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: 7373500
• 负责人: Harold George Monbouquette
• 金额: $ 22.87万
• 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 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7373500
• 关键词: Anabolism; Archaea; Archaeal Genes; Archaeoglobus fulgidus; Area; Aromatic Amino Acids; Bacteria; Binding; Binding Proteins; Biochemical; Biochemistry; Bioinformatics; Biological; Biological Assay; Biological Process; Biosensor; Carotenoids; Chemicals; Chemistry; Class; Complex; Condition; Coupled; DNA Microarray Chip; DNA Microarray format; DNA Sequence; Data; Doctor of Philosophy; Dolichol; Drug Delivery Systems; Education; Engineering; Enzymes; Escherichia coli; Esters; Ethers; Ethyl Ether; Film; Food; Funding; Gene Expression Profile; Genes; Genetic Transcription; Genome; Glycerol; Health; Heat-Shock Response; Human Resources Development; Hydrocarbons; Inorganic Sulfates; Joints; Kinetics; Knowledge; Link; Lipids; Liposomes; Louisiana; Malignant Neoplasms; Membrane; Membrane Lipids; Metabolic; Metabolic Pathway; Microarray Analysis; Modeling; Molecular; Monitor; Mutase; Nucleic Acids; Numbers; Operon; Organism; Outcomes Research; Oxidoreductase; Paclitaxel; Pathway interactions; Phospholipids; Pigments; Polymerase Chain Reaction; Preparation; Prephenate Dehydratase; Prephenate Dehydrogenase; Proteins; Proteomics; Publications; Quinones; Radiolabeled; Range; Regulation; Regulon; Research; Route; S-Lim; Salmon; Science; Sesquiterpenes; Sterols; Structure; Students; System; Technology; Unspecified or Sulfate Ion Sulfates; Woman; Work; aminoacid biosynthesis; antimicrobial drug; antitumor agent; astaxanthine; base; benzoquinone; cDNA Arrays; capsidiol; commercial application; design; enzyme activity; expression cloning; extreme thermophile; gene cloning; hyperthermophile; improved; insight; ipomeamarone; isoprenoid; lipid biosynthesis; liquid chromatography mass spectrometry; member; membrane reconstitution; novel; polyol; protein purification; radiotracer; reconstruction; shikimate; symposium; tool; transcriptomics

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 C2ophytanyl 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 C4obiphytanyl 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 DMAmicroarrays, 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.

类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、杜仲醇、类胡萝卜素、倍半萜和苯二酚。生物衍生的异戊二烯类化合物构成了 广泛的与健康有关的重要产品，包括抗肿瘤药物(紫杉醇)、抗菌药物 和食用色素(虾青素)。然而，由于只有低水平的异戊二烯或其 细菌和真核生物的生物学功能通常需要长时间的缓慢积累， 相应的酶机械处于低浓度和/或具有低周转数的特点。这个 古生菌必然具有更高产量的类异戊二烯生物合成机制，因为它们的膜脂是基于 仅在支链饱和碳氢化合物上与多元醇(通常是甘油)基头基团缩合。多数 通常，脂类碳氢化合物是C2Phytanyl链；它们通过乙醚偶联到甘油基部分 与细菌和真核生物中不太稳定的酯连接膜脂不同，细菌和优加拉植物的膜脂不稳定。许多古生菌，特别是 超嗜热菌还含有由C4双植酸链两端偶联的多元醇组成的类异戊二烯类脂 给出大环双极四醚类脂类。古生物类异戊二烯醚联磷脂的生物化学 膜通常基于二植酸甘油二醚和二植酸二甘油四醚，是 古生菌领域；四醚-脂生物合成的后几步需要前所未有的类异戊二烯生物化学。这个 类异戊二烯醚脂的化学稳定性以及它们形成更稳定的类脂膜结构的能力 导致了这些分子物种的一些重要的新兴应用，包括在药物脂质体中的应用 膜结合蛋白系统的传递和重建，以及用于生物传感器应用的平面脂膜。 古生菌合成醚类脂肪代谢途径的阐明及其新型酶的特性 这将为工业上有用的类异戊二烯醚脂的合成和生物合成提供新的生物合成工具 可获得的异戊二烯化学的扩展，用于广泛的商业应用。本项目的重点是 一种极端嗜热的异养硫酸盐还原考古子的类异戊二烯生物合成机制， 其基因组已被测序。该项目需要新的、协调的应用 生化研究技术，即各种条件下的极端嗜热菌培养，蛋白质纯化和 酶分析(包括放射性标记和LC/MS的使用)、全基因组DNA微阵列、基因克隆和 表达，(1)鉴定或确认假设酶活性，(2)赋予这些酶DNA序列 酶，(3)研究它们的动力学和调节，(4)洞察起源的代谢能力 生物体，以及(5)探索新发现的酶的合成用途。 该项目将为合成一类复杂的分子发现新的生物学路线，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新药输送的设计中有用。 车辆和用于健康监测的生物传感器。