Panwar Alka, Martins Berta M, Sommer Frederik, Schroda Michael, Dobbek Holger, Iobbi-Nivol Chantal, Jourlin-Castelli Cécile, Leimkühler Silke
Department of Molecular Enzymology, Institute of Biochemistry and Biology, University of Potsdam, Karl-Liebknecht Str. 24-25, 14476 Potsdam, Germany.
Department of Biology, Humboldt-Universität zu Berlin, Unter den Linden, 10999 Berlin, Germany.
Int J Mol Sci. 2024 Dec 12;25(24):13331. doi: 10.3390/ijms252413331.
The enterobacterium present in the human gut can reduce trimethylamine N-oxide (TMAO) to trimethylamine during anaerobic respiration. The TMAO reductase TorA is a monomeric, bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor-containing enzyme that belongs to the dimethyl sulfoxide reductase family of molybdoenzymes. TorA is anchored to the membrane via TorC, a pentahemic -type cytochrome which receives the electrons from the menaquinol pool. Here, we designed an expression system for the production of a stable soluble form of multiheme-containing TorC, providing, for the first time, the purification of a soluble pentahemic cytochrome- from . Our focus was to investigate the interaction between TorA and soluble TorC to establish the electron transfer pathway. We solved the X-ray structure of TorA and performed chemical crosslinking of TorA and TorC. Another goal was to establish an activity assay that used the physiological electron transfer pathway instead of the commonly used unphysiological electron donors methylviologen or benzylviologen. An AlphaFold model including the crosslinking sites provided insights into the electron transfer between TorC and the active site of TorA.
存在于人体肠道中的肠杆菌在无氧呼吸过程中可将氧化三甲胺（TMAO）还原为三甲胺。TMAO还原酶TorA是一种单体酶，含有双钼蝶呤鸟嘌呤二核苷酸（bis-MGD）辅因子，属于含钼酶的二甲基亚砜还原酶家族。TorA通过TorC锚定在膜上，TorC是一种五血红素型细胞色素，从甲萘醌池接收电子。在此，我们设计了一种表达系统，用于生产稳定的可溶性多血红素TorC形式，首次从[具体来源未给出]中纯化出可溶性五血红素细胞色素。我们的重点是研究TorA与可溶性TorC之间的相互作用，以建立电子传递途径。我们解析了TorA的X射线结构，并对TorA和TorC进行了化学交联。另一个目标是建立一种活性测定方法，该方法使用生理电子传递途径，而不是常用的非生理电子供体甲基紫精或苄基紫精。包含交联位点的AlphaFold模型为TorC与TorA活性位点之间的电子传递提供了见解。
