Abstract

Arsinothricin is a potent antibiotic secreted by soil bacteria. The biosynthesis of arsinothricin was proposed to involve a C–As bond formation between trivalent As and the 3-amino-3-carboxypropyl (ACP) group of S-adenosyl-l-methionine (SAM), which is catalyzed by the protein ArsL. However, ArsL has not been characterized in detail. Interestingly, ArsL contains a CxxxCxxC motif and thus belongs to the radical SAM enzyme superfamily, the members of which cleave SAM and generate a 5′-deoxyadenosyl radical. Here, we found that ArsL cleaves the Cγ,Met–S bond of SAM and generates an ACP radical that resembles Dph2, a noncanonical radical SAM enzyme involved in diphthamid biosynthesis. As Dph2 does not contain the CxxxCxxC motif, ArsL is a unique radical SAM enzyme that contains this motif but generates a noncanonical ACP radical. Together with the methyltransferase ArsM, we successfully reconstituted arsinothricin biosynthesis in vitro. ArsL has a conserved RCCLKC motif in the C-terminal sequence and belongs to the RCCLKC-tail radical SAM protein subfamily. By truncation and mutagenesis, we showed that this motif plays an important role in binding to the substrate arsenite and is highly important for its activity. Our results suggested that ArsL has a canonical radical SAM enzyme motif but catalyzes a noncanonical radical SAM reaction, implying that more noncanonical radical SAM chemistry may exist within the radical SAM enzyme superfamily.