Each year microorganisms produce more than 400 million tons of methane gas, which is both a potential source of energy and a greenhouse gas. Methanogens use a complex biochemical pathway that requires eight dedicated enzymes and seven coenzymes to reduce carbon dioxide (or other single carbon compounds) to produce methane gas. The terminal methyl carrying coenzyme in this pathway is coenzyme M (CoM; 2-mercaptoethanesulfonic acid). This project examines how a biosynthetic pathway evolved to produce CoM and how genes recruited from this pathway function in some non-methanogenic microbes. Because the enzymes for CoM biosynthesis are dissimilar to previously characterized proteins, tertiary structural models are required to identify their provenance. Genes encoding phosphosulfolactate phosphatase and sulfopyruvate decarboxylase from the hydrothermal vent-dwelling methanogen, Methanococcus jannaschii, will be heterologously expressed in Escherichia coli to produce soluble fusion proteins. Protein crystals will be formed from chromatographically purified enzyme for X-ray diffraction and structural analysis. Structural models of phosphosulfolactate phosphatase will establish the relationship between this Mg(II)-dependent acid phosphatase and well-characterized analogous phosphatases. A model of sulfopyruvate decarboxylase will test that enzyme's proposed relationship to pyruvate decarboxylase. Models of both enzymes will be used to infer their mechanisms of sulfonate substrate binding and to guide the design of highly specific inhibitors of CoM biosynthesis. Although the spore-forming soil bacterium Bacillus subtilis produces neither methane nor CoM, it has homologs of the first three genes required for CoM biosynthesis. Heterologously expressed proteins will be tested as catalysts of sulfolactate biosynthesis during sporulation. Combined with chromatographic analysis of sulfolactate production by mutants of B. subtilis missing these genes, these results will link the function of three previously unidentified genes to sulfolactate biosynthesis. Together, results from studies of the methanogen and bacterial proteins will clarify the evolutionary origins of CoM biosynthesis.
Broader impacts: Understanding the methanogenic process is important to control anthropogenic methane formation associated with agriculture and waste processing. CoM is essential for methane production from all known carbon substrates and is evolutionarily fundamental to methanogenesis. Therefore inhibitors of CoM biosynthesis will be specific tools to control methanogenesis. Knowledge of the structure and function of CoM biosynthetic enzymes will establish the evolutionary and mechanistic relationship of those proteins to previously characterized enzymes, revealing the catalytic potential of these protein families. This research will provide hands-on molecular biology, enzymology, and molecular evolution training for undergraduate and graduate students. The relevant academic programs in biochemistry, microbiology, cell and molecular biology at UT-Austin attract a geographically diverse group of students with a high level of participation of under-represented groups.
