The global dissemination, potential activity in diverse species and broad resistance spectrum conferred by the
aminoglycoside-resistance
ribosomal RNA methyltransferases make them a significant potential new threat to the efficacy of
aminoglycoside antibiotics in the treatment of serious
bacterial infections. The N1 methylation of
adenosine 1408 (m(1)A1408) confers resistance to structurally diverse
aminoglycosides, including
kanamycin,
neomycin and
apramycin. The limited analyses to date of the
enzymes responsible have identified common features but also potential differences in their molecular details of action. Therefore, with the goal of expanding the known
16S rRNA (m(1)A1408)
methyltransferase family as a platform for developing a more complete mechanistic understanding, we report here the cloning, expression and functional analyses of four hypothetical
aminoglycoside-resistance rRNA
methyltransferases from recent genome sequences of diverse bacterial species. Each of the genes produced a soluble, folded
protein with a secondary structure, as determined from circular dichroism (CD) spectra, consistent with
enzymes for which high-resolution structures are available. For each
enzyme,
antibiotic minimum inhibitory concentration (MIC) assays revealed a resistance spectrum characteristic of the known
16S rRNA (m(1)A1408)
methyltransferases and the modified
nucleotide was confirmed by reverse transcription as A1408. In common with other family members, higher binding affinity for the methylation reaction by-product
S-adenosylhomocysteine (SAH) than the cosubstrate
S-adenosyl-L-methionine (SAM) was observed for three
methyltransferases, while one unexpectedly showed no measurable affinity for SAH. Collectively, these results confirm that each hypothetical
enzyme is a functional
16S rRNA (m(1)A1408)
methyltransferase but also point to further potential mechanistic variation within this
enzyme family.