Calorimetric characterization of the stability and activity of trimethylamine‐N‐oxide ( <scp>TMAO</scp> ) demethylase from <i>Methylocella silvestris</i> <scp>BL2</scp>

Trimethylamine-N-oxide (TMAO) is an organic osmolyte found in numerous species and is known to have a range of biological effects. TMAO has recently garnered attention in the medical field due to its association with cardiovascular diseases, underscoring the need for its reliable detection and quantification. Current methods for TMAO analysis often rely on hazardous reagents or costly analytical instrumentation. In this study, we focus on Methylocella silvestris BL2, which produces a TMAO-demethylase (Tdm), with the aim of developing a direct enzymatic assay for TMAO detection. We report on the bioinformatic analysis, expression, purification, and calorimetric characterization of Tdm. Structural predictions generated by AlphaFold suggest that the protein, previously described as hexameric, is organized as a trimer of dimers. The 3D model reveals that the binding sites for the metal cofactors Zn²⁺ and Fe²⁺ are located in close proximity. Differential scanning calorimetry (DSC) experiments show an irreversible unfolding behavior with two independent endothermic transitions, consistent with a two-state model. Isothermal titration calorimetry (ITC) was employed in a time-resolved manner to determine the enzyme's optimal reaction pH and substrate detection limit. The assay revealed an optimal pH of 7.0, a minimum effective enzyme concentration of 100 nM, and a TMAO detection limit of 10 μM. Kinetic parameters were also precisely measured using ITC, with the highest observed k_cat value being 15.47 s^-1 at 100 nM Tdm concentration. Overall, these findings support the potential application of Tdm as a sensitive and direct tool for the detection and quantification of the medically relevant biomarker TMAO.