Sgm (Sisomicin-gentamicin methyltransferase) from antibiotic-producing bacterium Micromonospora zionensis is an enzyme that confers resistance to aminoglycosides like gentamicin and sisomicin by specifically methylating G1405 in bacterial 16S rRNA. Sgm belongs to the aminoglycoside resistance methyltransferase (Arm) family of enzymes that have been recently found to spread by horizontal gene transfer among disease-causing bacteria. Structural characterization of Arm enzymes is the key to understand their mechanism of action and to develop inhibitors that would block their activity. Here we report the structure of Sgm in complex with cofactors S-adenosylmethionine (AdoMet) and S-adenosylhomocysteine (AdoHcy) at 2.0 and 2.1 Å resolution, respectively, and results of mutagenesis and rRNA footprinting, and protein-substrate docking. We propose the mechanism of methylation of G1405 by Sgm and compare it with other m7G methyltransferases, revealing a surprising diversity of active sites and binding modes for the same basic reaction of RNA modification. This analysis can serve as a stepping stone towards developing drugs that would specifically block the activity of Arm methyltransferases and thereby re-sensitize pathogenic bacteria to aminoglycoside antibiotics.

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Methyltransferases from the Erm family catalyze S-adenosyl-L-methionine-dependent modification of a specific adenine residue in bacterial 23S rRNA, thereby conferring resistance to clinically important macrolide, lincosamide, and streptogramin B antibiotics. Thus far, no inhibitors of these enzymes have been identified or designed that would effectively abolish the resistance in vivo. We used the crystal structure of ErmC' methyltransferase as a target for structure-based virtual screening of a database composed of 58,679 lead-like compounds. Among 77 compounds selected for experimental validation (63 predicted to bind to the catalytic pocket and 14 compounds predicted to bind to the putative RNA binding site), we found several novel inhibitors that decrease the minimal inhibitory concentration of a macrolide antibiotic erythromycin toward an Escherichia coli strain that constitutively expresses ErmC'. Eight of them have IC(50) values in the micromolar range. Analysis of docking models of the identified inhibitors suggests a novel strategy to develop potent and clinically useful inhibitors.

Ribosome-targeting antibiotics block protein synthesis by binding at functionally important regions of the bacterial rRNA. Resistance is often conferred by addition of a methyl group at the antibiotic binding site within an rRNA region that is already highly modified with several nucleotide methylations. In bacterial rRNA, each methylation requires its own specific methyltransferase enzyme, and this raises the question as to how an extra methyltransferase conferring antibiotic resistance can be accommodated and how it can gain access to its nucleotide target within a short and functionally crowded stretch of the rRNA sequence. Here, we show that the Sgm methyltransferase confers resistance to 4,6-disubstituted deoxystreptamine aminoglycosides by introducing the 16S rRNA modification m 7 G1405 within the ribosomal A site. This region of Escherichia coli 16S rRNA already contains several methylated nucleotides including m 4 Cm1402 and m 5 C1407. Modification at m 5 C1407 by the methyltransferase RsmF is impeded as Sgm gains access to its adjacent G1405 target on the 30S ribosomal subunit. An Sgm mutant (G135A), which is impaired in S-adenosylmethionine binding and confers lower resistance, is less able to interfere with RsmF methylation on the 30S subunit. The two methylations at 16S rRNA nucleotide m 4 Cm1402 are unaffected by both the wild-type and the mutant versions of Sgm. The data indicate that interplay between resistance methyltransferases and the cell's own indigenous methyltransferases can play an important role in determining resistance levels.