In this study, we purified and analyzed enzymatic activities of two Radical SAM methyltransferases that operate upon the same nucleotide residue in a 3000 nucleotide-long RNA. RlmN introduces a “native” C2 methylation of A2503 in the bacterial 23S rRNA, whereas Cfr methylates the C8 position and confers antibiotic resistance. Unlike methylation of other RNA bases via a polar mechanism (for example, enzymatic mono- and dimethylation of A2058 in 23S rRNA that leads to macrolide resistance54
), the modifications of A2503 are unique as they are carried out on amidine carbons of the substrate. These carbon-carbon bond forming methyl transfer reactions are catalyzed by two methyl transferases, RlmN and Cfr, both members of the Radical SAM enzyme superfamily (). While members of this family have been shown to catalyze a number of transformations, to date no methyltransferase activity has been demonstrated in vitro
. By showing that purified and in vitro
reconstituted RlmN and Cfr catalyze methylation of 23S rRNA and by obtaining experimental evidence consistent with the radical mechanism, we now add C-methyltransferase activity to the inventory of transformations catalyzed by Radical SAM enzymes.
23S rRNA modifications catalyzed by RlmN and Cfr.
In this study, RlmN and Cfr were cloned and expressed as His-tagged fusions. Both enzymes were purified under anaerobic conditions and their iron-sulfur clusters reconstituted. Both enzymes were capable of methylation of 23S rRNA. The nature of the enzymatic products was established by co-elution of these products, digested to single nucleosides, with synthetic standards. These experiments showed that while RlmN catalyzes methylation at position C2 of the substrate, Cfr could catalyze methylation at two distinct positions of the substrate adenine nucleotide (C2 and C8), with the physiologically relevant C8 as the preferred substrate. This observation also indicates that RlmN action is not a prerequisite for Cfr activity and that the two enzymes may even compete for the unmethylated rRNA substrate during ribosomal assembly (see below). Our observation that the C2 methylation is catalyzed by RlmN, while the C8 methylation is performed by Cfr is in agreement with in vivo
studies in which the nature of modification was determined by TLC analysis and mass spectrometry.5,11
In addition, our studies unambiguously established that SAM is a methyl donor for both enzymes, as radioactivity was transferred to the RNA substrate when [3
H-methyl]-SAM was used in the reaction.
The most intriguing aspect of the RlmN and Cfr-catalyzed methylations is the ability of the enzymes to append a methyl group to aromatic carbon centers. Such a reaction requires high activation energy, as the homolytic bond dissociation energy for hydrogen atom abstraction from the C2 carbon of adenosine is approximately 98 kcal·mol−1
, among the highest known for anaerobic C-H bond cleavage.18,55
These enzymes catalyze adenosine methylation by using a radical mechanism for substrate activation. The radical chemistry is enabled by the [4Fe-4S] cluster, the presence of which was established by UV-Vis spectroscopy and the iron content determination of the purified and reconstituted proteins. Mutation of any of the conserved cysteines within the characteristic CX3
C abolished methyltransferase activity for both RlmN and Cfr. This is consistent with the in vivo
observation that mutants of the iron-sulfur cluster-coordinating cysteine residues in Cfr fail to protect bacterial cells from antibiotic action.5
We observed that the enzymatic methylation requires the presence of sodium dithionite, a common one-electron donor in the reactions catalyzed by Radical SAM enzymes and a surrogate for physiological reductant, likely necessary for the reduction of the iron-sulfur cluster to the +1 oxidation state, and initiation of the reaction cycle ().
In addition to the involvement of the [4Fe-4S] cluster, a hallmark of Radical SAM enzymes is the reductive cleavage of the cofactor SAM to generate methionine and a 5′-deoxyadenosyl radical. Substrate activation is achieved by 5′-deoxyadenosyl radical-mediated hydrogen atom abstraction, leading to generation of 5′-deoxyadenosine as one of reaction products ().16–18
By using [3
H-methyl]-SAM and [2,8-3
H-adenosyl]-SAM, we demonstrated that RlmN- and Cfr-catalyzed methyl transfer reactions are accompanied by the production of methionine and 5′-deoxyadenosine, confirming the role of the 5′-deoxyadenosyl radical in hydrogen atom abstraction. In addition to 5′-deoxyadenosine, RlmN- and Cfr-catalyzed methylations also generate S
-adenosylhomocysteine, suggesting that these methyltransferases consume two molecules of SAM per every methyl group introduced. The evidence that methionine, 5′-deoxyadenosine and SAH are formed on the productive pathway was obtained by monitoring their time-dependent formation in the RlmN-catalyzed reaction, which indicated a strong correlation between the formation of 2-methyladenosine and methionine, as well as 5′-deoxyadenosine and SAH. As methionine and 5′-deoxyadenosine are both formed as a result of the homolytic cleavage of SAM, these experiments provided a link between the introduction of the methyl group into the RNA and consumption of two molecules of SAM.
Based on these findings, we postulate a methylation mechanism depicted in . In the first step, the [4Fe-4S]1+
cluster donates one electron to SAM1
to form the 5′-deoxyadenosyl radical. This highly reactive radical then abstracts a hydrogen atom from the substrate (the C2 or C8 position of the adenosine) to generate a substrate-centered radical. In the subsequent steps, a methyl group is transferred to the substrate from SAM2
, forming the methylated product. The exact nature of the substrate-derived species that is subject to methylation is unknown, and its identification is hampered by the tremendous complexity of the rRNA substrate. Binding of two molecules of SAM per each molecule of the methyltransferase is in agreement with the recent structural modeling of Cfr.56
Given their requirement for two distinct roles of SAM in catalysis, as a source of the hydrogen atom-abstracting 5′-deoxyadenosyl radical and as a donor of the methyl group, RlmN and Cfr resemble MiaB and RimO, members of the methylthiotransferase subclass of the Radical SAM superfamily.36–39
Further similarity between MiaB and the methyltransferases described in this study is evident in the conserved first catalytic step, where all three enzymes use the 5′-deoxyadenosyl radical to abstract a hydrogen atom from an sp2
-hybridized carbon center of an adenosine-derivative nucleotide in the substrate rRNA.
