Abstract 1196 Structural and functional characterization of Trimethylamine N-oxide Demethylase

Trimethylamine and its derivative trimethylamine N-oxide are widely present in marine and gut environments. These toxins, introduced from animal-derived food intake, have been linked to various health issues such fish odor syndrome, cardiovascular diseases, and gastrointestinal cancer. The microbiome has evolved into a metabolism that efficiently processes TMA as an energy and a carbon source, which has significant implications for bioenergy conversion and medical detoxification. However, the molecular mechanism of the process of turning over TMA in bacteria remains unclear due to the lack of structural insights. In this study, we obtained high-quality cryo-EM structures of trimethylamine N-oxide demethylase from Paracoccus sp. (paraTDM) in both apo- and substrate-bound conformations at 2.6 and 2.4 Å resolution, respectively. The paraTDM structure contains three distinct domains: the N-terminal, Core, and C-terminal domains. The N-terminal domain is involved in structural stability and assembly, while the C-terminal domain is a tetrahydrofolate-binding protein. The core domain binds to the substrate and to two metal ions, Zinc and Iron. The structures revealed that paraTDM adopts 2 + 21/2 complexation. The two half-domains of the complex contain only the C-terminal domain. Further biophysical and biochemical assays showed that paraTDM triggers breaking carbon-nitrogen bonds at the metal iron site. Overall, our study elucidated the molecular mechanism of the ubiquitous enzyme system involved in single-carbon transfer in prokaryotes and eukaryotes.