2.

Proposed mechanism of choline cleavage by CutC. The reaction begins with a thiyl radical abstracting a hydrogen atom from the C1 position of choline, forming an α-hydroxyalkyl radical intermediate. This radical can then follow two possible pathways to generate trimethylamine (TMA) and acetaldehyde in the CutC active site. In Pathway 1, the α-hydroxyalkyl radical undergoes base-catalyzed direct elimination of TMA via a spin-center shift, with protonation of the departing TMA possibly involving a proton relay and rearrangement of active site residues. In Pathway 2, there is a 1,2-migration of the trimethylammonium group, producing a carbinolamine intermediate that spontaneously decomposes (1,2-elimination) to release TMA and acetaldehyde. The experimentally determined turnover number (k _cat) of 157 ± 2 s^–1 corresponds to an activation free energy (ΔG ^⧧) of 14.6 kcal mol^–1 at 300 K.