Mechanistic Insights into Choline Degradation Catalyzed by the Choline Trimethylamine-Lyase CutC

Choline trimethylamine-lyase (CutC) is a prominent glycyl radical enzyme that catalyzes the degradation of choline into nitrogenous metabolites trimethylamine (TMA) and acetaldehyde. Choline and TMA are crucial nitrogen-containing compounds and play essential roles in various biological pathways, including neurotransmission and global metabolic functions. Although many experimental studies have been dedicated to elucidating the function of CutC, its exact catalytic mechanism remains elusive. Herein, we employed molecular dynamics (MD) simulations and quantum mechanics/molecular mechanics (QM/MM) methodologies to investigate the reaction mechanism of CutC in detail. Our calculation results reveal that the enzymatic reaction is initiated by a two-step hydrogen atom transfer (HAT) mechanism, a typical process mediated by a cysteine radical in glycyl radical enzymes (GREs). Significantly, in our suggested reaction mechanism, unlike the previously proposed 1,2-elimination pathway, a more favorable stepwise 1,2-migration of the TMA group occurs after the formation of a substrate radical. This migration of the TMA group leads to the formation of a hemiaminal intermediate, which is likely to be eliminated outside of CutC. Furthermore, our mechanistic investigations indicate that the residue Glu440, adjacent to the choline substrate, plays a pivotal role in helping substrate binding through a hydrogen bond rather than serving as a general base for proton abstraction. These findings provide deeper insights into the catalytic strategy that CutC employs for C-N bond cleavage in choline metabolism and broaden the mechanistic repertoire documented for glycyl radical enzymes in mediating elimination reactions.