Photoaffinity SAM analogues for the identification of SAM-binding proteins.

S-Adenosylmethionine (SAM) serves as an important substrate in a variety of biochemical reactions, and it is important to identify unknown SAM-binding proteins to fully understand the biological functions of SAM. Previous studies on SAM-binding proteins used S-Adenosylhomocystein (SAH)-analogues, which mainly identified SAM dependent methyltransferases. Here, we developed and validated three SAM photoaffinity probes to label and enrich SAM-binding proteins. These probes efficiently labeled the known SAM-binding protein Dph2 involved in diphthamide biosynthesis from cell lysates. Using these probes, we enriched SAM-binding proteins from the cell lysates of Burkholderia gladioli and Saccharomyces cerevisiae. In addition, we validated five SAM binders and revealed the SAM cleavage activities of three of them, including the radical SAM enzyme ArsL, which cleaves SAM to generate methylthioadenosine (MTA), and AcnA and EDD84_07545, which generate S-adenosyl-l-homocysteine (SAH). Therefore, our SAM-based photoaffinity probes are promising tools for the identification of SAM-binding proteins.