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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 pKa of Tyr353 will affect the deuterium exchange rate. Indeed, the 3,5-difluoro tyrosine-containing EanB catalyzes the deuterium exchange reaction with a kex ∼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. 

Protein chemical modifications are important tools for elucidating chemical and biological functions of proteins. Several strategies have been developed to implement these modifications, including enzymatic tailoring reactions, unnatural amino acid incorporation using the expanded genetic codes, and recognition-driven transformations. These technologies have been applied in metalloenzyme studies, specifically in dissecting their mechanisms, improving their enzymatic activities, and creating artificial enzymes with non-natural activities. Herein, we summarize some of the recent efforts in these areas with an emphasis on a few metalloenzyme case studies. 

Ergothioneine, a natural longevity vitamin and antioxidant, is a thiol-histidine derivative. Recently, two types of biosynthetic pathways were reported. In the aerobic ergothioneine biosyntheses, non-heme iron enzymes incorporate a sulfoxide into an sp2 C–H bond from 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 the 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.