Naslov
Mutational analysis defines the roles of conserved residues in the predicted catalytic pocket and suggests a model of protein-RNA interactions of the methyltransferase ErmC’

Autori
Maravić, Gordana ; Bujnicki, Janusz M. ; Feder, Marcin ; Pongor, Sandor ; Flogel, Mirna

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
Zbornik sažetaka, Prvi kongres hrvatskih znanstvenika iz domovine i inozemstva / Kniewald, Zlatko - Zagreb : Akademija tehničkih znanosti Hrvatske (HATZ), 2004

Skup
Prvi kongres hrvatskih znanstvenika iz domovine i inozemstva

Mjesto i datum
Zagreb, Hrvatska, 15.11.2004. - 19.11.2004

Vrsta sudjelovanja
Poster

Vrsta recenzije
Nije recenziran

Ključne riječi
antibiotic resistance; rRNA methyltransferase; Erm; mutagenesis; inhibitor

Sažetak
Methyltransferases from the Erm family catalyze S-adenosyl-L-methionine-dependent modification of a specific adenine residue in bacterial 23S rRNA (A2058 in E. coli), thereby conferring resistance to clinically important macrolide-lincosamide-streptogramin B antibiotics. Despite the available structural data and functional analyses on the level of the RNA substrate, the knowledge about the mechanism of rRNA:adenine-N6 methylation is still very limited, while the substrate-binding site and the amino acids involved in RNA recognition by the Erm MTases remain completely unknown. 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:m6A methyltransferases. It has also been postulated that the small C-terminal domain functions as a target-binding module, but none of aforesaid predictions has been tested experimentally. To validate both the structure-based predictions of presumably essential residues in the catalytic pocket of ErmC’ , and to identify the area of protein-RNA interactions 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:m6A MTases is much more tolerant to amino acid substitutions than the active site of DNA:m6A 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’ . Furthermore, our results suggest that the key RNA-binding residues are located not in the small domain, but in the large catalytic domain, facing the cleft between the two domains. Based on the mutagenesis data, we were able to construct the preliminary three-dimensional model of ErmC’ complexed with the minimal substrate. The identification of the RNA-binding site of ErmC' may be useful for the structure-based design of novel drugs that do not necessarily bind to the cofactor-binding site common to many AdoMet-dependent MTases, but specifically block the substrate-binding site of MTases from the Erm family.

Izvorni jezik
Engleski

Znanstvena područja
Biologija

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