Active Site in RrmJ, a Heat Shock-induced Methyltransferase

The heat shock protein RrmJ (FtsJ), highly conserved from eubacteria to eukarya, is responsible for the 2′- O -ribose methylation of the universally conserved base U2552 in the A-loop of the 23 S rRNA. Absence of this methylation, which occurs late in the maturation process of the ribosome, appears to cause the destabilization and premature dissociation of the 50 S ribosomal subunit. To understand the mechanism of 2′- O -ribose methyltransfer reactions, we characterized the enzymatic parameters of RrmJ and conducted site-specific mutagenesis of RrmJ. A structure based sequence alignment with VP39, a structurally related 2′- O -methyltransferase from vaccinia virus, guided our mutagenesis studies. We analyzed the function of our RrmJ mutants in vivo and characterized the methyltransfer reaction of the purified proteins in vitro . The active site of RrmJ appears to be formed by a catalytic triad consisting of two lysine residues, Lys-38 and Lys-164, and the negatively charged residue Asp-124. Another highly conserved residue, Glu-199, that is present in the active site of RrmJ and VP39 appears to play only a minor role in the methyltransfer reaction in vivo . Based on these results, a reaction mechanism for the methyltransfer activity of RrmJ is proposed.