Proposed mechanism for methylation catalyzed by the Radical SAM methyltransferase RlmN.
We have determined that neither enzyme can act on the assembled 50S subunit or the 70S ribosome, but protein-free 23S rRNA proved to be a good substrate. This observation is fully compatible with the location of A2503 deep in the peptidyl-transferase cavity of the mature large ribosomal subunit, where it is poorly accessible to the modification enzymes. Thus, RlmN- and Cfr- catalyzed methylation of A2503 most likely takes place during ribosome assembly.57
Many intermediate assembly steps separate naked 23S rRNA and the mature 50S subunit in the assembly pathway, thus limiting the knowledge of the precise ribosomal assembly step at which the two enzymes may act. However, the observation that the enzymes are not functional with the mature 50S subunit substrate indicates that there is only a narrow time-frame during the course of the subunit assembly when A2503 can be modified. This conclusion has important ramifications for the Cfr-mediated mechanism of antibiotic resistance because the extent of modification (and thus, the extent of resistance) may critically depend on the rate of ribosomal assembly. Other factors, such as pre-treatment of cells with antibiotics that inhibit ribosome biogenesis or the activity of other modification enzymes that utilize adjacent segments of rRNA in the heavily-modified peptidyl transferase center, may influence the window of opportunity for the Cfr and RlmN enzymes to act. The environmental conditions and growth phase of the cell can also have effects on the extent of A2503 modification, since ribosomes need to be actively assembling in order for Cfr and RlmN to methylate the rRNA.
Following this logic, it is also possible that organisms with different rates of ribosomal biogenesis may then be modified at A2503 to different extents. Association of cfr
with mobile genetic elements such as plasmids and transposons makes it potentially prone to rapid horizontal transfer between bacterial species.8,58
Investigation of the species-specific variation of Cfr-mediated A2503 modification and its correlation with pre-treatment with other antibiotics may pave the way for better antibiotic regimens and new approaches for combating antibiotic resistance.
Studies of the RNA substrate of RlmN and Cfr suggest that the 23S rRNA segment required for moderate methylation of A2503 is limited to helix system H90-H92, and the adjacent single-stranded stretch of RNA that includes A2503 (). Removal of helices 90–92 from the RNA substrate precluded both RlmN and Cfr from modifying A2503, indicating that this helical structure is the key recognition element for both enzymes. At the late steps of ribosomal assembly, proper folding of this structure may be assisted by an RNA helicase DbpA, which directly interacts with helix 92.59,60
Therefore, depending on the sequence of events, DbpA could also influence the extent of A2503 modification by RlmN and/or Cfr. Nevertheless, we did not observe any difference in resistance to florfenicol when Cfr was expressed in wild type or in dbpA−
cells (LaMarre and Mankin, unpublished) and thus concluded that either Cfr (and probably RlmN) acts upon RNA prior to DbpA action or that the 90–92 helical element of 23S rRNA can be recognized by the methyltransferases irrespective of its DbpA-mediated transformation. The knowledge of the minimal RNA substrate of the RlmN and Cfr radical SAM methyltransferases should facilitate subsequent structural and kinetic studies of both enzymes.
The role of the RlmN-catalyzed C2 methylation of A2503 in the process of translation is unknown. Previous work has demonstrated that while growth rates of wild type and ΔrlmN
cells are comparable, cells lacking the RlmN methyltransferase slowly lose in growth competition with wild type cells.11
gene is present in many bacterial species and its homologs are found in some single-celled eukaryotes and archaea, indicating that the A2503 modification may assist ribosome functions in all three major evolutionary domains. A2503 is located at the junction between the peptidyl transferase center and the nascent peptide exit tunnel and is apparently critical for the ability of the ribosome to sense and respond to specific nascent peptide sequences (Vazquez-Laslop and Mankin, in preparation). Posttranscriptional modification of A2503 may be required to optimize this function. In addition, H92, which is located close to A2503 and is a part of an rRNA element recognized by RlmN, contains the highly conserved A-loop, which makes critical contacts with the A-site tRNA.60
It is therefore possible that the A2503 C2 methylation serves as one of the check-points of proper ribosome assembly and gives a stamp of approval for the subsequent assembly steps (possibly involving DbpA-mediated refolding of helix 92). The role of posttranscriptional modifications as indicators of correct assembly has been proposed in the case of some modifying enzymes such as KsgA and RluD, highlighting the possibility that the endogenous C2 methylation might play a similar function.61,62
In summary, we have successfully reconstituted two methylation events of an adenosine nucleotide in the complex 23S rRNA substrate. These methyl transfer reactions are catalyzed by the Radical SAM methyltransferases RlmN and Cfr. We demonstrate that these enzymes require an intact [4Fe-4S] cluster for catalysis, and that SAM serves both as a source of the 5′-deoxyadenosyl radical and the methyl donor. We further demonstrated the RNA substrate requirements of both enzymes. To our knowledge, this study represents the first in vitro description of a methyl transfer catalyzed by a member of Radical SAM superfamily and adds a new catalytic function to this diverse enzyme class. Our work represents a starting point for future studies into the mechanism of the Radical SAM enzymes mediated methyl transfer reactions.