TDM catalyzes two sequential reactions.

(A) Steady-state kinetic analysis of TDM - producing HCHO, in response to varying concentrations of the substrate TMAO (n=3). (B) Representative HPLC-MS spectrum of DMA product from TDM:TMAO reaction. The standard curve for DMA is shown in green, and that for TMAO is shown in orange. The red trace shows the product of the reaction when TMAO is added to TDM, indicating the formation of DMA. Counts in the Y-axis. (C) Linear regression analysis demonstrates a concentration-dependent decrease in HCHO production when THF is included in the reaction mixture, suggesting THF-dependent consumption or conversion of HCHO. (D) Representative HPLC-MS confirming the formation of MTHF adduct as a product of the TDM:TMAO:THF reaction. The red, gray, and green lines represent the standard curves for THF, MTHF, and the reaction product when TMAO and THF were present with TDM, respectively.

Cryo-EM data collection, refinement, validation and statistics

Overall architecture of TDM determined by cryo-electron microscopy.

(A) Representative cryo-electron microscopy (EM) maps. (B) Overall structure of the complex, with each subunit colored differently. TDM adopts a 2 + 21/2 complexation. The two half-domains of the complex are the C-terminal domains. (C) (C) Schematic illustration of TDM complexation. TDM monomers with N-terminal, Core, and C-terminal domains are colored forest, orange, and blue, respectively. The same coloring is used in panels A and B. (D) Close-up view of the 3Cys:Zn2+ binding motif. (E) Close-up view of the interactions between DMA and HCHO products with the 3Cys:Zn2+ motif. (F) The TMAO substrate is trapped in the 3Cys:Zn2+ active site of the double D220A/D367A mutant. The density maps are shown and contoured at 3.0 σ with a carve of 2 Å. Bound small molecules are shown as ball-and-stick representations, amino acid residues as sticks, and hydrogen bonds as dashed lines. (G) Relative activity of TDM variants.

Mechanism of Zn2+-dependent TMAO demethylation by TDM.

Step 1: Water activation. Zn2+ coordinates to OH− from water, which is deprotonated by the carboxylate group of D220, increasing its nucleophilicity. Step 2: Substrate binding. The oxygen of TMAO (O−) can coordinate with Zn2+, activating the N–CH3 bond for a nucleophilic attack. Step 3: Electron/bond rearrangement and formation of the tetrahedral intermediate. Upon methyl group transfer to OH−, the positive charge on the N atom in the (CH3)2NH-O-Zn2+ transition state is neutralized, leading to N-O bond collapse. Step 4: Product formation and HCHO release. The released DMA binds to D367 via a hydrogen bond, and water binds to Zn2+, regenerating the active site.

THF binding to TDM.

(A) The THF-binding domain forms a cloverleaf of three subdomains (I-III), creating a central cavity that accommodates THF. EM density for THF is shown as a surface. (B) Close-up of the binding pocket highlighting hydrophobic and hydrogen-bonding residues that stabilize THF, colored according to their subdomain location in panel A. (C) ITC analysis confirms specific THF binding to TDM (blue), with negligible signal in buffer control (gray).

HCHO channels from the active site to the THF-binding site in TDM complex.

(A, B) Surface representations of the channels within the TDM complex colored according to either electrostatic potential or radius. (C) Tunnel analysis of a CG MD simulation suggests that HCHO in each chain can access the THF binding sites in chains A and B. The five top-ranked paths for chain A are shown in different colors. THF, DMA, and HCHO are depicted as spheres colored by the atom type. TA, TB, TC, and TD: THF molecules in chains A, B, C, and D, respectively; HA: chain A, HCHO.

The N-terminal domain is involved in the structural stability and assembly of proteins.

(A) The evolutionary conservation of residue positions on the TDM structure is estimated based on phylogenetic connections among homologous sequences. (B) N-terminal domain was depicted as forest. Residues ranging from 1-20, 150-158 were highlighted. (C) Relative TDM activity of TDM mutants compared to that of WT variant.