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

产甲烷菌和其他古细菌中一碳和二碳代谢界面的独特生物化学

• 批准号: 0923766
• 负责人: David Grahame
• 金额: $ 61.34万
• 依托单位: Henry M Jackson Fdn for Advmt of Military Medicine
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0923766&HistoricalAwards=false
• 关键词: Unique; Biochemistry; Interface; Two; Carbon

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.

该项目正在提供一种独特的多酶复合体--乙酰辅酶A脱羧酶/合成酶复合体(ACDS)--如何在产甲烷菌和古生菌其他物种中发挥作用的生化理解。ACDS络合物催化了一种不寻常的、产生能量的、基于金属的脱碳反应，在该反应中，乙酰辅酶A中乙酰基的C-C和C-S键都被打断或形成。ACDs在产甲烷菌将醋酸盐分解为二氧化碳和甲烷的过程中发挥着核心作用，地球上每年约三分之二的甲烷生产来自这一过程。在消耗二氧化碳和氢的产甲烷菌中，ACDs相反地作用于合成乙酰基，以便在生长过程中碳同化。因此，ACDs在全球碳循环中扮演着重要的角色，既可以释放二氧化碳和甲烷形式的碳，也可以参与二氧化碳的自养固定。乙酰基的合成和裂解发生在与蛋白质β亚基结合的含Ni-Ni和Fe/S的活性中心(A簇)。人们对了解该反应的不寻常的生物无机机理很感兴趣，本项目重点分析了一种新分离的在A簇处形成的乙酰镍有机金属中间体的结构、光谱和生化性质，该中间体对乙酰辅酶A的合成和切割机理至关重要。正在对乙酰化形式的ACDSβ亚基进行合作X射线结晶学分析，以提供结构信息，这些信息将有助于对酶如何能够利用不寻常的有机金属化学来实现一碳和两碳底物的相互转化提供新的见解。正在对ACDSβ亚基酶-乙酰中间体进行补充生化和FTIR光谱分析，以提供定义A簇乙酰基-镍物种的反应性和功能性质的信息。此外，该项目正在测试由结构域特定的构象变化所产生的空间效应如何影响A簇乙酰基镍中间体的配位几何和稳定性。对乙酰基C-C和C-S键激活机制的新见解是通过对几个结构域截断和单点定向突变体的鉴定来确定不同的蛋白质构象如何强制特定的金属中心配位几何来促进C-1和C-2中间体的逐步相互转化。本研究涉及组成生命第三域的生物体--古生菌的生化和生理学基础知识。古生菌占海洋微生物总生物量的20%，在反刍动物的消化系统中发现的产甲烷菌数量特别多，在土壤和水环境中也广泛分布。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，更好地了解甲烷生物化学对社会开发替代能源的努力具有影响。因此，这项工作的结果产生了广泛的影响，不仅对于工农业目的有益地开发古生物的代谢潜力，而且对于更好地了解世界的生态和环境也是如此。这一项目的重要教育和教学组成部分进一步扩大了其更广泛的影响。所有级别的学生(从高中到博士后)和技术人员都在积极参与，通过最先进的生化、光谱、酶和分子生物学技术来表征氧敏感的多酶系统，该项目也接纳了代表性不足和处境不利的学生的参与。此外，在这项研究中，建立了新的合作关系，拓宽了几个机构的学生和博士后助理的跨学科知识和技能集。这大大有助于加强研究和教育的基础设施。