Unique Isoprenoid Biosynthesis Machinery of the Hyperthermophilic Archaea

超嗜热古菌独特的类异戊二烯生物合成机制

• 批准号: 7088312
• 负责人: Harold George Monbouquette
• 金额: $ 22.88万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-04 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7088312
• 关键词: Archaea; enzyme activity; isoprenoid; lipid biosynthesis; membrane lipids; microarray technology; microorganism culture; microorganism metabolism; phospholipids; protein purification

DESCRIPTION (provided by applicant): The isoprenoids constitute a large, diverse and important class of molecules that includes over 23,000 known members ncluding sterols, dolichols, carotenoids, sesquiterpenes, and quinones. Biologically derived isoprenoids form the basis of a broad spectrum of important health-related products including antitumor agents (taxol), antimicrobial agents (ipomeamarone and capsidiol), and food pigments (astaxanthin). However, since only low levels of isoprenoids or their slow accumulation over long periods typically are required for biological function in bacteria and eucarya, the corresponding enzymatic machinery is at low concentration and/or is characterized by low turnover number. The archaea necessarily have higher throughput isoprenoid biosynthesis machinery; as their membrane lipids are based solely on branched-chain, saturated hydrocarbons condensed with polyol (usually glycerol)-based headgroups. Most commonly, the lipid hydrocarbons are C2o phytanyl chains; and they are coupled to a glyceryl moiety through ether linkages as opposed to the less stable ester-linked membrane lipids of bacteria and eucarya. Many archaea, especially the hyperthermophiles, also contain isoprenoid lipids consisting of C4o biphytanyl chains coupled at both ends to polyols to give macrocyclic, bipolar tetraether lipids. The biochemistry of the isoprenoid ether-linked phospholipids of archaeal membranes, based typically on diphytanylglycerol diethers and dibiphytanyldiglycerol tetraethers, is unique to the archaeal domain; and the latter steps in tetraether-lipid biosynthesis entail unprecedented isoprenoid biochemistry. The chemical robustness of isoprenoid ether lipids as well as their ability to form more stable lipid membrane structures has led to a number of important emerging applications for these molecular species including usage in liposomes for drug delivery and reconstitution of membrane-bound protein systems, and in planar lipid films for biosensor applications. Elucidation of archaeal metabolic pathways for ether lipid synthesis and characterization of their novel enzymes therefore will provide new biosyntnetic tools for the synthesis of industrially useful isoprenoid ether lipids and for the expansion of accessible isoprenoid chemistry for wide-ranging commercial application. This project focuses on the isoprenoid biosynthetic machinery of an extremely thermophilic, heterotrophic sulfate-reducing archaeon, Archaeoglobus fulgidus, whose genome has been sequenced. The project entails the novel, coordinated application of biochemical research technology, i.e., extreme thermophile culture under varied conditions, protein purification and enzyme assays (including use of radiolabeling and of LC/MS), full-genome DMA microarrays, and gene cloning and expression, (1) to identify or to confirm hypothesized enzyme activities, (2) to assign DNA sequences to these enzymes, (3) to study their kinetics and regulation, (4) to gain insight into the metabolic capabilities of the originating organism, and (5) to explore the synthetic utility of the novel enzymes discovered. This project will uncover new biological routes for the synthesis of a complex class of molecules that includes potent anti-cancer and anti-microbial drugs. These molecules also may prove useful in the design of new drug delivery vehicles and of biosensors for health monitoring.

描述（由申请人提供）：类异型构成了一类大，多样而重要的分子，其中包括23,000多个已知成员，含有固醇，多乙基醇，类胡萝卜素，倍苯二酚和奎因酮。生物学衍生的类异戊二烯构成了广泛的重要健康相关产品的基础，包括抗肿瘤剂（紫杉醇），抗微生物剂（ipomeamarone and Capsidiol）和食物色素（astaxanthin）。但是，由于通常在细菌和欧洲菌中生物学功能通常需要长时间的类吸收或它们的缓慢积累，因此相应的酶促机器在低浓度下和/或以低离职数为特征。古细菌必然具有较高的多透产类吸收性生物合成机制。由于它们的膜脂质仅基于分支链，饱和烃与多醇（通常是甘油）的头组凝结。最常见的是，脂质烃是C2O植物链。它们通过以太链接耦合到甘油部分，而不是细菌和桉树的较不稳定的酯连接的膜脂质。许多古细菌，尤其是热疗中心，还含有由双二烷基链组成的类异磷酸脂质，两端偶联到多醇，以产生宏观的双极三乙醚脂质。古细胞膜的异烷基醚连接的磷脂的生物化学通常基于二羟基甘油二维体和二苯甲酰甘油甘油三乙醇，是古细胞领域所特有的；后者的四链脂质生物合成的步骤需要前所未有的类异戊二烯生物化学。异荷乙醚脂质的化学鲁棒性及其形成更稳定的脂质膜结构的能力，导致了这些分子物种的许多重要新兴应用，包括在脂质体中使用脂质体和膜结合蛋白质系统的药物递送和重新重构的脂质蛋白质系统，以及用于生物传感器应用的平面脂质膜中。因此，阐明用于醚脂质合成的古细胞代谢途径和新型酶的表征将为合成工业有用的类异丙酸酯脂质的合成以及扩展可访问的类异源性化学以用于广泛的商业应用提供新的生物环节性工具。该项目的重点是极嗜热的，异嗜性硫酸盐的古老的古老的古氏菌，Archaeoglobus fulgidus的类异型生物合成机制，其基因组已被测序。该项目需要新颖的生化研究技术的应用，即在各种条件下极端的热嗜热培养，蛋白质纯化和酶测定（包括使用放射性映射和LC/MS），全基因组DMA微阵列，以及基因的克隆和表达，以确认这些活动（1）来确认这些活动（2），以确认这些活动（2），并确认这些Zyme-enzeme，2），2） （3）研究其动力学和调节，（4）以深入了解原始生物的代谢能力，（5）探索发现的新酶的合成效用。 该项目将发现新的生物学途径，用于合成一类复杂的分子，其中包括有效的抗癌和抗微生物药物。这些分子也可能在设计新药递送车和生物传感器的设计中有用。