Naslov
Identification of essential residues in novel aminoglycoside resistance methyltransferase NpmA

Autori
Babić, Fedora ; Čubrilo, Sonja ; Maravić Vlahoviček, Gordana

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

Izvornik
Book of abstracts / Strelec, Ivica ; Glavaš-Obrovac, Ljubica - Osijek : Hrvatsko društvo za biokemiju i molekularnu biologiju (HDBMB), 2008

ISBN
978-953-95551-2-0

Skup
Congress of the Croatian Society for Biochemistry and Molecular Biology with international participation

Mjesto i datum
Osijek, Hrvatska, 17.09.2008. - 20.09.2008

Vrsta sudjelovanja
Poster

Vrsta recenzije
Domaća recenzija

Ključne riječi
aminoglycoside antibiotics; microbial resistance; rRNA methylation; NpmA methyltransferase

Sažetak
The methylation of ribosomal RNA is conferred by various rRNA methyltransferases. Some of these enzymes are found in antibiotic producing bacteria, where they have a protective role against the action of their own antibiotics. A growing number of antibiotic resistant strains were also found to contain different rRNA methylases, which poses a serious threat to a successful clinical outcome in treating various bacterial infections. Arm and Kam family of enzymes cause high-level resistance against aminoglycoside antibiotics in bacteria by methylating a specific nucleotide in 16S rRNA. Recently a novel plasmid mediated methyltransferase NpmA has been detected in multiple aminoglycoside resistant clinical strain of Escherichia coli. NpmA is an adenine N-1 methyltransferase specific for the A1408 position at the A site of 16S rRNA and is a member of Kam family of enzymes. In this study, we focus on the functional characterisation of the methyltransferase NpmA. We cloned the npmA gene into pET-25b(+) vector and developed the assay for functional test in vivo. By site-directed alanine mutagenesis we investigated the evolutionary conserved amino acids. We transformed the susceptible E. coli cells with recombinant vector carrying mutant genes and determined the minimal inhibitory concentration for kanamycin and gentamicin. By comparing the in vivo activity of mutant variants with the action of the wild type enzyme we got a first insight into the localisation of the active site and cofactor S-adenosyl-methionine binding site. In addition to these results, the kinetic analysis that is currently in progress will enable us to define the mechanism of action of NpmA enzyme. Our results could therefore set a basis in possible finding of a specific inhibitor of NpmA methyltransferase, which would in turn block this new type of clinical aminoglycoside resistance.

Izvorni jezik
Engleski

Znanstvena područja
Biologija

POVEZANOST RADA