Roles of Conserved Cysteinyl Residues in the Radical SAM Methyltransferases RlmN and Cfr

One of the most exciting new discoveries involving the radical S‐adenosylmethionine (RS) superfamily of enzymes are reactions involving methylation of the C2 or C8 positions of adenosine 2503 in 23S rRNA. These reactions are catalyzed by the proteins RlmN and Cfr, respectively. Methylation at C2 by RlmN is an activity found throughout prokaryotes, and thought to serve a housekeeping function. Cfr from Staphyloccus aureus catalyzes methylation at C8 and is believed to have evolved from RlmN. As members of the RS superfamily, both enzymes reductively leave S‐adenosylmethione (SAM) to methionine and a 5′‐deoxyadenosyl radical (5′‐dA•), a common intermediate in this family of enzymes that initiates radical‐based turnover by abstracting a strategic hydrogen atom. These enzymes all require a [4Fe–4S] cluster in catalysis, which is ligated to the protein by three conserved cysteinyl residues typically lying in a CxxxCxxC motif. RlmN and Cfr, however, contain two additional Cys residues that are absolutely conserved. In this work, we provide biochemical spectroscopic data for unique catalytic roles for these two Cys residues, as well as a comprehensive working hypothesis for the catalytic mechanism of these two very interesting enzymes.