Journal
JOURNAL OF BIOLOGICAL INORGANIC CHEMISTRY
Volume 17, Issue 1, Pages 49-56Publisher
SPRINGER
DOI: 10.1007/s00775-011-0828-1
Keywords
Anaerobic metabolism; (R)-benzylsuccinate synthase; Glycyl-radical enzyme; Electron paramagnetic spectroscopy; Toluene; Thauera aromatica K172
Funding
- Deutsche Forschungsgemeinschaft (DFG)
- Landesoffensive zur Entwicklung wissenschaftlich-okonomischer Exzellenz (LOEWE)
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The anaerobic degradation pathway of toluene is initiated by the addition of the methyl group of toluene to the double bond of fumarate. This reaction is catalyzed by a novel glycyl-radical enzyme, (R)-benzylsuccinate synthase (BSS). The enzyme consists of three subunits, alpha, beta, and gamma, and differs from most other glycyl-radical enzymes in having additional cofactors. We have purified a Strep-tagged nonactivated BSS from recombinant Escherichia coli and identified the additional cofactors as FeS clusters by UV/vis, EPR, and Mossbauer spectroscopy. Analysis of the metal content as well as the EPR and Mossbauer spectra indicated that BSS contains magnetically coupled low-potential [4Fe-4S] clusters. Several enzyme preparations showed differing amounts of [3Fe-4S] clusters that could be reconstituted to [4Fe-4S] clusters, indicating that they arise from partial decay of the initial [4Fe-4S] clusters. The most likely location of these FeS clusters in the enzyme are the small beta and gamma subunits, which are unique for the BSS subfamily of glycyl-radical enzymes and contain conserved cysteines as potential ligands.
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