CAREER: Methanopterin Biosynthesis in Archaea and Methylotrophic Bacteria

古生菌是原核生物，在系统发育上不同于细菌领域的微生物。古古门中最具代表性的是产甲烷微生物（产甲烷菌），它们在全球碳循环、复杂生物质降解以及作为温室气体和能源的甲烷生产中发挥着关键作用。在这些严格的厌氧菌中发现了七种新的辅酶，包括改性叶酸甲烷蝶呤。直到最近，人们普遍认为甲蝶呤和其他改性叶酸只存在于古细菌中。然而，在甲基营养细菌methylobactenum extorquens （L. Chistoserdova， J. a . Vorholt， R. K. Thauer, and M. E. Lidstrom, 1998; Science: 281:99-102）中发现利用甲烷雀素的酶和修饰叶酸的发现，引发了关于修饰叶酸的进化起源和基因从一个结构域转移到另一个结构域的机制的争议性问题。提出的产甲烷菌甲烷蝶呤生物合成途径包括至少八个步骤。所有参与甲烷蝶呤生物合成的酶都没有被纯化到同质性，尽管有两种产甲烷古菌的完整基因组序列，但编码生物合成酶的基因都没有被鉴定出来。本研究的总体目标是利用生物化学和遗传相结合的方法来研究太古菌和甲基营养细菌中修饰叶酸生物合成的分子基础。研究项目的具体目标是：1。纯化并表征嗜热甲烷古菌中首个甲烷蝶呤合成特有酶b -核糖呋喃氨基苯5′-磷酸合成酶（B-RFA-P synthase）；鉴定嗜热产甲烷菌jannaschii产甲烷球菌和硫酸盐还原古菌fulgidus产考古舌菌基因组中B-RFA-P合成酶的编码基因，并对其在大肠杆菌中异种产生的酶进行鉴定；说谎。通过确定甲基蝶呤生物合成基因是否属于敲诈勒索甲基杆菌基因簇的一部分，研究细菌结构域修饰叶酸的生物合成，该基因簇编码与未识别的古细菌基因产物相似的蛋白质。这些结果将为阐明产甲烷菌和古菌中改性叶酸生物合成的酶学和遗传学基础奠定基础。比较古细菌和甲基营养细菌中甲烷蝶呤的生物合成将有助于确定未知的古细菌样蛋白在甲基营养细菌中的功能。相反，在古古菌中建立一个遗传系统可以帮助鉴定参与甲烷蝶呤生物合成的基因和酶。这些基因的表征将有助于发现未识别的古细菌蛋白质的功能，这些蛋白质与已知的细菌和真核蛋白质没有相似性（功能基因组学）。对甲烷蝶呤生物合成的深入了解将有助于深入了解产甲烷古菌的不同寻常的生物化学，携带C1化合物的辅酶的基本性质，以及细菌和古菌结构域之间存在的进化关系。除了为本科生、研究生和博士后学者提供研究培训机会外，拟议的研究还将成为一项教育计划的基础，该计划将使一名高中生和一名大学预科教师每年参加为期六周的暑期研究计划。将大学预科学生与科学发现的过程直接联系起来，通过积极参与拟议的研究，或者通过大学预科教师的影响，间接地联系起来，是提高公众科学素养和促进研究意识的一种手段，它是发现后来在课堂上教授的原则的一种手段。一项名为“万维网核磁共振谱仪”的创新技术将被运用在由首席研究员教授的本科讲座课程中，通过互联网远程启动实时核磁共振实验，并以动态的方式展示微生物学和科学研究的基本原理。

RascheThe Archaea are procaryotes that are phylogenetically distinct from microorganisms of the Bacteria domain. The most well characterized representatives of the Archaca are the methane-producing microorganisms (methanogens), which play critical roles in the global cycling of carbon the degradation of complex biomass, and the production of methane as both a greenhouse gas and energy source. Seven novel coenzymes, including the modified folate methanopterin, have been discovered in these strict anaerobes. Until recently it was generally accepted that methanopterin and other modified folates were restricted to the archaea. However, the discovery of methanopterin-utilizing enzymes and a modified folate in the methylotrophic bacteriumMethylobactenum extorquens (L. Chistoserdova, J. A. Vorholt, R. K. Thauer, and M. E. Lidstrom, 1998; Science 281:99-102) has raised provocative questions about the evolutionary origin of modified folates and the mechanisms by which genes can be transferred from one domain to another.The proposed pathway of methanopterin biosynthesis in methanogens consists of at least eight steps. None of the enzymes involved in methanopterin biosynthesis have been purified to homogeneity, and despite the availability of complete genome sequences for two methanogenic archaea, none of the genes encoding the biosynthetic enzymes have been identified. The overall goal of the proposed research is to utilize a combined biochemical and genetic approach to investigate the molecular basis of modified folate biosynthesis in archeea and methylotrophic bacteria. The specific objectives of the research project are 1. To purify and characterize B-ribofuranosylaminobenzene 5'-phosphate synthase (B-RFA-P synthase), the first unique enzyme of methanopterin biosynthesis in the archaeon Methanosarcina thermophila;2. To identify the gene(s) encoding B-RFA-P synthase in the genomes of the hyperthermophilic methanogen Methanococcus jannaschii and the sulfate-reducing archaeon Archaeoglobus fulgidus, and to characterize the enzymes produced heterologously in Escherichia coli; and3. To investigate the biosynthesis of modified folates in the Bacteria domain by determining if methanopterin biosynthetic genes are part of a Methylobacterium extorquens gene cluster that encodes proteins with similarity to unidentified archaeal gene products.These results will serve as a foundation for elucidating the enzymological and genetic basis of modified folate biosynthesis in methanogens and archaea. Comparisons of methanopterin biosynthesis in archaea and methylotrophic bacteria will contribute to identifying the functions of unknown archaeal-like proteins in methylotrophic bacteria. Conversely, the availability of a genetics system in M extorquens could facilitate the identification of genes and enzymes involved in methanopterin biosynthesis in archaca. Characterization of these genes will contribute to the broader goal of discovering the functions of unidentified archaeal proteins that exhibit no similarity to known bacterial and eucaryal proteins (functional genomics). A deeper understanding of methanopterin biosynthesis will provide insight into the unusual biochemistry of methanogenic archaea, the fundamental nature of coenzymes that carry C1 compounds, and the evolutionary relationships that exist between the Bacteria and Archaea domains.In addition to providing research training opportunities for undergraduates, graduate students, and postdoctoral scholars, the proposed research will be the foundation for an educational program that will enable one high school student and one pre-college teacher per year to participate in a six-week summer research program. Connecting pre-college students with the process of scientific discovery either directly, through active participation in the proposed research, or indirectly, through the influence of pre-college teachers, is one means of increasing scientific literacy in the general public and facilitating an awareness of research as a means of discovering principles taught later in the classroom. A technological innovation called the World Wide Web Nuclear Magnetic Resonance Spectrometer will be utilized in the undergraduate lecture course taught by the principal investigator to initiate real-time NMR experiments from a remote location via the internet and demonstrate fundamental principles of microbiology and scientific research in a dynamic way.