Elucidation and Regulation of Rhodoquinone Biosynthesis in Rhodospirillum rubrum

红色红螺菌中红醌生物合成的阐明和调控

• 批准号: 8035700
• 负责人: Jennifer Niven Shepherd
• 金额: $ 24.14万
• 依托单位: GONZAGA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8035700
• 关键词: Aerobic; Amino Acids; Aminooxyacetate; Anabolism; Animal Model; Anthelmintics; Award; Bacteria; Benzoquinones; Bioinformatics; Biological Assay; Caenorhabditis elegans; Candidate Disease Gene; Catabolism; Collaborations; Data; Drug Delivery Systems; Drug Design; Drug resistance; Electron Transport; Electrons; Energy Metabolism; Enzymes; Gene Targeting; Genes; Genome; Helminths; Human; In Vitro; Investigation; Label; Laboratories; Lead; Length; Life; Lipids; Livestock; Mammals; Metabolism; Methods; Methyltransferase; Methyltransferase Gene; Multienzyme Complexes; Mutation; Nematoda; Nitrogen; O-Demethylating Oxidoreductases; Organic Chemistry; Oxygen; Parasites; Parasitology; Pathway interactions; Pharmaceutical Preparations; Point Mutation; Preparation; Process; Proteins; Quinones; Regulation; Research Personnel; Resistance development; Respiratory Chain; Rhodospirillum rubrum; Role; Side; Source; Staging; Structure; Techniques; Testing; Toxic effect; Transaminases; Ubiquinone; Universities; Work; amino group; analog; base; cofactor; design; drug development; drug discovery; drug testing; feeding; gabaculine; genome sequencing; in vitro Assay; in vivo; inhibitor/antagonist; mutant; novel; overexpression; polypeptide; research study; rhodoquinone

DESCRIPTION (provided by applicant): Rhodoquinone (RQ) is an essential cofactor used in the anaerobic energy metabolism of species such as the parasitic helminths, the free-living nematode Caenorhabditis elegans (C. elegans), and the purple non-sulfur bacterium, Rhodospirillum rubrum (R. rubrum). RQ is not synthesized or used in humans and other mammals with a primarily aerobic energy metabolism. However, RQ is structurally similar to ubiquinone (coenzyme Q or Q), an important lipid component involved in the aerobic respiratory chain. Both RQ and Q have a fully substituted benzoquinone ring and a polyisoprenoid side chain of varying length (depending on species). The only difference between the structures is that RQ has an amino group (NH2) instead of a methoxy group (OCH3) on the quinone ring. Therefore, the biosynthetic pathways of RQ and Q are proposed to be similar and may diverge from common precursors. The biosynthesis of Q has been well-characterized in both eukaryotic and prokaryotic species. It has recently been shown in my laboratory that the catabolism of Q is required for RQ biosynthesis in R. rubrum. A mutant strain (F11) of R. rubrum has also been identified which can synthesize Q, but not RQ, and therefore cannot grow anaerobically. The whole genomes of the mutant F11 and its spontaneous revertant RF110 were recently sequenced. The main focus of this proposal is to identify candidate gene(s) and polypeptide(s) responsible for RQ biosynthesis using the model organism R. rubrum. Selective inhibition of a unique enzyme target used in RQ biosynthesis (e.g. an aminotransferase) may lead to highly specific antihelminthic drugs that do not have a toxic effect on the host. There are three specific aims proposed for completion within the three-year award period. Specific Aim 1 involves the characterization of the conversion of Q to RQ in R. rubrum. Both in vivo and in vitro assays will be developed to identify new RQ biosynthetic intermediates, and the source of the amino group in RQ. Inhibition assays that target RQ biosynthesis will also be performed with commercially available aminotransferase inhibitors. Specific Aim 2 focuses on characterization of the F11 mutant using complementation experiments. From sequencing data, it was determined that the F11 mutation occurs in a putative methyltransferase gene. The corresponding gene product will be overexpressed and characterized. Specific Aim 3 will take advantage of bioinformatics to identify candidate aminotransferase and O-demethylase genes involved in RQ biosynthesis in R. rubrum. Deletion mutants will be prepared from the strongest candidates to determine their role in RQ biosynthesis. Unique RQ biosynthetic enzyme targets that are identified will ultimately be used for antihelminthic drug design. PUBLIC HEALTH RELEVANCE: Rhodoquinone (RQ) is an essential aminoquinone used in the anaerobic energy metabolism of the helminth parasites and it is not found or required in mammalian hosts. Characterization of genes and gene products in the RQ biosynthetic pathway in Rhodospirillum rubrum is the focus of this proposal, and may identify a parasite-specific target for drug development.

描述（由申请人提供）：红醌（RQ）是一种必需的辅助因子，用于物种的厌氧能量代谢，如寄生蠕虫、自由生活的线虫秀丽隐杆线虫（C. elegans）和紫色非硫细菌红红螺旋菌（R. rubrum）。RQ在人类和其他以有氧能量代谢为主的哺乳动物中不被合成或使用。然而，RQ在结构上与泛醌（辅酶Q或Q）相似，泛醌是参与有氧呼吸链的重要脂质成分。RQ和Q都有一个完全取代的苯醌环和一个不同长度的聚异戊二烯侧链（取决于物种）。唯一的区别是RQ在醌环上有一个氨基（NH2）而不是一个甲氧基（OCH3）。因此，RQ和Q的生物合成途径被认为是相似的，可能与共同前体不同。Q的生物合成在真核生物和原核生物中都有很好的特征。最近在我的实验室中已经证明，红毛蕊的RQ生物合成需要Q的分解代谢。另外，还鉴定出一株能合成Q而不能合成RQ的突变株（F11），因此不能厌氧生长。最近对突变体F11及其自发反转体RF110的全基因组进行了测序。本提案的主要重点是确定使用模式生物红毛霉进行RQ生物合成的候选基因和多肽。选择性抑制RQ生物合成中使用的独特酶靶标（例如转氨酶）可能导致高度特异性的抗蠕虫药物，而对宿主没有毒性作用。建议在三年的资助期内完成三项具体目标。具体目标1涉及表征的Q转化为RQ红。将开发体内和体外测定方法，以确定新的RQ生物合成中间体，以及RQ中氨基的来源。针对RQ生物合成的抑制试验也将使用市售的转氨酶抑制剂进行。特异性目标2侧重于利用互补实验表征F11突变体。从测序数据，确定F11突变发生在一个假定的甲基转移酶基因。相应的基因产物会被过表达和表征。Specific Aim 3将利用生物信息学的优势，鉴定红毛鼠RQ生物合成中涉及的候选转氨酶和o -去甲基化酶基因。删除突变体将从最强的候选者中制备，以确定它们在RQ生物合成中的作用。鉴定出的独特RQ生物合成酶靶点将最终用于抗蠕虫药物设计。

项目成果

Microbial eukaryotes have adapted to hypoxia by horizontal acquisitions of a gene involved in rhodoquinone biosynthesis.

• DOI: 10.7554/elife.34292
• 发表时间: 2018-04-26
• 期刊: eLife
• 影响因子: 7.7
• 作者: Stairs CW;Eme L;Muñoz-Gómez SA;Cohen A;Dellaire G;Shepherd JN;Fawcett JP;Roger AJ
• 通讯作者: Roger AJ