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: 7195122
• 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 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7195122
• 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

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类庞大、多样且重要的分子，其中包括 23,000 多种已知成员，其中包括甾醇、多醇、类胡萝卜素、倍半萜和醌。生物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）、抗菌剂（ipomemarone 和辣椒二醇）和食品色素（虾青素）。然而，由于细菌和真核植物的生物功能通常只需要低水平的类异戊二烯或其长期缓慢积累，因此相应的酶促机制处于低浓度和/或以低周转数为特征。古细菌必然具有更高通量的类异戊二烯生物合成机制；因为它们的膜脂仅基于与多元醇（通常是甘油）基头基缩合的支链饱和烃。最常见的是，脂烃是C20植烷基链；它们通过醚键与甘油基部分偶联，这与细菌和真核生物的不太稳定的酯连接膜脂不同。许多古细菌，特别是超嗜热菌，还含有类异戊二烯脂质，其由两端与多元醇偶联的C 40 联植烷基链组成，得到大环、双极四醚脂质。古菌膜的类异戊二烯醚连接磷脂的生物化学通常基于二植烷基甘油二醚和二二植烷基二甘油四醚，是古菌领域所独有的；四醚脂质生物合成的后面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导致了这些分子种类的许多重要的新兴应用，包括用于药物递送和膜结合蛋白系统重建的脂质体，以及用于生物传感器应用的平面脂质膜。因此，阐明醚脂合成的古菌代谢途径及其新型酶的表征将为合成工业上有用的类异戊二烯醚脂和扩展可获取的类异戊二烯化学以实现广泛的商业应用提供新的生物合成工具。该项目重点研究极端嗜热、异养硫酸盐还原古菌——Archaeoglobus fulgidus 的类异戊二烯生物合成机制，其基因组已被测序。该项目需要生物化学研究技术的新颖、协调应用，即不同条件下的极端嗜热培养、蛋白质纯化和酶测定（包括放射性标记和LC/MS的使用）、全基因组DMA微阵列以及基因克隆和表达，（1）识别或确认假设的酶活性，（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可用于设计新的药物输送载体和用于健康监测的生物传感器。