In vitro and in vivo characterization and protein-bound structural elucidation of three microbial choline TMA lyase inhibitors

Microbial choline TMA lyases (CutC, catalytic polypeptide) are glycyl radical-dependent enzymes that catalyze the biochemical transformation of choline into both trimethylamine (TMA) and acetaldehyde. Present in intestinal microbiota, this gut microbial enzyme is a major contributor to TMA production, the precursor for host-generated trimethylamine N-oxide (TMAO). Both clinical and mechanistic studies reveal TMAO as a contributor to host cardiometabolic diseases, making CutC a target for the development of small molecule inhibitors. Here, we characterize the inhibitory potency of three choline analog-based CutC inhibitor tool drugs (cyclopropylcholine, CpC; fluoromethylcholine, FMC; and difluoromethylcholine, FMF) against phylogenetically distant cloned and expressed microbial CutC, whole cell microbial monocultures, both human and mouse polymicrobial fecal cultures, and in mice following an oral [d9]-choline challenge. Finally, we resolved the crystal structures of CutC from Klebsiella pneumoniae with each of the three inhibitors bound to the enzyme and demonstrate the molecular basis for their interaction with active site amino acid residues critical for enzyme activity.