Mutational analysis defines the roles of conserved amino acid residues in the predicted catalytic pocket of the rRNA:m6A methyltransferase ErmC'.
Methyltransferases (MTases) from the Erm family catalyze S-adenosyl-L-methionine-dependent modification of a specific adenine residue in bacterial 23S rRNA, thereby conferring resistance to clinically important macrolide, lincosamide and streptogramin B antibiotics. Despite the available structural data and functional analyses on the level of the RNA substrate, still very little is known about the mechanism of rRNA:adenine-N(6) methylation. Only predictions regarding various aspects of this reaction have been made based on the analysis of the crystal structures of methyltransferase ErmC' (without the RNA) and their comparison with the crystallographic and biochemical data for better studied DNA:m(6)A MTases. To validate the structure-based predictions of presumably essential residues in the catalytic pocket of ErmC', we carried out the site-directed mutagenesis and studied the function of the mutants in vitro and in vivo. Our results indicate that the active site of rRNA:m(6)A MTases is much more tolerant to amino acid substitutions than the active site of DNA:m(6)A MTases. Only the Y104 residue implicated in stabilization of the target base was found to be indispensable. Remarkably, the N101 residue from the "catalytic" motif IV and two conserved residues that form the floor (F163) and one of the walls (N11) of the base-binding site are not essential for catalysis in ErmC'. This somewhat surprising result is discussed in the light of the available structural data and in the phylogenetic context of the Erm family.
Maravić
Gordana
G
Protein Structure and Bioinformatics Group, International Centre for Genetic Engineering and Biotechnology, Padriciano 99, 34012 Trieste, Italy. gordana@pharma.hr
Feder
Marcin
M
Pongor
Sándor
S
Flögel
Mirna
M
Bujnicki
Janusz M
JM
D018829:Adhesins, Bacterial / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry*; D020134:Catalytic Domain; D004352:Drug Resistance, Microbial; D008780:Methyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D016415:Sequence Alignment
D018829:Adhesins, Bacterial; D000596:Amino Acids; D012313:RNA; D008780:Methyltransferases; C035181:rRNA (adenosine-O-2'-)methyltransferase