Cysteine methylation controls radical generation in the Cfr radical AdoMet rRNA methyltransferase

• Published in: PloS one 2013-07, Vol.8 (7), p.e67979-e67979
• Main Authors: Challand, Martin R, Salvadori, Enrico, Driesener, Rebecca C, Kay, Christopher W M, Roach, Peter L, Spencer, James
• Format: Article
• Language: English
• Subjects: Adenosine; Affinity; Antibiotics; Bacteria; Biochemistry; Biology; Clusters; Cysteine; Cysteine - metabolism; Cystine; Deoxyadenosine; Deoxyadenosines - biosynthesis; Drug resistance; E coli; Electron paramagnetic resonance; Electron Spin Resonance Spectroscopy; Enzymes; Escherichia coli; Escherichia coli Proteins - biosynthesis; Escherichia coli Proteins - genetics; Free Radicals - metabolism; Ligands; Linezolid; Methicillin; Methionine; Methylation; Methyltransferases - biosynthesis; Methyltransferases - genetics; Molecular biology; Nanotechnology; Oxidation; Peptidyltransferase; Polypeptides; Protection and preservation; Protein Binding; Recombinant Proteins; Ribonucleic acid; RNA; rRNA (adenosine-2'-0'-)-methyltransferase; rRNA 23S; rRNA methyltransferase; S-Adenosylmethionine - metabolism; Spectroscopy; Staphylococcus aureus; Substrates; Sulfur; Thiols; Transferases
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0067979

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.