Bifunctional architecture enables substrate catalysis and channeling in Paracoccus TMAO demethylase.

Substrate channeling enhances efficiency and prevents toxicity by directing unstable intermediates between active sites. Trimethylamine N-oxide demethylase (TDM) degrades trimethylamine N-oxide (TMAO) to dimethylamine and formaldehyde (HCHO), but the fate of HCHO has remained unclear. We report cryo-EM structures of TDM in apo, substrate-, and product-bound states that reveal a previously unknown channeling pathway. Combined structural, biochemical, and target molecular dynamics analyses show that HCHO is generated in a catalytic core and guided through a tunnel to a remote tetrahydrofolate (THF)-binding site, where it forms methylene-THF. Thus, TDM emerges as a bifunctional enzyme that unites TMAO demethylation with one-carbon transfer, providing a mechanistic explanation for its role in metabolic efficiency and detoxification.