Abstract
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 2ʹO-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 (5ʹ) 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.
Highlights
The covalent modification of nucleic acids, proteins and small molecules through the addition of methyl groups is an important part of cellular metabolism
To investigate whether dimeric nosiheptide resistance methyltransferase (NHR) is required for RNA binding, we prepared NHR protein containing two different N-terminal affinity tags; His-tagged NHR (His-NHR) and thioredoxin-tagged NHR (Trx-NHR)
Electrophoretic mobility shift assays (EMSA) of 58 or 29 nt RNA substrates show that binding by His-NHR or TrxNHR caused a significant retardation to the RNA protein complexes in the gel relative to unbound RNA, and that the RNA-His-NHR and RNA-Trx-NHR complexes can be resolved in the gel system in proportion to the relative sizes of each affinity tag (Fig 2)
Summary
The covalent modification of nucleic acids, proteins and small molecules through the addition of methyl groups (methylation) is an important part of cellular metabolism. In most cases substrate molecules are methylated through methyl group transfer from the donor molecule S-adenosylmethionine (SAM) by methyltransferase enzymes. The basic chemistry of methylation by SAM–dependent methyltransferases normally proceeds by direct transfer of the methyl group from SAM to a nucleophillic substrate by an SN-2 like mechanism [12,13], a class of SAM–dependent methyltransferases that methylate non-nucleophilic carbon atoms through an intermediate 50-deoxyadenosyl 50- radical has recently been identified [14]. 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 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
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