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

9304637 Grahame该项目将获得确定多酶复合物的四级结构所需的信息，该复合物催化产甲烷细菌Methanosarcina barkeri中乙酸盐的代谢。 提出的结构研究是基本的，以实现详细的了解的机制，多酶复合物催化裂解的乙酰辅酶A乙酸碳-碳键。 本研究的具体目标如下。 1)通过动态光散射、电子显微镜、速率区带超离心和凝胶过滤等物理方法确定蛋白质复合物的分子量、亚基结构和整体对称性。 2)鉴定在乙酰辅酶A裂解过程中特异性结合用于甲基转移的类咕啉（维生素B12样）辅因子的亚基。 将使用破坏性最小的方法，然后进行色谱法，以获得结合类咕啉辅因子的单个亚基或蛋白质组分。3)鉴定结合四氢沙那蝶呤的成分。 该组分将从破坏的蛋白质复合物的色谱级分中纯化，并鉴定为催化甲基-类咕啉和四氢-sarcinapterin之间甲基转移的酶。4)从完整复合物的化学交联实验中获得关于亚基类型之间最近邻关系的额外信息。 研究的一个主要成果将是开发一个复杂的三维模型。 整体结构将显示类咕啉和四氢-sarcinapterin-结合亚基彼此之间以及与复合物的所有其他亚基之间的相对位置接近。 在自然界中，涉及乙酸生产和消耗的微生物转化过程是非常重要的生物反应。 据估计，在自然界中每年产生1010公吨乙酸。 乙酸随后转化为气态产物如二氧化碳和甲烷发生在自然生态系统和由人类创建的用于废物处理和生物质转化的系统中。 产甲烷八叠球菌在乙酸转化为甲烷和二氧化碳的过程中起着重要的作用。 本研究旨在了解M.巴克里在分子水平上工作。 物理、生物化学和免疫学方法用于确定蛋白质复合物结构中蛋白质亚基和某些有机辅因子结合位点的排列。 从这项研究提供的结构细节将提供所需的基本信息的乙酸裂解的分子机制的完整理解。 ***

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. ***