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(申请人提供)：异戊二烯构成了一个大的、多样的和重要的分子类别，包括超过23,000个已知成员，包括甾醇、杜松醇、类胡萝卜素、倍半萜和苯二酮。生物衍生的异戊二烯类化合物构成了一系列与健康有关的重要产品的基础，包括抗肿瘤药物(紫杉醇)、抗菌剂(异丙咪唑酮和虾青醇)和食品色素(虾青素)。然而，由于细菌和真核生物的生物学功能通常只需要低水平的异戊二烯或其长期缓慢积累，相应的酶机械处于低浓度和/或以低周转数为特征。古生菌必然有更高的异戊二烯生物合成机制；因为它们的膜脂只基于支链饱和碳氢化合物与多元醇(通常是甘油)为基础的头基缩合。最常见的是脂类碳氢化合物是C2O植酸链；它们通过乙醚连接到甘油基部分，而不是细菌和真核生物中不太稳定的酯连接膜脂。许多古菌，特别是嗜热菌，也含有由C4o双植酸链两端连接到多元醇的异戊二烯类脂，从而得到大环双极四醚类脂类。古生物膜的异戊二烯醚联磷脂的生物化学是古生物领域所独有的，通常基于二植酸甘油二醚和二植酸二甘油四醚；四醚-脂生物合成的后几步需要史无前例的类异戊二烯生物化学。异戊二烯醚脂的化学稳定性以及它们形成更稳定的脂膜结构的能力导致了这些分子物种的一些重要的新兴应用，包括用于药物输送的脂质体和膜结合蛋白系统的重建，以及用于生物传感器的平面脂膜。因此，阐明古生代合成异戊二烯的代谢途径及其新型酶的特性，将为合成工业上有用的类异戊二烯醚脂和扩大可获得的类异戊二烯化学的广泛商业应用提供新的生物合成工具。本项目主要研究一种极端嗜热、异养硫酸盐还原古生体的类异戊二烯生物合成机制，它的基因组已经被测序。该项目需要生物化学研究技术的新颖和协调应用，即在不同条件下的极端嗜热培养，蛋白质纯化和酶分析(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)鉴定或确认假想的酶活性，(2)为这些酶分配DNA序列，(3)研究它们的动力学和调节，(4)深入了解起源生物的代谢能力，以及(5)探索所发现的新酶的合成用途。 该项目将为合成一类复杂的分子开辟新的生物学路线，其中包括有效的抗癌和抗微生物药物。这些分子还可能被证明在设计新的药物输送工具和用于健康监测的生物传感器方面很有用。