Spectroscopic and Biochemical Characterization of the Noncanonical Radical SAM Enzyme ArsL, Involved in Arsinothricin Biosynthesis.

Radical SAM enzymes are the most widespread biocatalysts. These metalloenzymes, using -adenosyl-l-methionine (SAM) and a [4Fe-4S] cluster as central cofactors, catalyze a broad range of chemically challenging transformations. The vast majority of radical SAM enzymes initiate their reaction by the homolytic cleavage of the SAM C5'-S bond and the generation of the central 5'-deoxyadenosyl radical (5'-dA·). In this study, by combining spectroscopic approaches with labeling and biochemical analyses, we show that ArsL, the key enzyme in the biosynthesis of the arsenic-containing antibiotic arsinothricin, catalyzes a unique reaction: the addition of the 3-amino-3-carboxypropyl radical (ACP·) to As(III). Remarkably, by exploiting several radical trapping strategies, we demonstrate that in sharp contrast to canonical radical SAM enzymes ArsL cleaves the SAM Cγ-S bond. In addition, using electron paramagnetic resonance (EPR) and hyperfine sublevel correlation (HYSCORE) spectroscopies, we establish that ArsL has a unique SAM binding mode, consistent with its catalytic properties and predicted structure. Notably, EPR and HYSCORE analyses support that SAM interacts with the radical SAM [4Fe-4S] cluster in an uncharacteristic conformation to form ACP·. Collectively, our study reveals that members of the superfamily of radical SAM enzymes are able to finely tune the binding of the SAM cofactor in order to perform unique chemistries.