Critical Residues for Cofactor Binding and Catalytic Activity in the Aminoglycoside Resistance Methyltransferase Sgm

J Bacteriol. 2008 Sep;190(17):5855-61. doi: 10.1128/JB.00076-08. Epub 2008 Jun 27.

Abstract

The 16S rRNA methyltransferase Sgm from "Micromonospora zionensis" confers resistance to aminoglycoside antibiotics by specific modification of the 30S ribosomal A site. Sgm is a member of the FmrO family, distant relatives of the S-adenosyl-L-methionine (SAM)-dependent RNA subfamily of methyltransferase enzymes. Using amino acid conservation across the FmrO family, seven putative key amino acids were selected for mutation to assess their role in forming the SAM cofactor binding pocket or in methyl group transfer. Each mutated residue was found to be essential for Sgm function, as no modified protein could effectively support bacterial growth in liquid media containing gentamicin or methylate 30S subunits in vitro. Using isothermal titration calorimetry, Sgm was found to bind SAM with a K(D) (binding constant) of 17.6 microM, and comparable values were obtained for one functional mutant (N179A) and four proteins modified at amino acids predicted to be involved in catalysis in methyl group transfer. In contrast, none of the G135, D156, or D182 Sgm mutants bound the cofactor, confirming their role in creating the SAM binding pocket. These results represent the first functional characterization of any FmrO methyltransferase and may provide a basis for a further structure-function analysis of these aminoglycoside resistance determinants.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aminoglycosides / pharmacology*
  • Anti-Bacterial Agents / pharmacology
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Binding Sites / genetics
  • Calorimetry
  • Catalysis
  • Circular Dichroism
  • Drug Resistance, Bacterial
  • Methylation
  • Methyltransferases / chemistry
  • Methyltransferases / genetics
  • Methyltransferases / metabolism*
  • Microbial Sensitivity Tests
  • Micromonospora / drug effects*
  • Micromonospora / genetics
  • Micromonospora / metabolism
  • Mutagenesis, Site-Directed
  • Mutation
  • Protein Binding
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Ribosome Subunits, Small, Bacterial / genetics
  • Ribosome Subunits, Small, Bacterial / metabolism
  • Sequence Homology, Amino Acid
  • Structural Homology, Protein

Substances

  • Aminoglycosides
  • Anti-Bacterial Agents
  • Bacterial Proteins
  • Methyltransferases