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

在甲烷菌中，醋酸酯降解途径的中心反应是由一种独特的多酶复合物（ACDS，即acetyl- coa脱碳酶/合成酶）进行的，该复合物可裂解乙酰辅酶a乙酰基的C-S和C-C键。这种关键酶的代谢功能是基于无机的金属化学，这在大多数生物体和它们所含的酶中是非常不寻常的。尽管这种酶复合物在全球具有重要意义，但人们对其催化的反应仍然知之甚少，对其三维结构知之甚少，对复合物内各种亚基和辅酶组分之间的相互作用几乎一无所知。该项目的长期目标是在分子水平上详细了解产甲烷菌醋酸裂解代谢途径中的酶和调控机制。本研究的目的是为理解M. barkeri ACDS复合体的结构和功能提供坚实的基础，这是该途径的核心。为了实现这一目标，将追求以下三个具体目标：(1)将彻底确定ACDS亚组分蛋白的物理化学性质(2)将对乙酰转移酶（乙酰辅酶a切割整个过程中的关键部分反应）的动力学进行详细研究，(3)将获得该复合物的三维模型，显示参与辅酶、辅因子和底物结合的所有亚基和结构域的位置。在这个项目完成后，酶复合物的整体结构将会得到。此外，将测定关键乙酰酶中间体的化学反应性——实际的，但尚未表征的经历C-C键切割的物质。产生甲烷的古细菌种类，如Methanosarcinae，是厌氧微生物分解中极其重要的终端成分（全球主要的例子包括反刍动物消化，几乎所有淡水环境中的腐烂，以及各种人类用途，如生物质转化过程和废物处理）。在我们的社会中，甲烷作为一种温室气体带来了潜在的问题，但同时也提供了一种非常有用的清洁燃烧燃料。相反，大自然选择了甲烷（它对进一步的厌氧反应是惰性的）作为厌氧环境中代谢产生的还原力的最终储存库。虽然产甲烷菌能够利用多种单碳化合物，但生产甲烷的主要生物途径是通过全面发酵裂解二碳底物醋酸酯：CH3COOH（CH4 + CO2）。然而，只有相当有限的几种产甲烷菌能够以醋酸盐作为碳和能量的唯一来源生长。该研究项目将直接有助于了解自然界中醋酸盐的厌氧代谢。此外，该项目将对广泛相关领域的进展产生重大影响，如金属酶的生物化学，酶底物和辅因子的氧化还原状态和平衡的调节意义，生物系统中不寻常的碳-碳键重排和羰基插入反应的化学，多酶复合物的结构和调节。以及产甲烷古生菌和其他生活在极端或不寻常环境中的生物的能量转导生理学。＊＊＊

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. ***