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）：类异戊二烯构成了一个大的、多样的和重要的分子类别，其包括超过23，000个已知成员，包括甾醇、长萜、类胡萝卜素、倍半萜和醌。生物衍生的类异戊二烯形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和capsidiol）和食品色素（虾青素）。然而，由于细菌和桉树中的生物学功能通常仅需要低水平的类异戊二烯或它们在长时间内的缓慢积累，因此相应的酶机制处于低浓度和/或特征在于低周转数。古生菌必然具有更高通量的类异戊二烯生物合成机制;因为它们的膜脂质仅基于与基于多元醇（通常是甘油）的头基缩合的支链饱和烃。最常见的是，脂质烃是C20植烷基链;并且它们通过醚键与甘油基部分偶联，这与细菌和桉树的较不稳定的酯连接的膜脂质相反。许多古生菌，尤其是超嗜热菌，也含有类异戊二烯脂质，所述类异戊二烯脂质由在两端与多元醇偶联以得到大环双极性四醚脂质的C40联植烷基链组成。类异戊二烯醚连接的磷脂的古菌膜，通常基于二植烷基甘油二醚和dibiphytanyldiglycerol四醚的生物化学，是独特的古菌域;和四醚脂质生物合成的后一个步骤需要前所未有的类异戊二烯生物化学。类异戊二烯醚脂质的化学稳定性以及它们形成更稳定的脂质膜结构的能力已经导致这些分子种类的许多重要的新兴应用，包括在脂质体中用于药物递送和膜结合蛋白质系统的重构，以及在平面脂质膜中用于生物传感器应用。因此，阐明古菌代谢途径的醚脂合成和表征其新的酶，将提供新的生物合成工具，用于合成工业上有用的类异戊二烯醚脂和扩大可访问的类异戊二烯化学广泛的商业应用。该项目的重点是一个极端嗜热，异养硫酸盐还原古菌，闪烁古生菌，其基因组已被测序的类异戊二烯生物合成机制。该项目需要生物化学研究技术的新的、协调的应用，即，不同条件下的极端嗜热菌培养、蛋白质纯化和酶测定（包括使用放射性标记和LC/MS）、全基因组DMA微阵列以及基因克隆和表达，（1）鉴定或确认假设的酶活性，（2）将DNA序列分配给这些酶，（3）研究它们的动力学和调节，（4）深入了解原始生物体的代谢能力，（5）探索所发现的新型酶的合成效用。 该项目将揭示合成一类复杂分子的新生物途径，其中包括有效的抗癌和抗微生物药物。这些分子也可能被证明在设计新的药物输送载体和用于健康监测的生物传感器方面是有用的。