Methanopterin Biosynthesis in Archaea and Methylotrophic Bacteria

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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.