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

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Abstract 9630488 Grahame In methanogens, the central reaction in the pathway of acetate degradation is carried out by a unique multienzyme complex (designated ACDS for acetyl-CoA decarbonylase/synthase) that brings about cleavage of the C-S and C-C bonds of the acetyl group of acetyl-CoA. The metabolic function of this key enzyme is based on inorganic, metal-based chemistry that is highly unusual among most organisms and the enzymes they contain. Despite the global importance of this enzyme complex, the reaction it catalyzes is still poorly understood, very little is known about its 3-dimensional structure, and almost nothing is known about the interactions between various subunits and coenzyme components within the complex. The long range goal of this project is to obtain a detailed understanding at the molecular level of the enzymatic and regulatory mechanisms that operate in the metabolic pathway of acetate cleavage in methanogens. The objective of this research is to provide a solid foundation for understanding the structure and function of the M. barkeri ACDS complex -- the centerpiece of this pathway. In order to accomplish this three specific aims will be pursued, as follows: (1) a thorough determination will be made of the physicochemical properties of the ACDS subcomponent proteins (2) a detailed study will be carried out on the kinetics of acetyltransferase (a critical partial reaction in the overall process of acetyl-CoA cleavage), and (3) a three-dimensional model of the complex will be obtained showing the location of all subunits and domains involved in coenzyme, cofactor, and substrate binding. At the completion of this project the overall structure of the enzyme complex will be at hand. In addition, a determination will be made of the chemical reactivity of the critical acetyl-enzyme intermediate -- the actual, but yet uncharacterized species that undergoes C-C bond cleavage. %%% Species of methane-forming archaebacteria, such as Methanosarcinae, are extremely imp ortant, terminal components in anaerobic microbial decomposition (major global examples include ruminant digestion, decay in virtually all freshwater environments, and various human uses such as biomass conversion processes and waste treatment). In our society, methane poses potential problems as a greenhouse gas, but at the same time provides an extremely useful clean-burning fuel. In contrast, nature has selected methane (which is inert to further anaerobic reaction) to serve as the ultimate repository for metabolically-generated reducing power in anaerobic environments. Although methanogens are able to utilize a variety of one-carbon compounds, the major biological pathway for production of methane is via cleavage of the two-carbon substrate, acetate, by overall fermentation as follows: CH3COOH(CH4 + CO2. Nevertheless, only a rather limited number of species of methanogens are capable of growth on acetate as a sole source for carbon and energy. This research project will directly contribute to the understanding of anaerobic metabolism of acetate in nature. Furthermore, the project will have substantial impact on progress in broadly related areas such as the biochemistry of metalloenzymes, the regulatory significance of redox states and equilibria of enzyme substrates and cofactors, the chemistry of unusual carbon-carbon bond rearrangements and carbonyl insertion reactions in biological systems, the structure and regulation of multienzyme complexes in general, and the physiology of energy transduction in the methanogenic Archaea and other organisms living in extreme or unusual environments. ***