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bioRxiv · 10.1101/2025.11.26.690739

Bifunctional Architecture Enables Substrate Catalysis and Channeling in Paracoccus TMAO Demethylase

Thach, T.·Dhanabalan, K.·Maurya, S.·Han-Hallett, Y.·Quan, S.·Allison, J.·Ramanathan, G.·Subramanian, R.
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Abstract

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.

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Thach, T., Dhanabalan, K., Maurya, S., Han-Hallett, Y., Quan, S., Allison, J., Ramanathan, G., Subramanian, R.. 2025-11-29. Bifunctional Architecture Enables Substrate Catalysis and Channeling in Paracoccus TMAO Demethylase. https://doi.org/10.1101/2025.11.26.690739

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