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 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 ∼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.