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Probing the role of active site residues in an antibiotic‐resistance methyltransferase (584.9)

Many antibiotics target the bacterial ribosome to inhibit the translation of bacterial proteins, resulting in decreased bacterial growth. As pathogenic bacteria become progressively resistant to antibiotics, the need for in‐depth understanding of resistance‐conferring mechanisms becomes more critical. Streptomyces azureus expresses thiostrepton‐resistance methyltransferase (Tsr), an enzyme that confers resistance by modifying its antibiotic binding site, specifically catalyzing 2’‐hydroxyl‐methylation of adenosine 1067 on 23S rRNA. Six amino acids surrounding the S‐adenosyl‐L‐methionine (SAM) cofactor, conserved in SAM‐dependent methyltransferases, are believed to play a critical role in cofactor binding and/or catalysis. Mutating these residues was hypothesized to disrupt methylation by Tsr. Residues in the catalytic domain were individually mutated using site‐directed mutagenesis. Tsr mutants were purified using fast protein liquid chromatography (FPLC). The formation of Tsr‐cofactor complex and the catalytic activity of Tsr were evaluated with isothermal titration calorimetry and a methylation assay respectively. All Tsr mutants showed reduced methylation, but similar binding affinity to the cofactor product as wild‐type Tsr. Therefore, the major effects of the mutated amino acids are on the Tsr catalytic mechanism. Elucidating the molecular mechanisms by which Tsr recognizes and methylates its substrate will provide insight into how Tsr and similar methyltransferases confer resistance to antibiotics.