Modeling and experimental analyses reveal a two-domain structure and amino acids important for the activity of aminoglycoside resistance methyltransferase Sgm.
Methyltransferases that carry out posttranscriptional N7-methylation of G1405 in 16S rRNA confer bacterial resistance to aminoglycoside antibiotics, including kanamycin and gentamicin. Genes encoding enzymes from this family (hereafter referred to as Arm, for aminoglycoside resistance methyltransferases) have been recently found to spread by horizontal gene transfer between various human pathogens. The knowledge of the Arm protein structure would lay the groundwork for the development of potential resistance inhibitors, which could be used to restore the potential of aminoglycosides to act against the resistant pathogens. We analyzed the sequence-function relationships of Sgm MTase, a member of the Arm family, by limited proteolysis and site-directed and random mutagenesis. We also modeled the structure of Sgm using bioinformatics techniques and used the model to provide a structural context for experimental results. We found that Sgm comprises two domains and we characterized a number of functionally compromised point mutants with substitutions of invariant or conserved residues. Our study provides a low-resolution (residue-level) model of sequence-structure-function relationships in the Arm family of enzymes and reveals the cofactor-binding and substrate-binding sites. These functional regions will be prime targets for further experimental and theoretical studies aimed at defining the reaction mechanism of m7 G1405 methylation, increasing the resolution of the model and developing Arm-specific inhibitors.
Maravić Vlahovicek
Gordana
G
Department of Biochemistry and Molecular Biology, Faculty of Pharmacy and Biochemistry, University of Zagreb, Ante Kovacića 1, 10000 Zagreb, Croatia. gordana@pharma.hr
Cubrilo
Sonja
S
Tkaczuk
Karolina L
KL
Bujnicki
Janusz M
JM
D000595:Amino Acid Sequence; D000617:Aminoglycosides / Q000494:pharmacology*; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D019295:Computational Biology; D024881:Drug Resistance, Bacterial; D005839:Gentamicins / Q000494:pharmacology; D007612:Kanamycin / Q000494:pharmacology; D008780:Methyltransferases / Q000737:chemistry* / Q000235:genetics; D008847:Micromonospora / Q000145:classification / Q000187:drug effects / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010802:Phylogeny; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D005839:Gentamicins; D007612:Kanamycin; D008780:Methyltransferases