Abstract
Cfr is a radical S-adenosylmethionine (RS) methylase that appends methyl groups to C8 and C2 of adenosine 2503 in 23S rRNA. Methylation of C8 confers resistance to several classes of antibiotics that bind in or near the peptidyltransferase center of the bacterial ribosome, including the synthetic antibiotic linezolid. The Cfr reaction requires the action of five conserved cysteines, three of which ligate a required [4Fe-4S] cluster cofactor. The two remaining cysteines play a more intricate role in the reaction; one (Cys338) becomes transiently methylated during catalysis. The function of the second (Cys105) has not been rigorously established; however, in the related RlmN reaction, it (Cys118) initiates resolution of a key protein-nucleic acid cross-linked intermediate by abstracting the proton from the carbon center (C2) undergoing methylation. We previously proposed that, unlike RlmN, Cfr would utilize a polyprotic base during resolution of the protein-nucleic acid cross-linked intermediate during C8 methylation and, like RlmN, use a monoprotic base during C2 methylation. We based this proposal on the fact that solvent hydrons could exchange into the product during C8 methylation, but not during C2 methylation. Herein, we show that Cys105 of Cfr has a function similar to that of Cys118 of RlmN while methylating C8 of A2503 and provide evidence for one molecule of water that is in close contact with it, which provides the exchangeable protons during catalysis.