Mechanistic Investigation of Radical SAM Methylases Involved in Tetrahydromethanopterin Biosynthesis

在化学系生命过程化学项目的支持下，弗吉尼亚理工大学的Kylie Allen博士和奥本大学的Evert Duin博士将研究这种名为MptM的酶的机制，MptM是自由基S-腺苷甲硫氨酸甲基酶家族的成员，参与四氢甲烷喋呤的生物合成。四氢甲烷蝶呤是叶酸(维生素B9)的改良版，对产甲烷微生物产生甲烷是必不可少的。该项目旨在揭示MptM催化的独特化学和生物学意义。通过这样做，这项研究有可能为更好地理解生物圈中甲烷的形成提供基础知识，对生物能源的应用具有潜在的影响。不同教育水平的学生将直接参与这项研究，从而激励下一代科学家，并提供重要的跨学科培训机会。作为这笔资金的结果，几项STEM(科学、技术、工程和数学)外联活动将得到扩大，包括为小学生举办的动手讲习班和为初中生举办的暑期项目。这个项目有可能通过对D类自由基SAM甲基酶的创始成员MptM的生化和光谱表征来促进我们对自由基SAM酶机制的了解。具体地说，本研究将确定甲基供体(S)，确定三个[4Fe-4S]簇的作用，并研究MptM催化的两个甲基化反应在产甲烷菌四氢生物蝶呤生物合成中的其他关键机制。还将进行遗传学研究，以确定两个同源自由基SAM甲基酶在模型产甲烷菌中的体内功能和重要性。综上所述，这些项目将有助于确定产甲烷菌中自由基SAM甲基酶的酶机制和生理功能，扩大不断增加的自由基SAM酶的催化谱系。在产甲烷菌之外发现了目前功能未知的相关D类自由基SAM甲基酶；因此，这项工作将有助于表征可能参与各种天然产品生物合成途径的一大组酶。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

With the support of the Chemistry of Life Processes Program in the Division of Chemistry, Dr. Kylie Allen from Virginia Tech and Dr. Evert Duin from Auburn University will study the mechanism of the enzyme known as MptM, a member of the radical S-adenosylmethionine (SAM) methylase family of enzymes, involved in tetrahydromethanopterin biosynthesis. Tetrahydromethanopterin is a modified version of folate (vitamin B9) that is essential for methane production by microorganisms known as methanogens. This project aims to uncover the unique chemistry and the biological significance of MptM catalysis. In doing so, this research has the potential to provide foundational knowledge toward a better understanding of the formation of methane in the biosphere, with potential implications for bioenergy applications. Students of various education levels will be directly engaged in this research, thus inspiring the next generation of scientists, and providing important interdisciplinary training opportunities. Several STEM(science, technology, engineering and mathematics) outreach activities will be expanded upon as a result of this funding, including hands-on workshops for elementary students and summer programs for middle and high school students. This project has the potential to advance our knowledge of radical SAM enzyme mechanisms through biochemical and spectroscopic characterization of MptM- the founding member of the class D radical SAM methylases. Specifically, this research will identify the methyl group donor(s), determine the roles of three [4Fe-4S] clusters, and examine other key mechanistic aspects of the two MptM-catalyzed methylation reactions in tetrahydrobiopterin biosynthesis in methanogens. Genetic studies will also be carried out to determine the in vivo functions and essentiality of two homologous radical SAM methylases in a model methanogen. Taken together, these projects will help to define the enzymatic mechanism and physiological functions of radical SAM methylases in methanogens, expanding the ever-increasing catalytic repertoire of radical SAM enzymes. Related putative class D radical SAM methylases with currently unknown functions are found outside of methanogens; therefore, this work will aid in characterizing a broad group of enzymes likely involved in diverse natural product biosynthetic pathways.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.