White 9311538 In this project, the biosynthetic pathway(s) for the methanogenic coenzymes 7-mercaptoheptanylthreonine-phosphate (HS-HTP) and methyl reducing cofactor (MRF) will be established. The first phase of work will be to establish the presence of HS-HTP and MRF in different species of methanogenic archaebacteria and to determine any possible precursor/product relationship between these coenzymes. The presence and interconversion of the molecules will be determined by fluorescence measurement of their monobromobimane derivatives using procedures recently developed by the author. The incorporation of individual subunit and groups of subunit molecules into HS-HTP and MRF will be determined. Both in vivo and in vitro experiments, using stable isotopically-labeled molecules and mass spectrometry to measure incorporation, will be performed to help define the biosynthetic pathways. %%% The biochemistry of the metabolic reduction of carbon dioxide to methane by the methanogenic archaebacteria represents an exciting area of microbiological metabolism. One important aspect of this metabolism is the discovery of seven new coenzymes. Many of the coenzymes in the methane pathway have chemical structures completely different from other well known coenzymes and vitamins. Because of this each structure represents a new biological compound with unusual functions to be discovered. In this award, the biosynthesis, structure, and to a limited extent, the distribution of two cofactors, 7-mercaptoheptanylthreonine-phosphate and methyl reducing cofactor, will be explored. Specific questions to be addressed are; how are each of these cofactors biosynthesized and assembled, and are they biosynthetically interrelated? Answers to these questions may complete another chapter in our understanding of the biosynthesis of the methanogenic coenzymes, and also shed more light on the microbial biochemistry of the formation of methane, an important energy source. ***
