白质复合物的形式存在。在Aim 2中，将利用在前期研究中获得的菌株、试剂和报告系统，利用几种互补的方法来鉴定IspH伴侣蛋白并研究其功能。

DESCRIPTION (provided by applicant): All isoprenoids are constructed by isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). The essential role played by the deoxyxylulose phosphate (DXP) pathway in prokaryotic IPP and DMAPP biosynthesis and the lack of this pathway in animals makes the DXP pathway enzymes ideal candidates for developing broad-spectrum antibiotics. The DXP pathway is also targeted for herbicide development because its plant mutants are not able to synthesize sufficient amounts of carotenoids and chlorophylls for normal growth. The low natural abundance of isoprenoids has also stimulated interest in their production through bioengineering. Because the limiting factor in bioengineering-based isoprenoid production is the inadequate supply of IPP and DMAPP, mechanistic studies of the DXP pathway will guide the construction of host strains for bioengineering-based isoprenoid production. The proposed project will study the reaction mechanism of one of the DXP pathway rate-limiting steps, a reductive dehydration reaction catalyzed by an iron-sulfur cluster containing IspH protein. Several major achievements accomplished in preliminary studies serve as the basis for the proposed work. In the preliminary studies, IspH activity was improved by 97-fold relative to that reported in the literature. In addition, using substrate analogs, several IspH mechanistic options were examined and narrowed down to a model that is consistent with all current data. [57Fe]-labeled IspH was isolated in large quantities (~500 mg from a one-day purification). Initial EPR and Mvssbauer characterizations demonstrated that the IspH protein developed has both a high degree of iron- sulfur cluster load and homogeneity. Based on these achievements, the team has acquired all the necessary materials and protocols for conducting the proposed studies. Specifically: In Aim 1, both enzyme- and substrate-based intermediates will be trapped and characterized using a combination of bioorganic and biophysical methods. Several lines of evidence indicate that IspH exists as protein complexes. In Aim 2, by making use of the strains, reagents, and reporter systems obtained in the preliminary studies, several complementary approaches will be utilized to identify IspH partner proteins and study their functions. PUBLIC HEALTH RELEVANCE: The proposed isoprenoid biosynthetic studies will guide the development of mechanism- based inhibitors of the DXP pathway enzymes, which can be used as broad-spectrum antibiotics. The public health benefit will result from the development of effective new treatments for drug-resistant strains of pathogens, currently of increasing concern worldwide.