Naslov
Site-directed mutagenesis of 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 / Vujaklija, Dušica ; Pigac, Jasenka ; Hađina, Suzana ; Kosalec, Ivan - Zagreb : Hrvatsko mikrobiološko društvo, 2008

ISBN
978-953-96567-7-3

Skup
4th Croatian Congress of Microbiology with international participation

Mjesto i datum
Zadar, Hrvatska, 24.09.2008. - 27.09.2008

Vrsta sudjelovanja
Poster

Vrsta recenzije
Domaća recenzija

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

Sažetak
Some antibiotic producing bacteria as well as growing number of antibiotic resistant strains contain different rRNA methylases, which protect their protein synthesis from the destructive action of ribosomal antibiotics. The high-level aminoglycoside resistance in bacteria is caused by the action of Arm and Kam family of enzymes that methylate 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. The aim of our study is to functionally characterise the methyltransferase NpmA. We cloned the npmA gene into pET-25b(+) vector and developed the assay for functional test in vivo. By site-directed mutagenesis we changed the evolutionary conserved amino acids into alanines. We transformed the susceptible E. coli cells with recombinant vector carrying mutant genes and determined the minimal inhibitory concentration for kanamycin and gentamicin. Comparision of the in vivo activity of mutant variants with the action of the wild type enzyme gave us the first insight into the localisation of the active site and cofactor S-adenosyl-methionine binding site. Obtained results together with the additional kinetic analysis that is currently in progress will enable us to define the mechanism of action of NpmA enzyme and investigate the possibilities of finding a specific inhibitor that would block this new type of clinical aminoglycoside resistance.

Izvorni jezik
Engleski

Znanstvena područja
Biologija

POVEZANOST RADA