Methanopterin Biosynthesis in Archaea and Methylotrophic Bacteria

产甲烷微生物(产甲烷菌)有助于厌氧环境中生物质的降解以及作为能源和温室气体的甲烷的生产。辅酶四氢甲烷蝶呤(H4MPT)是一种四氢叶酸类似物，在产甲烷古生菌的一碳(C1)代谢中起着重要作用。在非产甲烷菌中也发现了H4MPT类似物，包括甲基营养细菌。更全面地了解H4MPT生物合成途径中涉及的基因和酶将有助于阐明产甲烷的生化基础，并有助于我们了解使用这种特殊辅酶的细菌和古菌之间的进化关系。在18种已提出的H4MPT生物合成酶中，只有5种被鉴定。在之前的资助中，该项目研究了两种酶：催化H4MPT生物合成第一步的核呋喃糖氨基苯5‘-磷酸合成酶(RFAP合成酶)和细菌二氢甲烷蝶呤还原酶(DMRA)。本研究项目的总体目标是加深对H4MPT生物合成的生化基础的认识。这项研究的具体目标是(I)利用定点突变技术研究RFAP合成酶的结构和功能，(Ii)比较细菌(DMRA)和古生菌的二氢甲烷蝶呤还原酶(DMRA)的进化无关，以及(Iii)确定细菌脱磷-H4MPT生物合成所需的其他细菌基因的特定功能。广泛的影响：除了提供有关产甲烷生物化学的不寻常方面的基础知识外，对H4MPT生物合成酶的深入了解也可能有助于开发更有效的H4MPT生物合成抑制剂，作为减少反刍动物甲烷排放的策略。这项研究将有助于培养两名研究生和一名博士后研究员。该计划还将通过为微生物学实验室课程提供基础研究项目来整合研究和教育，该课程旨在增加本科生、高中生和高中教师对原创研究的贡献。

Methane-producing microorganisms (methanogens) contribute to the degradation of biomass in anaerobic environments and the production of methane as an energy source and greenhouse gas. The coenzyme tetrahydromethanopterin (H4MPT) is a tetrahydrofolate analog that plays an essential role in the one-carbon (C1) metabolism of methane-producing archaea. H4MPT analogs have also been identified in nonmethanogens, including methylotrophic bacteria. A more complete understanding of the genes and enzymes involved in the pathway of H4MPT biosynthesis will help to elucidate the biochemical basis of methanogenesis and contribute to our knowledge about evolutionary relationships among bacteria and archaea that use this specialized coenzyme.Of the eighteen proposed enzymes of H4MPT biosynthesis, only five have been characterized. During the previous funding, this project characterized two enzymes: ribofuranosyl aminobenzene 5'-phosphate synthase (RFAP synthase), which catalyzes the first committed step of H4MPT biosynthesis, and bacterial dihydromethanopterin reductase (DmrA). The overall goal of this research project is to deepen the knowledge of the biochemical basis of H4MPT biosynthesis. The specific objectives of the research are (i) to investigate the structure and function of RFAP synthases using site-directed mutagenesis, (ii) to compare the evolutionarily unrelated dihydromethanopterin reductases from bacteria (DmrA) and archaea, and (iii) to determine the specific functions of additional bacterial genes required for bacterial dephospho-H4MPT biosynthesis.Broader impacts: In addition to providing fundamental knowledge about unusual aspects of methanogen biochemistry, a deeper understanding of H4MPT biosynthesis enzymes may also assist in the development of more effective H4MPT biosynthesis inhibitors as a strategy for decreasing methane emissions from ruminant animals. The research will contribute to the education of two graduate students and a postdoctoral researcher. The program will also integrate research and education by providing the foundational research project for a microbiology laboratory course designed to increase the contributions of undergraduates, high school students, and high school teachers to original research.