All Roads Lead to Carbinolamine: QM/MM Study of Enzymatic C-N Bond Cleavage in Anaerobic Glycyl Radical Enzyme Choline Trimethylamine-Lyase (CutC).

The anaerobic glycyl radical enzyme choline trimethylamine-lyase (CutC) is produced by multiple bacterial species in the human gut microbiome and catalyzes the conversion of choline to trimethylamine (TMA) and acetaldehyde. CutC has emerged as a promising therapeutic target due to its role in producing TMA, which is subsequently oxidized in the liver to form trimethylamine--oxide (TMAO). Elevated TMAO levels are associated with several human diseases, including atherosclerosis and other cardiovascular disorders─a leading cause of mortality worldwide. Understanding the catalytic mechanism of this enzyme should aid successful design of potent inhibitors. Here, we employed extensive molecular dynamics (MD) simulations to reveal that hydrogen bonding within the CutC active site plays a crucial role in orienting choline for the initial -S hydrogen abstraction, leading to the formation of the α-hydroxy radical. The reaction mechanism was explored with quantum mechanics/molecular mechanics (QM/MM). The performance of three density functionals (B3LYP-D3, ωB97X-D3, and M06-2X) was tested against DLPNO-CCSD(T) ab initio calculations. These results indicate that choline cleavage occurs via TMA migration leading to a stable product carbinolamine which likely undergoes 1,2-elimination to acetaldehyde and TMA in water. Mechanistic insights consistently support the TMA migration pathway over direct TMA elimination, providing clear evidence for the preferred reaction mechanism. Two distinct mechanistic pathways were identified: one with a relatively high activation energy barrier, and the other with a lower barrier which is in a good agreement with the previously reported experimental kinetic parameters. QM/MM MD simulations further confirm that Glu491 functions as a catalytic base, abstracting a proton from the α-hydroxy radical and thereby facilitating the experimentally observed C-N bond cleavage. The relative binding affinity of the reactant (choline) and product (carbinolamine) was estimated with alchemical relative binding free energy calculations, complemented by noncovalent interaction analysis. These results elucidate the molecular basis for differences in their interactions with CutC (particularly highlighting key electrostatic interactions with Asp216 and Glu491) providing insights for future inhibitor design.