Abstract
In the anaerobic ergothioneine biosynthetic pathway, a rhodanese domain containing enzyme (EanB) activates tne hercynine's sp2 ε-C-H Dona ana 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 analog of ergothioneine, selenoneine. However, in deuterated buffer, the perselenide-modified EanB catalyzes the deuterium exchange between hercynine's sp2 ε-C-H bond and D2O. Results from QM/MM 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 k ex of ~10-fold greater than the wild-type EanB (EanBWT). With regards to potential mechanisms, these results support the involvement of a carbene intermediate in EanB-catalysis, rendering EanB as one of the few carbene-intermediate involving enzymatic systems.