CAREER: Methanopterin Biosynthesis in Archaea and Methylotrophic Bacteria

RascheTheseea是原核生物，在遗传学上与细菌域的微生物不同。 Archaca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、复杂生物质的降解以及甲烷作为温室气体和能源的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，包括修饰的叶酸甲烷蝶呤。直到最近，人们才普遍接受甲氨蝶呤和其他修饰的叶酸仅限于古细菌。然而，在甲基营养细菌扭脱甲基杆菌（Methylobactenum extorquens）中发现了利用甲氨蝶呤的酶和修饰的叶酸。Chistoserdova，J. A.沃霍尔特河K. Thauer和M. E. Lidstrom，1998年; Science 281：99-102）已经提出了关于修饰的叶酸的进化起源和基因可以从一个域转移到另一个域的机制的挑衅性问题。参与甲烷蝶呤生物合成的酶都没有被纯化到同质，尽管两个产甲烷古菌的完整基因组序列的可用性，编码生物合成酶的基因都没有被确定。拟议研究的总体目标是利用生物化学和遗传学相结合的方法来研究古细菌和甲基营养细菌中修饰的叶酸生物合成的分子基础。研究项目的具体目标是 1.目的纯化并鉴定B-呋喃核糖氨基苯5 ′-磷酸合酶（B-RFA-P 合成酶），嗜热甲烷八叠球菌中甲烷蝶呤生物合成的第一个独特的酶;2.鉴定极端嗜热产甲烷菌詹氏甲烷球菌和硫酸盐还原古菌闪烁古球菌基因组中编码B-RFA-P合酶的基因，并对大肠杆菌中异源产生的酶进行表征; 3.通过确定甲烷蝶呤生物合成基因是否属于编码与古细菌基因产物相似蛋白质的扭脱甲基杆菌基因簇，研究细菌结构域中修饰叶酸的生物合成，为阐明甲烷菌和古细菌中修饰叶酸生物合成的酶学和遗传基础奠定基础。通过比较古菌和甲基营养菌中甲氨蝶呤的生物合成，将有助于确定甲基营养菌中未知的类古菌蛋白的功能。相反，在扭脱甲基杆菌的遗传系统的可用性，可以促进在archaca中的甲烷蝶呤生物合成的基因和酶的鉴定。这些基因的表征将有助于发现未知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，拟议的研究将成为一项教育方案的基础，该方案将使每年一名高中生和一名大学预科教师参加为期六周的夏季研究方案。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，或者间接地，通过大学预科教师的影响，是提高公众科学素养的一种手段，并促进研究意识，作为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光谱仪的技术创新将在主要研究者教授的本科课程中使用，通过互联网从远程位置启动实时核磁共振实验，并以动态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.