6 research outputs found
Characterization of a Cross-Linked Protein–Nucleic Acid Substrate Radical in the Reaction Catalyzed by RlmN
RlmN
and Cfr are methyltransferases/methylsynthases that belong
to the radical <i>S</i>-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, <sup>13</sup>C hyperfine interactions
between the radical and the methylene carbon of the formerly [<i>methyl</i>-<sup>13</sup>C]ÂCys355 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
Further Characterization of Cys-Type and Ser-Type Anaerobic Sulfatase Maturating Enzymes Suggests a Commonality in the Mechanism of Catalysis
The anaerobic sulfatase-maturating
enzyme from <i>Clostridium
perfringens</i> (anSMEcpe) catalyzes the two-electron oxidation
of a cysteinyl residue on a cognate protein to a formylglycyl residue
(FGly) using a mechanism that involves organic radicals. The FGly
residue plays a unique role as a cofactor in a class of enzymes termed
arylsulfatases, which catalyze the hydrolysis of various organosulfate
monoesters. anSMEcpe has been shown to be a member of the radical <i>S</i>-adenosylmethionine (SAM) family of enzymes, [4Fe-4S] cluster-requiring
proteins that use a 5′-deoxyadenosyl 5′-radical (5′-dA<sup>•</sup>) generated from a reductive cleavage of SAM to initiate
radical-based catalysis. Herein, we show that anSMEcpe contains in
addition to the [4Fe-4S] cluster harbored by all radical SAM (RS)
enzymes, two additional [4Fe-4S] clusters, similar to the radical
SAM protein AtsB, which catalyzes the two-electron oxidation of a
seryl residue to a FGly residue. We show by size-exclusion chromatography
that both AtsB and anSMEcpe are monomeric proteins, and site-directed
mutagenesis studies of AtsB reveal that individual Cys → Ala
substitutions at seven conserved positions result in an insoluble
protein, consistent with those residues acting as ligands to the two
additional [4Fe-4S] clusters. Ala substitutions at an additional conserved
Cys residue (C291 in AtsB and C276 in anSMEcpe) afford proteins that
display intermediate behavior. These proteins exhibit reduced solubility
and drastically reduced activity, behavior that is conspicuously similar
to that of a critical Cys residue in BtrN, another radical SAM dehydrogenase
[Grove, T. L., et al. (2010) <i>Biochemistry</i> <i>49</i>, 3783–3785]. We also show that wild-type anSMEcpe
acts on peptides containing other oxidizable amino acids at the target
position. Moreover, we show that the enzyme will convert threonyl
peptides to the corresponding ketone product, and also <i>allo</i>-threonyl peptides, but with a significantly reduced efficiency,
suggesting that the <i>pro-S</i> hydrogen atom of the normal
cysteinyl substrate is stereoselectively removed during turnover.
Lastly, we show that the electron generated during catalysis by AtsB
and anSMEcpe can be utilized for multiple turnovers, albeit through
a reduced flavodoxin-mediated pathway