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

职业：微生物生物合成途径中酶促甲基转移的机制研究

• 批准号: 0953721
• 负责人: Susan Wang
• 金额: $ 60万
• 依托单位: Washington State University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-02-01 至 2016-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0953721&HistoricalAwards=false
• 关键词: CAREER; Mechanistic; Investigations; Enzymatic; Methyl

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.

从微生物中分离出来的酶在日常生活中发挥着重要的作用，从藻类的光合作用和产氧到乳杆菌和乳球菌的酸奶形成。拟议的研究将调查一种独特的、鲜为人知的机制，即链霉菌中酶所使用的非活性碳和磷原子的甲基化。链霉菌是一大类有益的细菌，天然地产生许多商业上使用的抗生素、抗癌和除草化学品。到目前为止，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能被亲核激活，这表明需要和使用另一种催化机制。将使用生化、光谱和合成有机技术来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-蛋氨酸和维生素B12的衍生物甲钴胺生成有机自由基。了解这些反应的机理将为设计这些酶和相关酶以产生新的碳-碳和/或碳-磷-碳键提供一个框架。这项研究中研究的酶、辅因子和微生物将与一项针对农村农业帕卢兹地区K-5年级儿童的教育推广计划有关，帕卢兹地区距离最近的大都市地区300英里。与帕劳斯发现科学中心合作，将向该地区的家庭和班级介绍有益微生物和酶化学的互动演示。赠品将与评估相结合，以提高留存率并获得改进反馈。还将以无障碍在线形式提供包含实验结果的类似演示文稿，以接触到更多的受众。这项建议的最终目标是提高对微生物酶化学的全面了解，并鼓励儿童从事未来的微生物学和/或生物化学职业。