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.

类异型构成了一个大型，多样而重要的分子，其中包括23,000多个已知成员 N乳清固醇，多利果醇，类胡萝卜素，倍半萜和奎因酮。生物学衍生的类异戊二烯形成基础 广泛的重要健康相关产品，包括抗肿瘤剂（紫杉醇），抗菌剂 （ipomeamarone和capsidiol）和食物色素（astaxanthin）。但是，由于只有低水平的类异戊二烯或它们 长时间的缓慢积累通常是细菌和Eucarya中的生物功能所必需的， 相应的酶促机制为低浓度和/或以低周转数为特征。这 古细菌必然具有较高的吞吐量类异型生物合成机制；因为它们的膜脂质是基于的 仅在带有多元醇（通常）基于甘油的头组的分支链，饱和的碳氢化合物上。最多 通常，脂质烃是C2ophytanyl链。然后它们通过以太耦合到甘油部部分 与细菌和桉树的酯连接膜脂质较不稳定的膜相反。尤其是许多古细菌 高疗量还含有由C4obiphytanyl链组成的类异丙脂质，两端耦合到多元醇 给出大环，双极分四脂质。原始乙醚连接的古细胞的生物化学 膜通常基于二羟基甘油二维体和二氨基甘油甘油二甲虫，是独一无二的 古细菌域；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。这 异胞素醚脂质的化学鲁棒性以及它们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，包括在药物中使用脂质体 膜结合的蛋白质系统的递送和重构，以及用于生物传感器应用的平面脂质膜。 阐明古细胞代谢途径，用于醚脂质合成和新型酶的表征 因此，将为合成工业有用的类异丙醚脂质提供新的生物态工具，并为 扩展可访问的类异丙化学用于广泛的商业应用。该项目着重于 极嗜热的，异营养的硫酸盐的古代的异on-生物合成机制 Archaeoglobus fulgidus，其基因组已被测序。该项目需要小说的协调应用 生化研究技术，即在各种条件下的极端嗜热培养，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Dmamicroars和Gene Cloning和Gene Cloning和 表达，（1）识别或确认假设的酶活性，（2）将DNA序列分配给这些序列 酶，（3）研究其动力学和调节，（4）以深入了解起源的代谢能力 有机体和（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，用于合成一个复杂类别的分子，包括有效的分子 抗癌和抗菌药物。这些分子也可能对新药的设计有用 用于健康监测的车辆和生物传感器。