Reaction of AdoMet with ThiC generates a backbone free radical - PubMed (original) (raw)

Reaction of AdoMet with ThiC generates a backbone free radical

N Cecilia Martinez-Gomez et al. Biochemistry. 2009.

Abstract

ThiC is an [4Fe-4S] cluster protein that catalyzes the formation of 4-amino-5-hydroxymethyl-2-methylpyrimidine. EPR spectroscopic studies demonstrate that, upon interaction with AdoMet, active ThiC from Salmonella enterica generates a persistent free radical on the alpha-carbon of an amino acid residue. The EPR properties of the radical are consistent with any residue other than a Gly or Ala. Exposure to oxygen was accompanied by a fission of the radical-carrying polypeptide chain between the Gly436 and His437 residues in ThiC. Regardless of whether the backbone radical is part of the catalytic machinery, its presence provides evidence that ThiC employs free radical chemistry as expected for radical SAM enzymes.

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Figures

Figure 1

Figure 1

EPR spectrum of ThiC backbone radical. A) EPR spectrum at 40 K of reconstituted ThiC in the presence of AdoMet. The spectrum is an average of 4 scans with microwave power 4 mW, modulation amplitude 4 Gauss. B) Resolution enhanced version of spectrum A. C) Simulation of B assuming coupling to two β-protons (Aiso H1 = 15 G, Aiso H2 = 0.2 G); one β-amide 14N (A14N [0 G, 15 G, 0 G]; and one β-amide proton (A1H [8.4 G, 7.4 G, 0.2 G]. Additional details regarding the fitting parameters are given in supporting information.

Figure 2

Figure 2

Oxygen Fragmentation of the polypeptide chain. Reduced ThiC+AdoMet gassed with O2 for 20 minutes or kept under anoxic conditions as indicated. The samples were resolved by SDS-PAGE. A) for the high molecular weight bands (4-12%), and B) for the low molecular weight bands (12%). 2 μg protein was applied per lane, visualization was by Commassie blue staining. The sequence of ThiC surrounding the cleavage site is depicted. Starred residues are conserved in over 100 ThiC homologs. The line between Gly436 and His437 indicates the site of oxygen dependent cleavage.

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