Cysteine Methylation Controls Radical Generation in the Cfr Radical AdoMet rRNA MethyltransferaseReportar como inadecuado

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The ‘radical S-adenosyl-L-methionine AdoMet’ enzyme Cfr methylates adenosine 2503 of the 23S rRNA in the peptidyltransferase centre P-site of the bacterial ribosome. This modification protects host bacteria, notably methicillin-resistant Staphylococcus aureus MRSA, from numerous antibiotics, including agents e.g. linezolid, retapamulin that were developed to treat such organisms. Cfr contains a single 4Fe-4S cluster that binds two separate molecules of AdoMet during the reaction cycle. These are used sequentially to first methylate a cysteine residue, Cys338; and subsequently generate an oxidative radical intermediate that facilitates methyl transfer to the unreactive C8 and-or C2 carbon centres of adenosine 2503. How the Cfr active site, with its single 4Fe-4S cluster, catalyses these two distinct activities that each utilise AdoMet as a substrate remains to be established. Here, we use absorbance and electron paramagnetic resonance EPR spectroscopy to investigate the interactions of AdoMet with the 4Fe-4S clusters of wild-type Cfr and a Cys338 Ala mutant, which is unable to accept a methyl group. Cfr binds AdoMet with high ∼ 10 µM affinity notwithstanding the absence of the RNA cosubstrate. In wild-type Cfr, where Cys338 is methylated, AdoMet binding leads to rapid oxidation of the 4Fe-4S cluster and production of 5-deoxyadenosine DOA. In contrast, while Cys338 Ala Cfr binds AdoMet with equivalent affinity, oxidation of the 4Fe-4S cluster is not observed. Our results indicate that the presence of a methyl group on Cfr Cys338 is a key determinant of the activity of the enzyme towards AdoMet, thus enabling a single active site to support two distinct modes of AdoMet cleavage.

Autor: Martin R. Challand, Enrico Salvadori, Rebecca C. Driesener, Christopher W. M. Kay , Peter L. Roach , James Spencer



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