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.
