Energy conservation in methanogenic archaea associated with the human gut

人类微生物组相关甲烷古菌可能参与疾病和健康，需要进一步研究以了解其生理和代谢。因此，本提案的主要目的是阐明在人类肠道中发现的产甲烷生物体中的能量守恒机制，所述产甲烷生物体专门使用H2和甲醇或甲胺作为底物。实验将基于模式生物甲烷微球菌（M.）鲁米尼人这种生物体不符合目前的甲烷菌如何从其底物中获得能量的方案。M. Luminyensis含有典型的氢化酶/异二硫化物氧化还原酶复合物（HdrABC/MvhADG），如在氢营养产甲烷菌中发现的，但不存在能量保存的甲基-H4 MPT辅酶M甲基转移酶复合物。所以问题就来了，能量在这个有机体中是如何守恒的。在之前的资助期间，我们可以证明可溶性HdrABC/MvhADG复合物在M.鲁米尼人在反应过程中，铁氧还蛋白起电子受体的作用。此外，我们可以证明，在重组HdrD存在下，经洗涤的细胞质膜使用还原的铁氧还蛋白（Fdred）来还原异二硫化物CoM-S-S-CoB。我们目前的假设是，Fdred被F420 H2脱氢酶（无头Fpo复合物）的截短形式再氧化，该形式缺失F420 H2氧化亚基FpoF。因此，电子被转移到HdrD，HdrD直接与无头Fpo复合物相互作用。反过来，HdrD催化CoM-S-S-CoB的还原。整个过程可以耦合到离子易位（H+或Na+）和生成的电化学离子梯度的ATP合成通过A1 A0型ATP合酶。为了验证这一假设，计划分析HdrD和Fdred与无头Fpo复合物的相互作用。该方法包括Fpo复合物或Fpo亚复合物的部分纯化以重建厌氧电子传递链。蛋白质：蛋白质相互作用将被分析，以阐明电子的路线，并确定亚基，参与电子转移从Fdred到CoM-S-S-CoB作为最终的电子受体的建议的新的节能电子传递链在M。鲁米尼人

The possible involvement of human microbiome-associated methanoarchaea in disease and health warrants further investigation to understand their physiology and metabolism. Therefore, the major aim of this proposal is to elucidate the energy conservation mechanism in methanogenic organisms found in the human gut which exclusively use H2 and methanol or methylamines as substrates. The experiments will be based on the model organism Methanomassiliicoccus (M.) luminyensis. This organism does not fit into the current scheme of how methanogens gain energy from their substrates. M. luminyensis contains the typical hydrogenase/ heterodisulfide oxidoreductase complex (HdrABC/MvhADG) as found in hydrogenotrophic methanogens but the energy conserving methyl-H4MPT coenzyme M methyltransferase complex is absent. So the question arises how energy is conserved in this organism. In the previous funding period we could show that the soluble HdrABC/MvhADG complex was highly active in the cytoplasmic compartment of M. luminyensis. In the course of the reaction ferredoxin functions as electron acceptor. Furthermore, we could show that reduced ferredoxin (Fdred) is used by washed cytoplasmic membranes to reduce the heterodisulfide CoM-S-S-CoB in the presence of recombinant HdrD. Our current hypothesis is that Fdred is reoxidized by a truncated form of the F420H2 dehydrogenase (headless Fpo complex) which misses the F420H2-oxidizing subunit FpoF. Accordingly, electrons are transferred to HdrD that directly interacts with the headless Fpo complex. In turn, HdrD catalyzes the reduction of CoM-S-S-CoB. The overall process could be coupled to ion translocation (H+ or Na+) and the generation of an electrochemical ion gradient for the synthesis of ATP via a A1A0 type ATP synthase. To verify this hypothesis, it is planned to analyze the interaction of HdrD and Fdred with the headless Fpo complex. The approach includes the partial purification of the Fpo complex or Fpo subcomplexes to reconstitute the anaerobic electron transport chain. Also protein: protein interaction will be analyzed to elucidate the route of electrons and to identify the subunit(s) which are involved in the electron transfer from Fdred to CoM-S-S-CoB as final electron acceptor of the proposed novel energy-conserving electron transport chain in M. luminyensis.