EAGER: Coupling Electron Transport and Metabolism using Biological Routers

产甲烷菌几乎存在于所有厌氧栖息地，是全球碳循环的关键参与者，每年产生10亿吨甲烷气体。产甲烷菌通过将低氧沉积物中的碳转化为甲烷气体而生存，然后在有氧环境中氧化为二氧化碳，从而在全球碳循环中发挥关键作用。最近的初步证据支持参与一个大的多酶复合物在生物甲烷生产和解决不明原因的突变体表型在文献中报道。多酶氧化还原复合物很重要，因为细胞必须协调氧化与还原电子载体的相对比例与参与中心代谢的酶的催化要求。该项目旨在阐明这种新型多酶复合物协调产甲烷菌代谢过程的基本生化原理。定义这种多酶复合物的功能机制也将有助于我们理解与其他古细菌，细菌或真核生物中的多酶氧化还原复合物相关的基本生物化学原理。该项目将支持学生在厌氧微生物生理学和生物物理生物化学方面的培训。 多酶复合物由三种酶组成，CoM-S-S-CoB杂二硫还原酶（HdrD）、乙酰辅酶A脱羰基酶/合酶（ACDS）和亚甲基四氢甲蝶呤还原酶（Mer）。 该奖项支持的研究将使用遗传学，生物物理生物化学和分子生物学技术来定义HdrD：ACDS：Mer复合物通过电子传递和代谢整合通量的机制。HdrD：ACDS：Mer多酶复合物根据细胞中电子载体的比率和氧化态将碳引导至生物合成或甲烷生成。该研究的具体目的包括通过体内交联、亲和纯化和所有组分亚基的质谱分析来验证HdrD：ACDS：Mer复合物的形成。还将进行实验以研究体内酶之间的氧化还原依赖性串扰。将通过UV/维斯分光光度法，在突变体细胞提取物中HdrD：ACDS：Mer复合物催化的偶联反应中测定乙酰辅酶A的产生和铁氧还蛋白、F420和CoM-S-S-CoB电子载体的甲基-四氢甲烷蝶呤依赖性还原。研究结果将通过在科学会议上的介绍和同行评审的出版物传播。

Methanogens, which are found in nearly all anaerobic habitats, are key players in the global carbon cycle, producing 1 gigaton of methane gas annually. Methanogens survive by converting carbon in low-oxygen sediments to methane gas, which is then oxidized to carbon-dioxide in the aerobic environment, thereby playing a key role in the global carbon cycle. Recent preliminary evidence supports the involvement of a large multienzyme complex in biological methane production and resolves unexplained mutant phenotypes reported in the literature. Multienzyme redox complexes are important because cells must coordinate the relative ratio of oxidized vs reduced electron carriers with the catalytic requirements of enzymes involved in central metabolism. This project seeks to elucidate the basic biochemical principles by which this novel multi-enzyme complex coordinates metabolic processes in methanogens. Defining the mechanism by which this multi-enzyme complex functions will also help us understand the basic underlying biochemical principles related to multienzyme redox complexes in other archaea, bacteria, or eukaryotes. The project will support student training in anaerobic microbial physiology and biophysical biochemistry. The multi-enzyme complex is comprised of three enzymes, CoM-S-S-CoB heterodisulfide reductase (HdrD), acetyl-CoA decarbonylase/synthase (ACDS), and methylene tetrahydromethanopterin reductase (Mer). The studies supported by this award will use genetics, biophysical biochemistry, and molecular biology techniques to define the mechanism by which the HdrD:ACDS:Mer complex integrates flux through electron transport and metabolism. The HdrD:ACDS:Mer multienzyme complex directs carbon to biosynthesis or methanogenesis dependent on the ratios and oxidation state of electron carriers in the cell. Specific aims for the study include verifying HdrD:ACDS:Mer complex formation by in vivo crosslinking, affinity purification, and mass spectrometry of all component subunits. Experiments will also be conducted to investigate redox-dependent crosstalk between enzymes in vivo. Acetyl-CoA production and methyl-tetrahydromethanopterin-dependent reduction of ferredoxin, F420, and CoM-S-S-CoB electron carriers will be measured in coupled reactions catalyzed by the HdrD:ACDS:Mer complex in mutant cell extracts by UV/Vis spectrophotometry. Results of the study will be disseminated through presentations at scientific conferences and through peer-reviewed publications.