title: Cysteine methylation controls radical generation in the Cfr radical AdoMet rRNA methyltransferase.
abstract: 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.
authors.affiliation: School of Cellular and Molecular Medicine, University of Bristol Medical Sciences Building, Bristol, United Kingdom.
authors.forename: Martin R
authors.initials: MR
authors.lastname: Challand
authors.forename: Enrico
authors.initials: E
authors.lastname: Salvadori
authors.forename: Rebecca C
authors.initials: RC
authors.lastname: Driesener
authors.forename: Christopher W M
authors.initials: CW
authors.lastname: Kay
authors.forename: Peter L
authors.initials: PL
authors.lastname: Roach
authors.forename: James
authors.initials: J
authors.lastname: Spencer
mesh_terms: D003545:Cysteine / Q000378:metabolism*; D003839:Deoxyadenosines / Q000096:biosynthesis; D004578:Electron Spin Resonance Spectroscopy; D029968:Escherichia coli Proteins / Q000096:biosynthesis* / Q000235:genetics; D005609:Free Radicals / Q000378:metabolism*; D008024:Ligands; D008745:Methylation; D008780:Methyltransferases / Q000096:biosynthesis* / Q000235:genetics; D011485:Protein Binding; D011994:Recombinant Proteins; D012436:S-Adenosylmethionine / Q000378:metabolism*
chemical_list: D003839:Deoxyadenosines; D029968:Escherichia coli Proteins; D005609:Free Radicals; D008024:Ligands; D011994:Recombinant Proteins; C033714:5'-deoxyadenosine; D012436:S-Adenosylmethionine; C534716:Cfr protein, E coli; D008780:Methyltransferases; D003545:Cysteine