Analysis of RNA methylation by phylogenetically diverse Cfr radical SAM enzymes reveals an iron-binding accessory domain in a clostridial enzyme.

Abstract

Cfr is a radical S-adenosylmethionine (SAM) RNA methylase linked to multi-drug antibiotic resistance in bacterial pathogens. It catalyzes a chemically challenging C-C-bond forming reaction to methylate C8 of A2503 [ Escherichia coli ( Ec) numbering) of 23 S ribosomal RNA during ribosome assembly. The cfr 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 in vitro. Here we show that Cfr homologs from Bacillus amyloliquefaciens, Enterococcus faecalis, Paenibacillus lautus, and Clostridioides difficile act as C8 adenine RNA methylases in biochemical assays. C. difficile Cfr contains an additional Cys-rich C-terminal domain that binds a mononuclear Fe2+ ion in a rubredoxin-type Cys4 motif. The C-terminal domain can be truncated with minimal impact on C. difficile Cfr activity, but turnover is diminished upon disruption of the Fe2+ binding site by Zn2+ 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 C. difficile Cfr Cys-rich domain shows that it is widespread (~1000 homologs) 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 in vitro C. difficile Cfr activity, genomic co-occurrence and high abundance in the human microbiome suggests a possible functional role for a specialized rubredoxin in certain radical-SAM RNA methylases with relevance to human health.