CAREER: Methanopterin Biosynthesis in Archaea and Methylotrophic Bacteria

职业：古细菌和甲基营养细菌中的甲烷蝶呤生物合成

• 批准号: 9876212
• 负责人: Madeline Rasche
• 金额: --
• 依托单位: University of Florida
• 依托单位国家: 美国
• 项目类别: Continuing grant
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至 2004-11-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9876212&HistoricalAwards=false
• 关键词: CAREER; Methanopterin; Biosynthesis; Archaea; Methylotrophic

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.

Raschethe Archaea是原核生物，在系统发育上不同于细菌领域的微生物。Archaca最具代表性的代表是甲烷产生微生物(产甲烷菌)，它们在全球碳循环、复杂生物质的降解以及作为温室气体和能源的甲烷生产中发挥关键作用。在这些严格的厌氧菌中，已经发现了七种新的辅酶，包括修饰的叶酸甲烷蝶呤。直到最近，人们还普遍认为，甲基蝶呤和其他经修饰的叶酸仅限于古生菌。然而，在甲基营养细菌(L.Chistoserdova，J.A.Vorholt，R.K.Thauer和M.E.Lidstrom，1998；Science 281：99-102)中发现的甲基喋呤利用酶和修饰的叶酸，提出了关于修饰的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挑衅性问题。参与甲烷蝶呤生物合成的酶都没有纯化到同质性，尽管有两个产甲烷古菌的完整基因组序列，但没有一个编码生物合成酶的基因被鉴定出来。这项拟议研究的总体目标是利用生化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。本研究的具体目标是：1.纯化和鉴定嗜热甲烷八叠胞菌中第一个独特的甲烷喋呤生物合成酶--B-呋喃糖氨基苯5‘-磷酸合成酶(B-RFA-P合成酶)；2.在嗜热产甲烷菌简氏甲烷球菌和硫酸盐还原古生菌的基因组中鉴定编码B-RFA-P合酶的基因(S)，并对在大肠杆菌中异源产生的酶进行鉴定；通过确定甲烷蝶呤生物合成基因是否属于一种编码与未知古生菌基因产物相似的蛋白质的基因簇，来研究细菌领域中修饰叶酸的生物合成。这些结果将为阐明产甲烷菌和古生菌中修饰叶酸生物合成的酶学和遗传学基础奠定基础。比较古细菌和甲基化细菌中甲基蝶呤的生物合成，将有助于鉴定甲基化细菌中未知的古菌样蛋白的功能。相反，在M extorquens中建立遗传系统可以帮助鉴定与Araca中甲烷喋呤生物合成有关的基因和酶。这些基因的特征将有助于更广泛的目标，即发现与已知细菌和真核生物蛋白没有相似之处的未知古菌蛋白的功能(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生物化学，携带C1化合物的辅酶的基本性质，以及细菌和古生菌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个教育项目的基础，该项目将使每年一名高中生和一名大学预科教师能够参加为期六周的暑期研究项目。通过积极参与拟议的研究，或通过大学前教师的影响，将大学预科学生直接与科学发现过程联系起来，是提高普通公众的科学素养，并促进将研究作为发现稍后在课堂上教授的原理的手段的意识的一种手段。一项名为万维网核磁共振波谱仪的技术创新将被用于由首席研究员讲授的本科讲座课程，通过互联网远程启动实时核磁共振实验，并以动态的方式演示微生物学和科学研究的基本原理。