engleski

Reaction mechanism and new approach towards the inhibition of the rRNA methyltransferase ErmC

Erm methyltransferases mediate the resistance to the macrolide-lincosamide-streptogramin B antibiotics via dimethylation of a specific adenine in 23 S rRNA (A2058 in E.coli). Despite the available structural data and functional analyses on the level of the RNA-substrate, the knowledge about the reaction mechanism is still very limited, while the substrate-binding site and residues involved in RNA-recognition by the Erm enzymes remain completely unknown. To validate 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 mutants in vitro and in vivo. Our results indicate that the active site of rRNA:m6A methyltransferases is much more tolerant to amino acid substitutions than the active site of DNA:m6A methyltransferases. Only Tyr104 implicated in stabilization of the target base was found to be indispensable. Remarkably, Asn101 from the “ catalytic” motif IV and conserved Phe163 and Asn11 that form the “ floor” and one of the “ walls” 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 3D model of ErmC’ complexed with the minimal substrate. The identification of the RNA-binding site of ErmC' may be useful for structure-based design of novel drugs that specifically block the substrate-binding site of Erm methyltransferases.

MLS resistance; Erm methyltransferases; RNA binding; catalytic mechanism; inhibitors

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