6.

Free energy landscapes (FEL) from MM MD simulations of CutC with (a) choline and (b) carbinolamine, showing dominant substrate conformations and key hydrogen bonds with active site residues. The FELs are constructed using two collective variables: the distances between the positively charged trimethylammonium group and the negatively charged residues Glu491 and Asp216, which are essential for substrate binding and catalysis. The FELs are evaluated at 300 K. Independent gradient model analysis based on sign(λ₂)­ρ from QM/MM MD snapshots (B3LYP-D3BJ/6-31G­(d)) visualizes noncovalent interactions of (c) choline and (d) carbinolamine with the active site as isosurfaces (isovalue 0.01).