Unique Biochemistry at the Interface of One- and Two-Carbon Metabolism in Methanogens and other Archaea

该项目提供了一个独特的多酶复合物，乙酰辅酶A脱羰酶/合酶（ACDS）复合物，在产甲烷菌和其他物种的生物化学的理解。 ACDS复合物催化一种不寻常的能量产生、基于金属的脱羰基反应，其中乙酰辅酶A中乙酰基的C-C和C-S键都断裂或形成。 ACDS在产甲烷菌将乙酸盐分解为CO2和甲烷的过程中发挥着核心作用，地球上每年约有三分之二的甲烷产量来自这一过程。 在消耗CO2和氢气的产甲烷菌中，ACDS在生长过程中反过来合成乙酰基用于碳同化。 因此，ACDS在全球碳循环中发挥着重要作用，以CO2和甲烷的形式释放碳，并参与CO2的自养固定。 乙酰基合成和裂解发生在与蛋白质β亚基结合的活性位点Ni-Ni-和Fe/S-含金属中心（A簇）处。 有相当大的兴趣在了解这个反应的不寻常的生物无机机制，这个项目的重点是分析的结构，光谱和生化性质的一个新分离的乙酰镍有机金属中间体形成的A簇，关键的乙酰辅酶A的合成和裂解的机制。 协作X射线晶体学分析正在进行的ACDS β亚基在其乙酰化的形式，以提供结构信息，将有助于新的洞察酶是如何能够利用不寻常的有机金属化学的相互转换的一个和两个碳底物。 互补的生物化学和红外光谱分析正在应用于ACDS β亚基酶-乙酰基中间体，以提供定义A簇乙酰镍物质的反应性和功能特性的信息。 此外，该项目正在测试由特定结构域构象变化产生的空间效应如何影响A簇乙酰镍中间体的配位几何形状和稳定性。 对乙酰基C-C和C-S键激活机制的新认识是从几个结构域截短和单位点定向突变体的表征中获得的，以确定不同的蛋白质构象状态如何执行特定的金属中心配位几何形状，以促进C-1和C-2中间体的逐步相互转化。更广泛的影响这项研究涉及到生物化学和生理学的基础知识，构成生命第三领域的生物体。 产甲烷菌占海洋微生物总生物量的20%，产甲烷菌在反刍动物的消化系统中数量特别多，并广泛分布于土壤和水生环境中。 产甲烷菌在用于缺氧废物的生物修复和解毒方面对社会有直接的好处。 此外，作为产甲烷代谢的最终产物产生的甲烷是一种清洁燃料，并且对甲烷生物化学的更好的基本理解对开发替代能源的社会努力具有影响。 因此，这项工作的结果产生了广泛的影响，不仅对工业和农业目的有用地利用了珊瑚的代谢潜力，而且对更好地了解世界生态和环境也有影响。 该项目的重要教育和教学部分进一步扩大了其更广泛的影响。 各级学生（高中到博士后）和技术人员积极参与通过最先进的生物化学，光谱，酶和分子生物学技术表征氧敏感的多酶系统，该项目也包括代表性不足和弱势学生的参与。 此外，在这项研究中，已经建立了新的合作，扩大了几个机构的学生和博士后的跨学科知识和技能。 这大大有助于加强研究和教育的基础设施。

This project is providing biochemical understanding of how a unique multienzyme complex, the acetyl-CoA decarbonylase/synthase (ACDS) complex, functions in methanogens and other species of Archaea. The ACDS complex catalyzes an unusual energy-yielding, metal-based decarbonylation reaction in which both the C-C and C-S bonds of the acetyl group in acetyl-CoA are broken, or formed. ACDS plays a central role in acetate decomposition to CO2 and methane by methanogens, with approximately two-thirds of the annual methane production on Earth being derived from this process. In methanogens consuming CO2 and hydrogen, ACDS acts in reverse to synthesize acetyl groups for carbon assimilation during growth. Thus, ACDS plays an important role in the global carbon cycle both in the release of carbon in the form of CO2 and methane, and by its involvement in the autotrophic fixation of CO2. Acetyl group synthesis and cleavage take place at an active site Ni-Ni- and Fe/S-containing metal center (the A cluster) bound to the protein beta subunit. There is considerable interest in understanding the unusual bioinorganic mechanism of this reaction, and this project is focused on analysis of the structure, spectroscopic, and biochemical properties of a newly isolated acetyl-nickel organometallic intermediate formed at the A cluster, critical to the mechanism of acetyl-CoA synthesis and cleavage. Collaborative X-ray crystallographic analyses are being carried out on the ACDS beta subunit in its acetylated form to provide structural information that will contribute new insight into how enzymes are able to exploit unusual organometallic chemistry for interconversion of one- and two-carbon substrates. Complementary biochemical and FTIR spectroscopic analyses are being applied to the ACDS beta subunit enzyme-acetyl intermediate to provide information defining the reactivity and functional properties of the A cluster acetyl-Ni species. In addition, this project is testing how steric effects generated by domain-specific conformational changes influence the coordination geometry and stability of the A cluster acetyl-Ni intermediate. New insight into the mechanism of acetyl C-C and C-S bond activation is being obtained from characterization of several domain-truncated and single site-directed mutants to determine how different protein conformational states enforce specific metal center coordination geometries to promote the stepwise interconversion of C-1 and C-2 intermediates.Broader Impacts This research is concerned with fundamental knowledge of the biochemistry and physiology of Archaea, organisms that constitute the third-domain of life. Archaea comprise as much as 20% of the total microbial biomass in the oceans and methanogens are found in especially high numbers in the digestive systems of ruminants, as well as being widely distributed in soil and aquatic environments. Methanogens present a direct advantage to society in their use for bioremediation and detoxification of anoxic wastes. In addition, methane produced as the end product of methanogenic metabolism is a clean fuel, and a better fundamental understanding of methane biochemistry has impact on societal efforts to develop alternative energy sources. Thus, the results from this work have a broad impact, not only for the useful exploitation of the metabolic potential of the Archaea for industrial and agricultural purposes, but also for a better understanding of the world's ecology and the environment. Important educational and teaching components of this project further extend its broader impact. Students at all levels (high school through postdoctoral) and technologists are actively engaged in characterizing oxygen-sensitive multi¬enzyme systems by state-of-the-art biochemical, spectroscopic, enzymatic, and molecular biological techniques, and the project embraces participation of underrepresented and disadvantaged students as well. Furthermore, in this research, new collaborations have been established that are broadening the interdisciplinary knowledge and skill sets of students and postdoctoral associates at several institutions. This contributes significantly to enhancement of infrastructure for research and education.