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PDBsum entry 4pl2
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References listed in PDB file
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Key reference
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Title
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Characterization of a cross-Linked protein-Nucleic acid substrate radical in the reaction catalyzed by rlmn.
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Authors
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A.Silakov,
T.L.Grove,
M.I.Radle,
M.R.Bauerle,
M.T.Green,
A.C.Rosenzweig,
A.K.Boal,
S.J.Booker.
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Ref.
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J Am Chem Soc, 2014,
136,
8221-8228.
[DOI no: ]
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PubMed id
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Abstract
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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, (13)C hyperfine interactions
between the radical and the methylene carbon of the formerly
[methyl-(13)C]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.
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