Implications for an Imidazole-2-yl Carbene Intermediate in the Rhodanase-Catalyzed C–S Bond Formation Reaction of Anaerobic Ergothioneine Biosynthesis

Abstract

In the anaerobic ergothioneine biosynthetic pathway, a rhodanese domain-containing enzyme (EanB) activates the hercynine’s sp2 ε-C–H bond and replaces it with a C–S bond to produce ergothioneine. The key intermediate for this trans-sulfuration reaction is the Cys412 persulfide. Substitution of the EanB-Cys412 persulfide with a Cys412 perselenide does not yield the selenium analogue of ergothioneine, selenoneine. However, in a deuterated buffer, perselenide-modified EanB catalyzes the deuterium exchange between hercynine’s sp2 ε-C–H bond and D2O. Results from quantum mechanics/molecular mechanics calculations suggest that the reaction involves a carbene intermediate and that Tyr353 plays a key role. We hypothesize that modulating the pKa of Tyr353 will affect the deuterium exchange rate. Indeed, the 3,5-difluoro tyrosine-containing EanB catalyzes the deuterium exchange reaction with a kex ∼10-fold greater than the wild-type EanB (EanBWT). With regard to potential mechanisms, these results support the involvement of a carbene intermediate in the EanB catalysis, rendering EanB as one of the few carbene intermediate-involving enzymatic systems.