Elucidation and Regulation of Rhodoquinone Biosynthesis in Rhodospirillum rubrum

描述（由申请人提供）：红醌（RQ）是寄生蠕虫、自由生活的线虫秀丽隐杆线虫（C. elegans）和紫色非硫细菌Rhodoacetyllum rubrum（R.红色）。RQ在人类和其他主要进行有氧能量代谢的哺乳动物中不合成或使用。然而，RQ在结构上类似于泛醌（辅酶Q或Q），一种参与有氧呼吸链的重要脂质成分。RQ和Q都具有完全取代的苯醌环和不同长度的聚类异戊二烯侧链（取决于物种）。结构之间的唯一区别是RQ在醌环上具有氨基（NH 2）而不是甲氧基（OCH 3）。因此，RQ和Q的生物合成途径被认为是相似的，并且可能与共同的前体不同。Q的生物合成在真核生物和原核生物中都得到了很好的表征。最近在我的实验室中发现，Q的催化作用是R.红色。对R.还鉴定了红色酵母，其可以合成Q，但不能合成RQ，因此不能厌氧生长。最近对突变体F11及其自发回复突变体RF 110的全基因组进行了测序。本研究的主要目的是利用模式生物R.红色。选择性抑制RQ生物合成中使用的独特酶靶标（例如氨基转移酶）可产生对宿主无毒性作用的高度特异性驱虫药。在三年的奖励期内，提出了三个具体目标。具体目标1涉及R中Q到RQ的转化的表征。红色。将开发体内和体外试验，以鉴定新的RQ生物合成中间体和RQ中氨基的来源。靶向RQ生物合成的抑制试验也将使用市售转氨酶抑制剂进行。具体目标2侧重于使用互补实验表征F11突变体。根据测序数据，确定F11突变发生在推定的甲基转移酶基因中。相应的基因产物将被过表达和表征。具体目标3将利用生物信息学的优势，确定参与RQ生物合成的候选转氨酶和O-脱甲基酶基因。红色。将从最强的候选物制备缺失突变体以确定它们在RQ生物合成中的作用。确定的独特RQ生物合成酶靶点最终将用于抗蠕虫药物设计。 公共卫生相关性：Rhodoquinone（RQ）是寄生蠕虫无氧能量代谢中必需的氨基醌，在哺乳动物宿主中不存在或不需要。本研究的重点是鉴定红杜鹃RQ生物合成途径中的基因和基因产物，并可能确定药物开发的寄生虫特异性靶点。

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.