Structural basis for the methylation of G1405 in 16S rRNA by aminoglycoside resistance methyltransferase Sgm from an antibiotic producer: a diversity of active sites in m 7 G methyltransferases

Abstract

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.

The aminoglycoside resistance methyltransferases from the ArmA/Rmt family operate late in the 30S ribosomal biogenesis pathway

“…1,2). A similarly ambiguous 16S rRNA footprint for the interaction of Sgm, an ArmA ortholog, with the 30S subunits was recently reported (Husain et al 2010). An attempt to measure a binding constant between ArmA and the 30S subunits failed in our hands (data not shown).…”

rRNA Methyltransferases and their Role in Resistance to Antibiotics

“…It has been established that both of them are composed of two domains, N-and C-terminus, with the same functions as in Sgm MTase. Not long after, the Sgm crystal structure was resolved, and it has become the first determined structure of 16S rRNA MTases from antibiotic producers (84). Also, for the first time the interactions between G1405 MTase and its substrate, the 30S subunit, were characterized (84).…”

“…Not long after, the Sgm crystal structure was resolved, and it has become the first determined structure of 16S rRNA MTases from antibiotic producers (84). Also, for the first time the interactions between G1405 MTase and its substrate, the 30S subunit, were characterized (84). It has been learned that conformational change of the 30S subunit upon Sgm binding involves the rearrangement of helix 44, and most likely baseflipping of G1405.…”

Aminoglycoside resistance 16S rRNA methyltransferases block endogenous methylation, affect translation efficiency and fitness of the host

“…Site-directed hydroxyl radical probing failed to define the regions of 16S rRNA involved in the interaction with Sgm and ArmA (Husain et al 2010;Zarubica et al 2011). Nucleotides found to be protected or deprotected in the presence of the methyltransferases are scattered over a wide area, suggesting that ArmA and Sgm induce global conformational changes in the 30S ribosomal subunit that allow both enzymes to access the buried nucleotide G1405.…”

“…Nucleotides found to be protected or deprotected in the presence of the methyltransferases are scattered over a wide area, suggesting that ArmA and Sgm induce global conformational changes in the 30S ribosomal subunit that allow both enzymes to access the buried nucleotide G1405. (Husain et al 2010). Zarubica et al (2011) hypothesize that the ArmA family of enzymes exploits a labile property of the mature 30S ribosomal subunit formation, rather than an intermediate on the pathway to subunit formation, to gain access to G1405.…”