m(5)C RNA and m(5)C DNA methyl transferases use different cysteine residues as catalysts

A family of RNA m(5)C methyl transferases (MTases) containing over 55 membe rs in eight subfamilies has been identified recently by an iterative search of the genomic sequence databases by using the known 16S rRNA m(5)C 967 MT ase. Fmu, as an initial probe. The RNA m(5)C MTase family contained sequenc e motifs that were highly homologous to motifs in the DNA m(5)C MTases. inc luding the ProCys sequence that contains the essential Cys catalyst of the functionally similar DNA-modifying enzymes; it was reasonable to assign the Cys nucleophile to be that in the conserved ProCys. The family also contai ned an additional conserved Cys residue that aligns with the nucleophilic c atalyst in m(5)U54 tRNA MTase. surprisingly, the mutant of the putative Cys catalyst in the ProCys sequence was active and formed a covalent complex w ith 5-fluorocytosine-containing RNA, whereas the mutant at the other conser ved Cys was inactive and unable to form the complex. Thus. notwithstanding the highly homologous sequences and similar functions, the RNA m(5)C MTase uses a different Cys as a catalytic nucleophile than the DNA m(5)C MTases. The catalytic Cys seems to be determined, not by the target base that is mo dified, but by whether the substrate is DNA or RNA. The function of the con served ProCys sequence in the RNA m(5)C MTases remains unknown.