Frontiers Science Center for Synthetic Biology, Key Laboratory of Systems Bioengineering (MOE), School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China.
Haihe Laboratory of Sustainable Chemical Transformations, Tianjin 300192, China.
J Am Chem Soc. 2024 Aug 7;146(31):21214-21219. doi: 10.1021/jacs.4c06403. Epub 2024 Jul 25.
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 -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 C-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.
砷硫菌素是一种由土壤细菌分泌的强效抗生素。砷硫菌素的生物合成被认为涉及三价砷与 -腺苷基-l-甲硫氨酸（SAM）的 3-氨基-3-羧基丙基（ACP）基团之间的 C-As 键形成，这是由 ArsL 蛋白催化的。然而， ArsL 尚未被详细表征。有趣的是，ArsL 含有一个 CxxxCxxC 基序，因此属于自由基 SAM 酶超家族，其成员裂解 SAM 并产生 5'-脱氧腺苷自由基。在这里，我们发现 ArsL 裂解 SAM 的 C-S 键并产生类似于 Dph2 的 ACP 自由基，Dph2 是一种参与二肽酰生物合成的非典型自由基 SAM 酶。由于 Dph2 不包含 CxxxCxxC 基序，因此 ArsL 是一种独特的自由基 SAM 酶，它包含该基序但产生非典型的 ACP 自由基。与甲基转移酶 ArsM 一起，我们成功地在体外重建了砷硫菌素的生物合成。ArsL 在 C 末端序列中具有保守的 RCCLKC 基序，属于 RCCLKC-尾自由基 SAM 蛋白亚家族。通过截短和突变，我们表明该基序在与底物亚砷酸盐结合中起重要作用，并且对其活性非常重要。我们的结果表明，ArsL 具有典型的自由基 SAM 酶基序，但催化非典型的自由基 SAM 反应，这表明在自由基 SAM 酶超家族中可能存在更多的非典型自由基 SAM 化学。
