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J Biol Chem. 2019 Oct 8. pii: jbc.RA119.011181. doi: 10.1074/jbc.RA119.011181. [Epub ahead of print]

Functionally critical residues in the aminoglycoside resistance-associated methyltransferase RmtC play distinct roles in 30S substrate recognition.

Author information

1
Emory University School of Medicine, United States.
2
Florida State University, United States.
3
Emory University School of Medicine.
4
Biochemistry, Emory University School of Medicine, United States.

Abstract

Methylation of the small ribosome subunit rRNA in the ribosomal decoding center results in exceptionally high-level aminoglycoside resistance in bacteria. 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 structure of the methyltransferase RmtC at 3.14 Å resolution, here we report a structure-guided functional study of 30S substrate recognition by the aminoglycoside resistance-associated 16S rRNA (m7G1405) methyltransferases. We found that the binding site for these enzymes in the 30S subunit directly overlaps with that of a second family of aminoglycoside resistance-associated 16S rRNA (m1A1408) methyltransferases, suggesting both groups of enzymes may exploit the same conserved rRNA tertiary surface for docking to the 30S. Within RmtC, we defined an N-terminal domain surface, comprising basic residues from both the N1 and N2 subdomains, that directly contributes to 30S-binding affinity. In contrast, additional residues lining a contiguous adjacent surface on the C-terminal domain were critical for 16S rRNA modification, but did not directly contribute to the binding affinity. The results from our experiments 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 for potential exploitation of unique aspects of substrate recognition in future therapeutic strategies.

KEYWORDS:

RNA methylation; RNA methyltransferase; RmtC; aminoglycoside antibiotics; antibiotic resistance; m1A1408; m7G1405; ribosomal ribonucleic acid (rRNA) (ribosomal RNA); ribosome

PMID:
31594862
DOI:
10.1074/jbc.RA119.011181
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