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

In the anaerobic ergothioneine biosynthetic pathway, a rhodanese domain-containing enzyme (EanB) activates the hercynine's sp(2) epsilon-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 sp(2) epsilon-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 pK(a) of Tyr353 will affect the deuterium exchange rate. Indeed, the 3,5-difluoro tyrosine-containing EanB catalyzes the deuterium exchange reaction with a k(ex) similar to 10-fold greater than the wild-type EanB (EanB(WT)). 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.

Automated summary

This study elucidates the trans-sulfuration reaction mechanism in the anaerobic ergothioneine biosynthetic pathway and identifies the influence of modulating the key amino acid Tyr353 on deuterium exchange rate. The results suggest that EanB may be one of the few enzymatic systems involving a carbene intermediate.