Ribosomes are responsible for synthesizing proteins by translating the genetic code into an amino acid sequence. The crucial step for establishing translational fidelity is ribosomal recognition of the codon-anticodon interaction performed at the A-site located on the 30S subunit. Aminoglycosides are bactericidal antibiotics, which interfere with the decoding of mRNA by binding to the A-site of 16S rRNA, hence causing errors in translation. One of the mechanisms of aminoglycoside resistance is modification of specific nucleotide residues in the aminoglycoside binding site via 16S rRNA methyltransferases, leading to decreased or lost affinity of the antibiotic for its target. The 16S rRNA aminoglycoside-resistance methyltransferases are classified into two groups based on the site of modification of 16S rRNA which can be N1 position of nucleotide A1408 or N7 position of nucleotide G1405. Methyltransferases studied in this work are KamB from the natural antibiotic producer Streptoalloteichus tenebrarius, and NpmA from the clinical E. coli strain ARS3. Both enzymes confer high-level resistance to 4,6- and 4,5-disubstituted 2-deoxystreptamines and to apramycin due to the methylation of the N1 position of A1408. The aim of this work was to observe the effects of single mutations in ribosomal A site on KamB and NpmA methyltransferase activity by determining their ability to methylate nucleotide A1408. The RNA used for a reverse transcriptase primer extension reaction was extracted from E. coli strain MC338 with either wild type 16S rRNA or mutant versions with single mutations U1406A, C1409U, G1491U, G1491C, G1491A, U1495A and expressing methyltransferase KamB or NpmA. Obtained extension fragments were separated by electrophoresis on 9% denaturing polyacrylamide gel. Analysis of the results suggests that nucleotide G1491 could be one of the important binding sites for both KamB and NpmA enzymes due to the decrease in their activity in the case of a single mutation at that position. Results also imply that methyltransferases KamB and NpmA could have a similar binding pattern to the A-site of small ribosomal subunit and thus similar interactions with nucleotides in 16S rRNA molecule. This work gives an insight into the KamB and NpmA interactions with the 16S rRNA molecule and hence provides a contribution to the study of their mechanisms of action, as well as to the study of highly conserved decoding site of small ribosomal subunit. Results obtained in this work can provide a basis for further research that could lead to the development of inhibitors that would specifically block the activity of this methyltransferases and thus prevent pathogenic bacterial resistance to aminoglycosides.