TY - JOUR T1 - Critical residues in the aminoglycoside-resistance 16S rRNA (m<sup>7</sup>G1405) methyltransferase RmtC play distinct roles in 30S substrate recognition JF - bioRxiv DO - 10.1101/712810 SP - 712810 AU - Meisam Nosrati AU - Debayan Dey AU - Atousa Mehrani AU - Sarah E. Strassler AU - Natalia Zelinskaya AU - Eric D. Hoffer AU - Scott M. Stagg AU - Christine M. Dunham AU - Graeme L. Conn Y1 - 2019/01/01 UR - http://biorxiv.org/content/early/2019/09/19/712810.abstract N2 - Methylation of the small ribosome subunit rRNA in the ribosomal decoding center results in exceptionally high-level aminoglycoside resistance. Enzymes that methylate 16S rRNA on N7 of nucleotide G1405 (m7G1405) have been identified in both aminoglycoside-producing and clinically drug-resistant pathogenic bacteria. Using a fluorescence polarization 30S binding assay and a new crystal of the methyltransferase RmtC, we report a structure-guided functional study of 30S substrate recognition by the aminoglycoside-resistance 16S rRNA (m7G1405) methyltransferases. We find that the 30S binding site for these enzymes directly overlaps that of a second family of aminoglycoside-resistance 16S rRNA (m1A1408) methyltransferases, suggesting both groups of enzymes may exploit the same conserved rRNA tertiary surface for docking on the 30S. Within RmtC we define an amino-terminal domain surface, comprising basic residues from both the N1 and N2 subdomains, that directly contribute to 30S binding affinity. In contrast, additional residues lining a contiguous adjacent surface on the CTD are found to be critical for 16S rRNA modification but do not directly contribute to binding affinity. Thus, our studies define the critical features of m7G1405 methyltransferase-substrate recognition and distinguish at least two distinct, functionally critical contributions of the tested enzyme residues: 30S binding affinity and stabilizing a binding-induced 16S rRNA conformation necessary for G1405 modification. Our study sets the scene for future high-resolution structural studies of the 30S-methyltransferase complex and potential exploitation of unique aspects of substrate recognition for future therapeutic purposes.AMEaminoglycoside modifying enzymeCA-MHBcation-adjusted Mueller-Hinton brothCTDcarboxy-terminal domainFPfluorescence polarizationh44(16S rRNA) helix 44MICminimum inhibitory concentrationNTDamino-terminal domainRmt(aminoglycoside) resistance methyltransferaseSAHS-adenosylhomocysteineSAMS-adenosyl-L-methionineTiinflection temperature ER -