Molecular Structure of the Multienzyme Complex Responsible for Acetate Cleavage in Methanosarcina barkeri

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9304637 Grahame This project will obtain information needed to define the quaternary structure of a multienzyme complex that catalyzes metabolism of acetate in Methanosarcina barkeri, a methanogenic bacterium . The structural study proposed is fundamental to achieve a detailed understanding of the mechanisms by which the multienzyme complex catalyzes cleavage of the acetyl-CoA acetate carbon-carbon bond. The specific objectives of this study follow. 1) Establish the molecular mass, subunit structure, and overall symmetry of the protein complex by physical methods of dynamic light scattering, electron microscopy, rate zonal ultracentrifugation and gel filtration. 2) Identify the subunit that specifically binds the corrinoid (vitamin B12-like) cofactor used for methyl-group transfer during acetyl-CoA cleavage. Minimally disruptive methods will be used, followed by chromatography, to obtain the single subunit, or protein component, that binds the corrinoid cofactor. 3) Identify the component that binds tetrahydro-sarcinapterin. This component will be purified from chromatographic fractions of the disrupted protein complex and identified as the enzyme that catalyzes methyl group transfer between methyl-corrinoid and tetrahydro-sarcinapterin. 4) Obtain additional information about nearest-neighbor relationships among subunit types from chemical cross-linking experiments on the intact complex. A major outcome of the research will be to develop a three-dimensional model of the complex. The overall structure will display the corrinoid and the tetrahydro-sarcinapterin-binding subunits placed in approximate relative position to one another and to all other subunits of the complex. %%% Microbial conversion processes involving both the production and the consumption of acetic acid in nature are biological reactions of major importance. It is estimated that in nature 1010 metric tons of acetic acid are produced per year. Subsequent conversion of acetic acid to gase ous products as carbon dioxide and methane occurs in both natural ecosystems and in systems created by man for waste treatment and biomass conversion. In conversion of acetic acid to methane and carbon dioxide, the methanogenic bacterium, Methanosarcina barkeri is very important. This research is designed to learn how a multienzyme structure responsible for acetic acid metabolism in M. barkeri works at the molecular level. Physical, biochemical, and immunological methods are used to determine the arrangement of protein subunits and certain organic cofactor binding sites within the architecture of the protein complex. The structural details provided from this research will provide fundamental information needed for a complete understanding of the molecular mechanism of acetate cleavage. ***