Mechanism of Sulfoxidation and C–S Bond Formation Involved in the Biosynthesis of Ergothioneine Catalyzed by Ergothioneine Synthase (EgtB)

Ergothioneine synthase (EgtB) is a unique non-heme mononuclear iron enzyme that catalyzes the sulfoxidation and C–S bond formation between γ-glutamyl cysteine (γGC) and N-α-trimethyl histidine (TMH) as a pivotal step in the ergothioneine biosynthesis. A controversy has arisen regarding the sequence of sulfoxidation and C–S bond formation in the catalytic cycle. To clarify this issue, the QM/MM approach has been employed to investigate the detailed mechanism of EgtB. Two binding modes of O2 to Fe(II) (“end-on” and “side-on”) have been identified. Within the present computational model, the end-on binding mode of O2 is preferred. The open-shell singlet is calculated to be the ground state, whereas the quintet is the most active state. Moreover, the sulfoxidation is prior to the formation of the C–S bond, and the reaction mainly occurs on the quintet state surface. Due to the electron transfer from the γGC to the ferric superoxide, the sulfur atom of γGC has partial radical characteristics, which facilitates the attack of the distal oxygen atom on the sulfur radical of γGC to form the sulfoxide. The formation of TMH C2 anion, i.e., the abstraction of the proton from the imidazole group in TMH by the Fe(IV)–oxo species, is the prerequisite for C–S bond formation, which is the rate-limiting step with an energy barrier of 21.7 kcal/mol. In addition, it is also found that although the resulting iron(III)–oxo can easily abstract a proton from Tyr377 to generate a phenolic hydroxyl anion, the subsequent proton transfer from C2 to Tyr377 is calculated to be difficult; thus, Tyr377 is not directly involved in the sulfoxidation and C–S bond formation. Our calculations also reveal that the side-on mode is not the catalytically relevant species. This work provides a direct comparison with previous experimental and theoretical studies, which is helpful for understanding the catalysis of ergothioneine synthase and related enzymes.