Arsinothricin Biosynthesis Involving a Noncanonical Radical SAM Enzyme for C-As Bond Formation

Abstract

Arsinothricin is a potent antibiotic secreted by soil bacteria. The biosynthesis of Arsinothricin was proposed to involve two steps. The first step is 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, the reaction has not been verified in vitro, and 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 C{gamma},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 noncanonical radical SAM enzyme that contains this motif but generates an 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, we showed that this motif binds to the substrate arsenite and is highly important for its activity. Our results suggested that ArsL is a noncanonical radical SAM enzyme with a canonical radical SAM enzyme motif, implying that more noncanonical radical SAM chemistry may exist within the radical SAM enzyme superfamily. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=40 SRC="FIGDIR/small/577332v1_ufig1.gif" ALT="Figure 1"> View larger version (8K): org.highwire.dtl.DTLVardef@14547e2org.highwire.dtl.DTLVardef@172753eorg.highwire.dtl.DTLVardef@16f4a99org.highwire.dtl.DTLVardef@18ab4c8_HPS_FORMAT_FIGEXP M_FIG C_FIG