Dataset Information

Substrate Recognition and Modification by a Pathogen-Associated Aminoglycoside Resistance 16S rRNA Methyltransferase.

ABSTRACT: The pathogen-associated 16S rRNA methyltransferase NpmA catalyzes m¹A1408 modification to block the action of structurally diverse aminoglycoside antibiotics. Here, we describe the development of a fluorescence polarization binding assay and its use, together with complementary functional assays, to dissect the mechanism of NpmA substrate recognition. These studies reveal that electrostatic interactions made by the NpmA ?2/3 linker collectively are critical for docking of NpmA on a conserved 16S rRNA tertiary surface. In contrast, other NpmA regions (?5/?6 and ?6/?7 linkers) contain several residues critical for optimal positioning of A1408 but are largely dispensable for 30S binding. Our data support a model for NpmA action in which 30S binding and adoption of a catalytically competent state are distinct: docking on 16S rRNA via the ?2/3 linker necessarily precedes functionally critical 30S substrate-driven conformational changes elsewhere in NpmA. This model is also consistent with catalysis being completely positional in nature, as the most significant effects on activity arise from changes that impact binding or stabilization of the flipped A1408 conformation. Our results provide a molecular framework for aminoglycoside resistance methyltransferase action that may serve as a functional paradigm for related enzymes and a starting point for development of inhibitors of these resistance determinants.

SUBMITTER: Vinal K

PROVIDER: S-EPMC5404524 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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Substrate Recognition and Modification by a Pathogen-Associated Aminoglycoside Resistance 16S rRNA Methyltransferase.

Vinal Kellie K Conn Graeme L GL

Antimicrobial agents and chemotherapy 20170424 5

The pathogen-associated 16S rRNA methyltransferase NpmA catalyzes m<sup>1</sup>A1408 modification to block the action of structurally diverse aminoglycoside antibiotics. Here, we describe the development of a fluorescence polarization binding assay and its use, together with complementary functional assays, to dissect the mechanism of NpmA substrate recognition. These studies reveal that electrostatic interactions made by the NpmA β2/3 linker collectively are critical for docking of NpmA on a co ...[more]

PMID: 28289026

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Project description:BackgroundThe proliferation of antibiotic resistant pathogens is an increasing threat to the general public. Resistance may be conferred by a number of mechanisms including covalent or mutational modification of the antibiotic binding site, covalent modification of the drug, or the over-expression of efflux pumps. The nosiheptide resistance methyltransferase (NHR) confers resistance to the thiazole antibiotic nosiheptide in the nosiheptide producer organism Streptomyces actuosus through 2'O-methylation of 23S rRNA at the nucleotide A1067. Although the crystal structures of NHR and the closely related thiostrepton-resistance methyltransferase (TSR) in complex with the cofactor S-Adenosyl-L-methionine (SAM) are available, the principles behind NHR substrate recognition and catalysis remain unclear.Methodology/principal findingsWe have analyzed the binding interactions between NHR and model 58 and 29 nucleotide substrate RNAs by gel electrophoresis mobility shift assays (EMSA) and fluorescence anisotropy. We show that the enzyme binds to RNA as a dimer. By constructing a hetero-dimer complex composed of one wild-type subunit and one inactive mutant NHR-R135A subunit, we show that only one functional subunit of the NHR homodimer is required for its enzymatic activity. Mutational analysis suggests that the interactions between neighbouring bases (G1068 and U1066) and A1067 have an important role in methyltransfer activity, such that the substitution of a deoxy sugar spacer (5') to the target nucleotide achieved near wild-type levels of methylation. A series of atomic substitutions at specific positions on the substrate adenine show that local base-base interactions between neighbouring bases are important for methylation.Conclusion/significanceTaken together these data suggest that local base-base interactions play an important role in aligning the substrate 2' hydroxyl group of A1067 for methyl group transfer. Methylation of nucleic acids is playing an increasingly important role in fundamental biological processes and we anticipate that the approach outlined in this manuscript may be useful for investigating other classes of nucleic acid methyltransferases.

Dataset Information

Substrate Recognition and Modification by a Pathogen-Associated Aminoglycoside Resistance 16S rRNA Methyltransferase.

Publications

Substrate Recognition and Modification by a Pathogen-Associated Aminoglycoside Resistance 16S rRNA Methyltransferase.

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