Cheng, RonghaiWu, LianLai, RuiPeng, ChaoNaowarojna, NathcharHu, WeiyaoLi, XinhaoWhelan, Stephen A.Lee, NormanLopez, JuanZhao, ChangmingYong, YouhuaXue, JiahuiJiang, XuefengGrinstaff, Mark W.Deng, ZixinChen, JieshengCui, QiangZhou, JiahaiLiu, Pinghua2022-06-032022-06-032020-08-21R. Cheng, L. Wu, R. Lai, C. Peng, N. Naowarojna, W. Hu, X. Li, S.A. Whelan, N. Lee, J. Lopez, C. Zhao, Y. Yong, J. Xue, X. Jiang, M.W. Grinstaff, Z. Deng, J. Chen, Q. Cui, J. Zhou, P. Liu. 2020. "Single-step Replacement of an Unreactive C-H Bond by a C-S Bond Using Polysulfide as the Direct Sulfur Source in Anaerobic Ergothioneine Biosynthesis.." ACS Catal, Volume 10, Issue 16, pp. 8981 - 8994. https://doi.org/10.1021/acscatal.0c018092155-5435https://hdl.handle.net/2144/44498Ergothioneine, a natural longevity vitamin and antioxidant, is a thiol-histidine derivative. Recently, two types of biosynthetic pathways were reported. In the aerobic ergothioneine biosynthesis, a non-heme iron enzyme incorporates a sulfoxide to an sp2 C-H bond in trimethyl-histidine (hercynine) through oxidation reactions. In contrast, in the anaerobic ergothioneine biosynthetic pathway in a green sulfur bacterium, Chlorobium limicola, a rhodanese domain containing protein (EanB) directly replaces this unreactive hercynine C-H bond with a C-S bond. Herein, we demonstrate that polysulfide (HSSnSR) is the direct sulfur-source in EanB-catalysis. After identifying EanB's substrates, X-ray crystallography of several intermediate states along with mass spectrometry results provide additional mechanistic details for this reaction. Further, quantum mechanics/molecular mechanics (QM/MM) calculations reveal that protonation of Nπ of hercynine by Tyr353 with the assistance of Thr414 is a key activation step for the hercynine sp2 C-H bond in this trans-sulfuration reaction.p. 8981 - 8994en-USAssaysCatalysisMonomersPeptides and proteinsSulfurArticle10.1021/acscatal.0c01809570301