CAREER: Mechanistic Investigations of Enzymatic Methyl Transfer in Microbial Biosynthetic Pathways

从微生物中分离的酶在日常生活中起着重要作用，从藻类的光合作用和氧气生产到乳酸杆菌和乳酸球菌的酸奶形成。 拟议的研究将调查链霉菌中酶使用的非反应性碳和磷原子的甲基化的独特的，不太了解的机制，链霉菌是一种天然产生许多抗生素，抗癌和除草化学品的有益细菌。 迄今为止，所有的酶促甲基转移反应都被表征为亲核取代反应。 然而，这些链霉菌甲基转移酶的底物不适合亲核活化，这表明需要和使用另一种催化机制。 生物化学，光谱和合成有机技术将用于确定这些酶是否催化甲基转移使用四铁，四硫簇从S-腺苷-L-甲硫氨酸（SAM）和维生素B12的衍生物甲钴胺产生有机自由基。 理解这些反应的机制将为工程化这些和相关的酶提供框架，以产生新的碳-碳和/或碳-磷-碳键。 本研究中研究的酶、辅因子和微生物将与一项针对农村、农业帕卢塞地区K-5年级儿童的教育推广计划相联系，该地区距离最近的大都市区300英里。 在与帕卢塞发现科学中心的合作伙伴关系，互动演示侧重于有益的微生物和酶化学将提交给整个地区的家庭和班级。 赠品将与评估相结合，以提高保留率，并获得改进的反馈。还将以可访问的在线格式提供包含实验结果的类似介绍，以接触更多的受众。 该提案的最终目标是提高对微生物酶化学的整体理解，并鼓励孩子们追求微生物学和/或生物化学的未来职业。

Enzymes isolated from microbes play significant roles in daily life, ranging from photosynthesis and oxygen production by algae to yogurt formation by Lactobacilli and Lactococci bacteria. The proposed research will investigate a unique, poorly-understood mechanism for the methylation of unreactive carbon and phosphorus atoms used by enzymes in Streptomyces, a large genus of beneficial bacteria that naturally produce many antibiotic, anticancer, and herbicidal chemicals used commercially. To date, all enzymatic methyl transfer reactions have been characterized as nucleophilic substitution reactions. However, the substrates for these Streptomyces methyltransferases are not amenable to nucleophilic activation, suggesting the need for and use of another catalytic mechanism. Biochemical, spectroscopic, and synthetic organic techniques will be used to determine whether these enzymes catalyze methyl transfer using a four-iron, four-sulfur cluster to generate organic radicals from S-adenosyl-L-methionine (SAM) and methylcobalamin, a derivative of vitamin B12. Understanding the mechanism of these reactions will provide a framework for engineering these and related enzymes to produce novel carbon-carbon and/or carbon-phosphorus-carbon bonds. The enzymes, cofactors, and microbes studied in this research will be linked to an educational outreach program targeting K-5th grade children of the rural, agricultural Palouse region, which is 300 miles from the nearest metropolitan area. In partnership with the Palouse Discovery Science Center, interactive presentations focusing on beneficial microbes and enzyme chemistry will be presented to families and classes throughout the area. Giveaways will be combined with assessments to improve retention and obtain feedback for improvements. Similar presentations incorporating experimental results will also be made available in an accessible online format to reach a larger audience. The ultimate goals of this proposal are to improve overall understanding of microbial enzyme chemistry and encourage children to pursue future careers in microbiology and/or biochemistry.