Chemistry Department Seminar: Squire Booker (Penn State)

“A Radical Approach to Antibiotic Resistance.”

RlmN catalyzes the methylation of C2 of adenosine 2503 (A2503) in bacterial 23S rRNA. This activity is widespread, if not ubiquitous, among bacteria, and enhances the efficiency of translation. An evolutionarily related enzyme, Cfr, preferentially catalyzes the methylation of C8 of the exact same nucleotide, but will also catalyze the methylation of C2. The methylation of C8 of A2503, which is located ultimately in the peptidyltransferase center of the ribosome, confers resistance to multiple classes of clinically relevant antibiotics. Recent studies indicate that RlmN is also responsible for the methylation of adenosine 37 in several tRNAs in Escherichia coli and most likely other bacteria.¹ Both RlmN and Cfr belong to the radical S-adenosylmethionine (SAM) superfamily of enzymes and operate via similar radical-based mechanisms of catalysis.  Key features of their mechanisms are: i) the transfer of a methyl group from SAM to a conserved C-terminal cysteine on the protein by a polar S_N2 process; ii) abstraction of a hydrogen atom from the resulting methylCys residue by a 5’-deoxyadenosyl 5’-radical generated from a second molecule of SAM; iii) addition of the resulting methylene radical to the carbon undergoing methylation to generate a paramagnetic protein–nucleic acid cross-linked intermediate; and resolution of the cross-linked intermediate by disulfide-bond formation.² All steps leading to formation of the cross-linked species are supported by strong biochemical, spectroscopic, and structural data, while those governing resolution of the cross-linked intermediate have been less well established.^3,4,5 In this lecture, evidence is presented for resolution of the cross-linked intermediate via a radical-dependent pathway in which a second, strictly conserved, cysteine plays a key role. In addition, in vitro evidence for the ability of RlmN to modify tRNA is presented.⁶