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

• Published in: bioRxiv
• Main Authors: Yao, Yadi, He, Jiale, Dong, Min
• Format: Paper
• Language: English
• Published: Cold Spring Harbor Cold Spring Harbor Laboratory Press 02.02.2024
• Subjects: Arsenite; Biosynthesis; Conserved sequence; Enzymes; Methionine; Methyltransferase; Soil microorganisms
• DOI: 10.1101/2024.02.01.577332

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 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 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 superfamilyCompeting Interest StatementThe authors have declared no competing interest.