Analysis of RNA Methylation by Phylogenetically Diverse Cfr Radical -Adenosylmethionine Enzymes Reveals an Iron-Binding Accessory Domain in a Clostridial Enzyme.

Cfr is a radical -adenosylmethionine (SAM) RNA methylase linked to multidrug antibiotic resistance in bacterial pathogens. It catalyzes a chemically challenging C-C bond-forming reaction to methylate C8 of A2503 ( numbering) of 23S rRNA during ribosome assembly. The gene has been identified as a mobile genetic element in diverse bacteria and in the genome of select Bacillales and Clostridiales species. Despite the importance of Cfr, few representatives have been purified and characterized . Here we show that Cfr homologues from , , , and act as C8 adenine RNA methylases in biochemical assays. Cfr contains an additional Cys-rich C-terminal domain that binds a mononuclear Fe ion in a rubredoxin-type Cys motif. The C-terminal domain can be truncated with minimal impact on Cfr activity, but the rate of turnover is decreased upon disruption of the Fe-binding site by Zn substitution or ligand mutation. These findings indicate an important purpose for the observed C-terminal iron in the native fusion protein. Bioinformatic analysis of the Cfr Cys-rich domain shows that it is widespread (∼1400 homologues) as a stand-alone gene in pathogenic or commensal Bacilli and Clostridia, with >10% encoded adjacent to a predicted radical SAM RNA methylase. Although the domain is not essential for Cfr activity, the genomic co-occurrence and high abundance in the human microbiome suggest a possible functional role for a specialized rubredoxin in certain radical SAM RNA methylases that are relevant to human health.