Cheng, RonghaiLai, RuiPeng, ChaoLopez, JuanLi, ZhihongNaowarojna, NathcharLi, KelinWong, ChristinaLee, NormanWhelan, Stephen A.Qiao, LuGrinstaff, Mark W.Wang, JiangyunCui, QiangLiu, Pinghua2022-06-032022-06-032021-03-19R. Cheng, R. Lai, C. Peng, J. Lopez, Z. Li, N. Naowarojna, K. Li, C. Wong, N. Lee, S.A. Whelan, L. Qiao, M.W. Grinstaff, J. Wang, Q. Cui, P. Liu. 2021. "Implications for an imidazol-2-yl carbene intermediate in the rhodanase-catalyzed C-S bond formation reaction of anaerobic ergothioneine biosynthesis.." ACS Catal, Volume 11, Issue 6, pp. 3319 - 3334. https://doi.org/10.1021/acscatal.0c048862155-5435https://hdl.handle.net/2144/44496In 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.p. 3319 - 3334en-USErgothioneineSelenoneineAnaerobic biosynthesisCarbene intermediateUnnatural amino acid (uAA)Article10.1021/acscatal.0c04886626892