biostudies-literatureSinner EKHHS | National Institutes of HealthNIAID NIH HHSNIGMS NIH HHSe2206494119https://www.ebi.ac.uk/biostudies/studies/S-EPMC9407657biostudies-literatureUnknown119(34)Complex carbapenems are important clinical antibiotics used to treat recalcitrant infections. Their biosynthetic gene clusters contain three essential B₁₂-dependent radical <i>S</i>-adenosylmethionine (rSAM) enzymes. The majority of characterized enzymes in this subfamily catalyze methyl transfer, but only one is required to sequentially install all methionine-derived carbons in complex carbapenems. Therefore, it is probable that the other two rSAM enzymes have noncanonical functions. Through a series of fermentation and in vitro experiments, we show that ThnL uses radical SAM chemistry to catalyze thioether bond formation between C2 of a carbapenam precursor and pantetheine, uniting initial bicycle assembly common to all carbapenems with later tailoring events unique to complexProceedings of the National Academy of Sciences of the United States of AmericaThnL, a B12-dependent radical <i>S</i>-adenosylmethionine enzyme, catalyzes thioether bond formation in carbapenem biosynthesis.PMC9407657GM080189T32 GM080189AI121072R01 AI121072Townsend CASinner EKMarous DRLi RfalseThnL, a B12-dependent radical <i>S</i>-adenosylmethionine enzyme, catalyzes thioether bond formation in carbapenem biosynthesis.Complex carbapenems are important clinical antibiotics used to treat recalcitrant infections. Their biosynthetic gene clusters contain three essential B₁₂-dependent radical <i>S</i>-adenosylmethionine (rSAM) enzymes. The majority of characterized enzymes in this subfamily catalyze methyl transfer, but only one is required to sequentially install all methionine-derived carbons in complex carbapenems. Therefore, it is probable that the other two rSAM enzymes have noncanonical functions. Through a series of fermentation and in vitro experiments, we show that ThnL uses radical SAM chemistry to catalyze thioether bond formation between C2 of a carbapenam precursor and pantetheine, uniting initial bicycle assembly common to all carbapenems with later tailoring events unique to complex2022-01-01T00:00:00Z2022 Aug2025-04-19T13:27:43.522Z2025-04-19T13:27:43.522ZS-EPMC94076573596979310.1073/pnas.2206494119