Characterization of a Cross-Linked Protein–Nucleic Acid Substrate Radical in the Reaction Catalyzed by RlmN

Abstract

RlmN and Cfr are methyltransferases/methylsynthases that belong to the radical S-adenosylmethionine superfamily of enzymes. RlmN catalyzes C2 methylation of adenosine 2503 (A2503) of 23S rRNA, while Cfr catalyzes C8 methylation of the exact same nucleotide, and will subsequently catalyze C2 methylation if the site is unmethylated. A key feature of the unusual mechanisms of catalysis proposed for these enzymes is the attack of a methylene radical, derived from a methylcysteine residue, onto the carbon center undergoing methylation to generate a paramagnetic protein–nucleic acid cross-linked species. This species has been thoroughly characterized during Cfr-dependent C8 methylation, but does not accumulate to detectible levels in RlmN-dependent C2 methylation. Herein, we show that inactive C118S/A variants of RlmN accumulate a substrate-derived paramagnetic species. Characterization of this species by electron paramagnetic resonance spectroscopy in concert with strategic isotopic labeling shows that the radical is delocalized throughout the adenine ring of A2503, although predominant spin density is on N1 and N3. Moreover, 13C hyperfine interactions between the radical and the methylene carbon of the formerly [methyl-13C]Cys355 residue show that the radical species exists in a covalent cross-link between the protein and the nucleic acid substrate. X-ray structures of RlmN C118A show that, in the presence of SAM, the substitution does not alter the active site structure compared to that of the wild-type enzyme. Together, these findings have new mechanistic implications for the role(s) of C118 and its counterpart in Cfr (C105) in catalysis, and suggest involvement of the residue in resolution of the cross-linked species via a radical mediated process.

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

“…Interestingly, the radical is observed in RlmN C118A or C118S variants (35). The EPR spectrum obtained with the C118S variant is consistent with the proposed radical species observed in the Cfr reaction, except the radical is more strongly coupled to C2 of the adenine ring rather than C8, and the majority of spin density is located on N3 rather than N7.…”

“…The EPR spectrum obtained with the C118S variant is consistent with the proposed radical species observed in the Cfr reaction, except the radical is more strongly coupled to C2 of the adenine ring rather than C8, and the majority of spin density is located on N3 rather than N7. Although the radical intermediate forms and decays on the time scale of product formation in the WT Cfr reaction, the radical species obtained with RlmN C118S, or a similar variant in Cfr (C105A), accumulates but does not decay to any significant extent (35). This observation suggests that this second conserved cysteine is necessary for decay of the radical intermediate in both the RlmN and Cfr reactions, implicating it in a role other than, or in addition to, formation of a disulfide bond as initially proposed.…”

“…In addition to the three cysteines that ligate the iron-sulfur (Fe/S) cluster cofactor, RlmN and Cfr contain two additional strictly conserved cysteines that are essential for catalysis (33)(34)(35). These additional cysteines distinguish Class A enzymes from other RS methylases and contribute to their unique reaction mechanism (11,33,35).…”

“…These additional cysteines distinguish Class A enzymes from other RS methylases and contribute to their unique reaction mechanism (11,33,35). Initial in vitro characterization of RlmN and Cfr by Fujimori and co-workers (36) demonstrated that both SAH and 5Ј-dA are produced during the reaction, suggesting that SAM is used both as a radical initiator and as the source of the methyl group.…”

“…In these scenarios, Cys-118 of RlmN (Cys-105 of Cfr) may (i) participate in a disulfide radical species with Cys-335 after deprotonation at C2 and radical fragmentation; (ii) act as the base that abstracts the C2 proton; or (iii) act in both capacities. The last step would be acquisition of an electron by the resulting thiyl radical or disulfide radical anion to regenerate the Cys-355 thiolate (35). Scheme 1 depicts the scenario that we currently favor, in which Cys-118 acts as the base that abstracts the C2 proton.…”