Mechanistic studies of enzymes in isoprenoid biosynthesis

说明书(申请人提供)：所有异戊二烯类化合物均由异戊烯基二磷酸(IPP)及其异构体二甲基烯丙基二磷酸(DMAPP)构成。脱氧果糖磷酸(DXP)途径在原核生物IPP和DMAPP的生物合成中起着重要的作用，而动物中缺乏这一途径，这使得DXP途径酶成为开发广谱抗生素的理想途径。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足够数量的类胡萝卜素和叶绿素来正常生长。异戊二烯的天然丰度很低，这也激发了人们对通过生物工程生产异戊二烯的兴趣。由于生物工程类异戊二烯生产的限制因素是IPP和DMAPP的供应不足，因此对DXP途径的机理研究将指导构建基于生物工程的类异戊二烯的宿主菌株。该项目将研究DXP途径限速步骤之一的反应机理，这是一种由含有IspH蛋白的铁-硫簇催化的还原脱水反应。在初步研究中取得的几项主要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，使用底物模拟，研究了几种ISPH机制选项，并缩小到一个与所有当前数据一致的模型。[57Fe]标记的IspH被大量分离(从一天的纯化中约500 mg)。初步的EPR和Mvssbauer表征表明，所开发的IspH蛋白既具有高度的铁-硫簇负载，又具有高度的均一性。在这些成果的基础上，该小组获得了开展拟议研究的所有必要材料和方案。具体地说：在目标1中，将使用生物有机和生物物理方法相结合的方法捕获和表征酶和底物中间体。多条证据表明IspH以蛋白质复合体的形式存在。在目标2中，通过利用在初步研究中获得的菌株、试剂和报告系统，将利用几种互补的方法来鉴定IspH伙伴蛋白并研究其功能。 公共卫生相关性：拟议的类异戊二烯生物合成研究将指导基于机理的DXP途径酶抑制剂的开发，这些抑制剂可用作广谱抗生素。对目前全世界日益关注的耐药病原体菌株开发有效的新治疗方法将使公共卫生受益。

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.