Single-Step Replacement of an Unreactive C–H Bond by a C–S Bond Using Polysulfide as the Direct Sulfur Source in the Anaerobic Ergothioneine Biosynthesis

Ergothioneine, a natural longevity vitamin and antioxidant, is a thiol-histidine derivative. Recently, two types of biosynthetic pathways were reported. In the aerobic ergothioneine biosyntheses, non-heme iron enzymes incorporate a sulfoxide into an sp² C–H bond from trimethyl-histidine (hercynine) through oxidation reactions. In contrast, in the anaerobic ergothioneine biosynthetic pathway in a green-sulfur bacterium, Chlorobium limicola, a rhodanese domain containing protein (EanB), directly replaces this unreactive hercynine C–H bond with a C–S bond. Herein, we demonstrate that polysulfide (HSS_nSR) is the direct sulfur source in EanB catalysis. After identifying EanB’s substrates, X-ray crystallography of several intermediate states along with mass spectrometry results provide additional mechanistic details for this reaction. Further, quantum mechanics/molecular mechanics (QM/MM) calculations reveal that the protonation of N_π of hercynine by Tyr353 with the assistance of Thr414 is a key activation step for the hercynine sp² C–H bond in this trans-sulfuration reaction.