Radical S -Adenosyl-l-Methionine Enzyme PylB: A C-Centered Radical to Convert l-Lysine into (3 R )-3-Methyl-d-Ornithine.

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.