Binding Induced RNA Conformational Changes Control Substrate Recognition and Catalysis by the Thiostrepton Resistance Methyltransferase (Tsr)

Kuiper, Emily G.; Conn, Graeme L.

doi:10.1074/jbc.m114.574780

Cited by 14 publications

(17 citation statements)

References 40 publications

(49 reference statements)

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“…The structures of both NHR and TSR in complex with SAM are dimeric and mutations to the interface between the dimer subunits are inactive or give low yields of soluble protein [ 36 ], suggesting that dimer formation stabilizes the active enzyme. RNA protein complexes of both enzymes migrate at high molecular weights in gel filtration and electrophoretic mobility shift assays [ 35 – 37 ], suggesting that the proteins may bind to the RNA as dimers. To investigate whether dimeric 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).…”

Section: Resultsmentioning

confidence: 99%

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Substrate Recognition and Modification by the Nosiheptide Resistance Methyltransferase

et al. 2015

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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.

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Section: Resultsmentioning

confidence: 99%

“…Both NHR and TSR resistance methyltransferases display similar specificities for a series of fragment and model rRNA substrates and share 74% sequence identity [ 35 – 37 ]. The crystal structures of NHR and TSR proteins in complex with SAM reveal them to have a similar tertiary fold.…”

Section: Introductionmentioning

confidence: 99%

Substrate Recognition and Modification by the Nosiheptide Resistance Methyltransferase

et al. 2015

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“…Moreover, considering that RNA binding to the Tsr dimer ostensibly follows that of SAM, it is possible that the catalytic site becomes designated from the association of RNA with the Tsr-SAM complex, which could allow for switching of the catalytic site when RNA substrates are encountered subsequent to dissociation of a methylated RNA product. Supporting evidence for this is seen by the demonstration of Tsr-induced structural changes within the RNA substrate that facilitate recognition [39]. Additional study is required to delineate the interplay between SAM and RNA binding on the catalytic activity of Tsr, particularly in the context of biologically relevant RNA acceptor substrates.…”

Section: Discussionmentioning

confidence: 97%

Functional roles inS‐adenosyl‐l‐methionine binding and catalysis for active site residues of the thiostrepton resistance methyltransferase

et al. 2015

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Resistance to the antibiotic thiostrepton, in producing Streptomycetes, is conferred by the S-adenosyl-l-methionine (SAM)-dependent SPOUT methyltransferase Tsr. For this and related enzymes, the roles of active site amino acids have been inadequately described. Herein, we have probed SAM interactions in the Tsr active site by investigating the catalytic activity and the thermodynamics of SAM binding by site-directed Tsr mutants. Two arginine residues were demonstrated to be critical for binding, one of which appears to participate in the catalytic reaction. Additionally, evidence consistent with the involvement of an asparagine in the structural organization of the SAM binding site is presented.

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“…TSR, a thiazole antibiotic, was first isolated and characterized from Streptomyces azureus (Cundliffe, 1971) in 1954 at the Squibb Institute (Pagano et al, 1956) and is used in veterinary medicine to treat mastitis, and as a topical agent for dogs. However, it has only found limited applications due to its poor solubility and toxicity (Kuiper and Conn, 2014). …”

Section: Introductionmentioning

confidence: 99%

A Cassette Containing Thiostrepton, Gentamicin Resistance Genes, and dif sequences Is Effective in Construction of Recombinant Mycobacteria

et al. 2017

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The genetic manipulation of Mycobacterium tuberculosis genome is limited by the availability of selection markers. Spontaneous resistance mutation rate of M. tuberculosis to the widely used kanamycin is relatively high which often leads to some false positive transformants. Due to the few available markers, we have created a cassette containing thiostrepton resistance gene (tsr) for selection in M. tuberculosis and M. bovis BCG, and gentamicin resistance gene (aacC1) for Escherichia coli and M. smegmatis mc2155, flanked with dif sequences recognized by the Xer system of mycobacteria. This cassette adds to the limited available selection markers for mycobacteria.

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Binding Induced RNA Conformational Changes Control Substrate Recognition and Catalysis by the Thiostrepton Resistance Methyltransferase (Tsr)

Cited by 14 publications

References 40 publications

Substrate Recognition and Modification by the Nosiheptide Resistance Methyltransferase

Substrate Recognition and Modification by the Nosiheptide Resistance Methyltransferase

Functional roles inS‐adenosyl‐l‐methionine binding and catalysis for active site residues of the thiostrepton resistance methyltransferase

A Cassette Containing Thiostrepton, Gentamicin Resistance Genes, and dif sequences Is Effective in Construction of Recombinant Mycobacteria

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