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PylB is a radical S -adenosyl-l-methionine (SAM) enzyme predicted to convert l-lysine into (3 R )-3-methyl-d-ornithine, a precursor in the biosynthesis of the 22nd proteogenic amino acid pyrrolysine. This protein highly resembles that of the radical SAM tyrosine and tryptophan lyases, which activate their substrate by abstracting a H atom from the amino-nitrogen position. Here, combining in vitro assays, analytical methods, electron paramagnetic resonance spectroscopy, and theoretical methods, we demonstrated that instead, PylB activates its substrate by abstracting a H atom from the Cγ position of l-lysine to afford the radical-based β-scission. Strikingly, we also showed that PylB catalyzes the reverse reaction, converting (3 R )-3-methyl-d-ornithine into l-lysine and using catalytic amounts of the 5'-deoxyadenosyl radical. Finally, we identified significant in vitro production of 5'-thioadenosine, an unexpected shunt product that we propose to result from the quenching of the 5'-deoxyadenosyl radical species by the nearby [Fe 4 S 4 ] cluster.

We report a mild and efficient electrochemical protocol to access a variety of vicinally C-O and C-N difunctionalized compounds from simple alkenes. Detailed mechanistic studies revealed a distinct reaction pathway from those previously reported for TEMPO-mediated reactions. In this mechanism, electrochemically generated oxoammonium ion facilitates the formation of azidyl radical via a charge-transfer complex with azide, TEMPO-N 3 . DFT calculations together with spectroscopic characterization provided a tentative structural assignment of this charge-transfer complex. Kinetic and kinetic isotopic effect studies revealed that reversible dissociation of TEMPO-N 3 into TEMPO • and azidyl precedes the addition of these radicals across the alkene in the rate-determining step. The resulting azidooxygenated product could then be easily manipulated for further synthetic elaborations. The discovery of this new reaction pathway mediated by the TEMPO + /TEMPO • redox couple may expand the scope of aminoxyl radical chemistry in synthetic contexts.