The goal of this project is to define the biochemistry of the anaerobic O-demethylation (AOD) reaction in Acetobacterium woodii, a homoactogenic bacterium. Initial studies will be undertaken to identify the product(s) of the reaction. To accomplish this, demethylation enzyme(s) will be partially purified to minimize interference by other competing cellular reactions that may act to modify the test substrate and/or transform reaction product(s), thereby obscuring the chemistry of the demethylation reaction itself. Currently, little is understood about A. woodii AOD, except that O-demethylation is stimulated by exogenous tetrahydrofolate (THF) and by ATP. To begin the understand the chemistry of the reaction, we will first identify reaction product(s) formed in the presence of THF and determine if other low-potential reductants also serve to stimulate the reaction. Our studies will continue on to determine the role played by ATP in O- demethylation. If time permits, and if it appears warranted, we will attempt to purify O-demetylation pathway proteins to homogeneity. To begin to learn about the regulation of the pathway, we will also initate studies to screen for transposon- generated AOD regulatory mutants cells. %%% Aromatic compounds containing O-methyl ethers are abundant in foods and wood processing wastes. The first step in biodegradation of these compounds is removal of their methyl group by the O- demethylation enzyme. The reaction has already been studied in aerobic organisms where it uses oxygen as a reactant. The mechanism of action of the analogous enzyme is not understood in anaerobic bacteria, where oxygen is not used. It is important that we understand how this anaerobic reaction also operates, because a major part of the earth's biosphere is anaerobic. For example, important anaerobic environments include the digestive tracts of animals, fresh water and marine sediments, and man-made anaerobic digestors. In our research program, we plan to determine how the mechanism of anaerobic O-demethylation (AOD) operates in Acetobacterium woodii, a common anaerobic bacterium. The results gained from our study will contribute to understanding the natural anaerobic process. In addition, we may discover ways to engineer the O-demethylation reaction in these anaerobic bacteria, and then use it commercially for the conversion of renewable biomass waste material and for the production of industrially useful products.
