Radical <i>S</i>-Adenosyl-<scp>l</scp>-Methionine Enzyme PylB: A C-Centered Radical to Convert <scp>l</scp>-Lysine into (3<i>R</i>)-3-Methyl-<scp>d</scp>-Ornithine

PylB is a radical <i>S</i>-adenosyl-l-methionine (SAM) enzyme predicted to convert l-lysine into (3<i>R</i>)-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 <i>in vitro</i> 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<i>R</i>)-3-methyl-d-ornithine into l-lysine and using catalytic amounts of the 5'-deoxyadenosyl radical. Finally, we identified significant <i>in vitro</i> 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 [Fe₄S₄] cluster.