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

PylB is a radical S-adenosyl-L-methionine (SAM) enzyme predicted to convert L-lysine into (3R)-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 C. position of L-lysine to afford the radical-based beta-scission. Strikingly, we also showed that PylB catalyzes the reverse reaction, converting (3R)-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 [Fe4S4] cluster.