Clostridium thermoaceticum and other acetogenic bacteria produce acetate as the sole product of growth on H2/CO2, CO, or organic substrates such as glucose. Acetate is formed via acetyl- CoA, which is the precursor for all the anabolic reactions in the acetogens. The acetyl-CoA pathway is a novel pathway of CO2 fixation in that the pathway is unidirectional (not cyclic) and the key intermediates (CH3-cobalt and nickel-iron-CO) are organometallic species which are enzyme-bound. Three enzymes (methyltransferase, a corrinoid protein, and CO dehydrogenase) are required to transfer the methyl group of methyl-THF to form the C-2 of acetyl-CoA. This proposal is concerned primarily with the mechanisms of methyltransferase (MeTr) and the corrinoid protein. These enzymes are involved in portions of the pathway which have not been studied in detail. The acetogenic methyl- THF:corrinoid protein MeTr is unique in that it does not contain a corrinoid and it catalyzes an intermolecular transfer of the methyl group of methyl-THF to the corrinoid of the corrinoid protein. This methyl is then transferred from the methylcorrinoid to CO dehydrogenase (CODH), and finally CODH condenses the methyl group with CO and CoA to form acetyl-CoA. The goal of the proposed research is to determine the detailed mechanisms by which MeTr and the corrinoid protein catalyze these methyl transfer reactions. (1) Kinetic and binding studies will be used to understand why the reaction of MeTr is irreversible with the corrinoid protein as substrate and reversible with B12. The mechanism and the stereochemistry of the transfer of the methyl group from methyl-THF to the corrinoid protein will be determined. (2) The primary structure of the corrinoid protein will be determined by cloning and sequencing the genes coding for the proteins. Preliminary X-ray analysis of the corrinoid protein will be performed. (3) The corrinoid protein is the first example of an iron-sulfur-containing corrinoid protein. The redox chemistry of the metal centers in the corrinoid protein will be determined by spectroelectrochemistry and by UV-visible, Mossbauer, and ESR spectroscopies. Transfer of the methyl of the methylcorrinoid to CODH will be studied by stereochemical analysis and by 13C-NMR, ESR, and Mossbauer spectroscopies.
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