Coenzyme B and Acetamidosugar Biosynthesis in Methanogens

排放到地球大气中的甲烷气体大部分是产甲烷微生物产生的。这种甲烷是一种潜在的能源，但也是一种温室气体和农业副产品，因此控制甲烷生成是废物处理和生物燃料研究的优先事项。这些微生物需要辅酶B(COB)来催化最终释放甲烷的反应。因此，抑制COB的生物合成会特别损害产甲烷菌。该项目将使用生物化学、基因组分析和遗传学来鉴定海洋产甲烷菌maripaludis所使用的COB生物合成途径。高乌头酸酶(HACN)是COB硫酰基部分生物合成的关键金属酶，将对其进行纯化和鉴定，以测试其立体化学和底物特异性(目标1)。这是第一个纯化的铁硫水解酶，它既能催化脱水反应，又能催化水合反应，从而生成均一异柠檬酸。实验将测试这种酶是否可以使用制造2-氧琥珀酸所需的所有三个伽玛-羧酸链长度类似物。HACNs还参与另一种赖氨酸生物合成途径，该途径是抗真菌药物开发的靶点。为了解决COB生物合成的后续步骤，将使用放射性标记的半胱氨酸、硫化物、谷氨酸和苏氨酸来追踪maripaludis途径(目标2)。这些研究将确定连接硫醇和苏氨酸基团所需的底物、中间体和辅因子。这些掺入研究的结果将有助于确定COB生物合成和硫代谢中的新酶。一份关于含有尿苷二磷酸二糖头基的延伸CoB结构的报告表明，CoB的形成可能类似于肽聚糖前体的生物合成。为了确定这种二糖在COB中的重要性，将在与乙酰胺糖生物合成有关的三个maripaludis基因上构建缺失突变(目标3)。除了探索COB的生物合成，这些突变体将是未来古细菌生物膜形成和糖基化研究的有价值的工具。这个项目是一个将比较基因组分析与实验方法相结合的模型，以确定具有新反应的复杂生化途径。该项目将培养本科生和研究生设计和执行代谢生物化学实验。这些学生具有不同的智力、种族和地理背景。这项工作还通过韦尔奇暑期学者和种子计划吸引了高中生。高级研究生和本科生通过协助培养应届研究生、本科生和高中生获得实践教学经验。通过结合基因组学、酶学、遗传学、分析化学和合成化学的跨学科领域，这项研究将提供广泛的基础和协作的学习经验。这里开发的方法和试剂将用于发展新生研究计划，向新生和未被充分代表的大学生介绍生物研究。这些实验的结果将被用来为以问题为基础的生物化学讲座课程创造新的问题，该课程定期为140名本科生授课。

Methanogenic microorganisms produce most of the methane gas that is released into Earth's atmosphere. This methane is a potential source of energy, but also a greenhouse gas and an agricultural byproduct, so controlling methanogenesis is a priority for waste processing and biofuel research. These microbes require coenzyme B (CoB) to catalyze the final reaction that releases methane. Therefore inhibiting CoB biosynthesis would specifically impair methanogens. This project will use biochemistry, genomic analysis and genetics to identify the CoB biosynthetic pathway used by the marine methanogen Methanococcus maripaludis. Homoaconitase (HACN), a key metalloenzyme in the biosynthesis of the thioacyl moiety of CoB, will be purified and characterized to test its stereochemistry and substrate specificity (Aim 1). This is the first purified iron-sulfur hydro-lyase that catalyzes both the dehydration and hydration reactions that make homoisocitrate. Experiments will test whether this enzyme can use all three gamma-carboxylate chain length analogs that are needed to make 2-oxosuberate. HACNs also participate in an alternative lysine biosynthesis pathway that is a target for anti-fungal drug development. To resolve the subsequent steps in CoB biosynthesis radiolabeled cysteine, sulfide, glutamate and threonine will be used to trace the M. maripaludis pathway (Aim 2). These studies will identify substrates, intermediates and cofactors required to attach the thiol and threonine groups. Results from these incorporation studies will help identify novel enzymes in CoB biosynthesis and sulfur metabolism. A report of an extended CoB structure containing a uridine diphosphate disaccharide headgroup suggests that CoB formation may resemble peptidoglycan precursor biosynthesis. To determine the significance of this disaccharide in CoB, deletion mutations will be constructed in three M. maripaludis genes implicated in acetamido sugar biosynthesis (Aim 3). Besides probing CoB biosynthesis, these mutants will be valuable tools for future research on biofilm formation and glycosylation in archaea. This project is a model for combining comparative genomic analysis with experimental methods to determine a complex biochemical pathway with novel reactions.Broader impacts. This project will train undergraduate and graduate students to design and execute experiments in metabolic biochemistry. These students have diverse intellectual, ethnic and geographic backgrounds. This work also engages high school students through the Welch Summer Scholars and SEED programs. Advanced graduate and undergraduate researchers gain practical teaching experience by assisting in training new graduate, undergraduate and high school students. By combining interdisciplinary fields of genomics, enzymology, genetics, analytical chemistry and synthetic chemistry, this research will provide a broad based and collaborative learning experience. Methods and reagents developed here will be used to develop freshman research initiatives to introduce new and underrepresented college students to biological research. Results from these experiments will be used to create new questions for a problem-based Biochemistry lecture course that the PI regularly teaches to 140 undergraduate students.