A substrate radical intermediate in catalysis by the antibiotic resistance protein Cfr

Abstract

Cfr-dependent methylation of C8 of adenosine 2503 (A2503) in 23S rRNA confers bacterial resistance to an array of clinically important antibiotics that target the large subunit of the ribosome, including the synthetic oxazolidinone antibiotic linezolid. The key element of the proposed mechanism for Cfr, a radical S-adenosylmethionine (SAM) enzyme, is the addition of a methylene radical — generated by hydrogen-atom abstraction from the methyl group of an S-methylated cysteine residue (mCys) — onto C8 of A2503 to form a protein – nucleic acid cross-linked species containing an unpaired electron. Herein we use continuous-wave and pulsed electron paramagnetic resonance (EPR) techniques to provide direct spectroscopic evidence for this intermediate, showing a spin-delocalized radical with maximum spin density at N7 of the adenine ring. In addition, we use rapid-freeze quench EPR to show that the radical forms and decays with rate constants that are consistent with the rate of formation of the methylated product.

Mechanistic Diversity of Radical S-Adenosylmethionine (SAM)-dependent Methylation

“…EPR spectroscopic studies of the WT Cfr reaction were also used to provide evidence for the crosslinked species and to show both its chemical and its kinetic competence in advancing to the products. When EPR samples containing Cfr, a 155-nucleotide rRNA substrate analog, and dithionite (a low potential reductant used to initiate reductive cleavage of SAM) were recorded, a paramagnetic signal characteristic of an organic radical strongly coupled to a single proton was observed (41). Additional studies using specific isotopically labeled substrates in concert with electron nuclear double resonance spectroscopy and density functional theory showed that the radical was delocalized throughout the adenine ring but had maximum spin density at N7 (41).…”

“…When EPR samples containing Cfr, a 155-nucleotide rRNA substrate analog, and dithionite (a low potential reductant used to initiate reductive cleavage of SAM) were recorded, a paramagnetic signal characteristic of an organic radical strongly coupled to a single proton was observed (41). Additional studies using specific isotopically labeled substrates in concert with electron nuclear double resonance spectroscopy and density functional theory showed that the radical was delocalized throughout the adenine ring but had maximum spin density at N7 (41). Moreover, the radical-containing species was shown to be chemically and kinetically competent using rapid-freeze-quench EPR in concert with mass spectrometry to monitor the formation of product, wherein the rate of formation of the m 8 A product was consistent with the formation and decay rates of the radical species.…”

“…Moreover, the radical-containing species was shown to be chemically and kinetically competent using rapid-freeze-quench EPR in concert with mass spectrometry to monitor the formation of product, wherein the rate of formation of the m 8 A product was consistent with the formation and decay rates of the radical species. Therefore, the radical species most likely represents a mechanistically relevant intermediate and provides a launching pad for probing downstream steps of the mechanism (41).…”

Investigation of Linezolid Resistance in Staphylococci and Enterococci

“…One example of nonmutational resistance is acquisition of the natural cfr (chloramphenicol-florfenicol resistance) gene, which is a plasmid-carried gene encoding a protein which catalyzes the posttranscriptional methylation of the C-8 atom of a key residue (A2503) in the 23S rRNA (8). cfr is a highly mobile genetic element that facilitates interspecies spread, and to date, cfr has been identified in staphylococci (both S. aureus and CoNS), enterococci, streptococci, and other, less common Gram-positive pathogens (5).…”

Determinants of tRNA Recognition by the Radical SAM Enzyme RlmN

“…A unique feature of these enzymes is their ability to utilize both homolytic and heterolytic reactivity of SAM to carry out methylation of the C2 and C8 amidine carbons of adenosine [19–21]. The first equivalent of SAM is used to methylate a conserved cysteine residue (C355), unassociated with the four iron-four sulfur ([4Fe-4S]) cluster, to form a protein-bound methyl thioether [19,22–24]. A second equivalent of SAM, coordinated by the [4Fe-4S] cluster in these proteins, is then cleaved homolytically to generate a 5′-deoxyadenosyl radical (5′-dA•), a canonical feature of radical SAM catalysis [25].…”