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Spectroscopic and Electrochemical Characterization of the Iron–Sulfur and Cobalamin Cofactors of TsrM, an Unusual Radical S-Adenosylmethionine Methylase
- Source :
- Journal of the American Chemical Society. 138:3416-3426
- Publication Year :
- 2016
- Publisher :
- American Chemical Society (ACS), 2016.
-
Abstract
- TsrM, an annotated radical S-adenosylmethionine (SAM) enzyme, catalyzes the methylation of carbon 2 of the indole ring of L-tryptophan. Its reaction is the first step in the biosynthesis of the unique quinaldic acid moiety of thiostrepton A, a thiopeptide antibiotic. The appended methyl group derives from SAM; however, the enzyme also requires cobalamin and iron-sulfur cluster cofactors for turnover. In this work we report the overproduction and purification of TsrM and the characterization of its metallocofactors by UV-visible, electron paramagnetic resonance, hyperfine sublevel correlation (HYSCORE), and Mössbauer spectroscopies as well as protein-film electrochemistry (PFE). The enzyme contains 1 equiv of its cobalamin cofactor in its as-isolated state and can be reconstituted with iron and sulfide to contain one [4Fe-4S] cluster with a site-differentiated Fe(2+)/Fe(3+) pair. Our spectroscopic studies suggest that TsrM binds cobalamin in an uncharacteristic five-coordinate base-off/His-off conformation, whereby the dimethylbenzimidazole group is replaced by a non-nitrogenous ligand, which is likely a water molecule. Electrochemical analysis of the protein by PFE indicates a one-electron redox feature with a midpoint potential of -550 mV, which is assigned to a [4Fe-4S](2+)/[4Fe-4S](+) redox couple. Analysis of TsrM by Mössbauer and HYSCORE spectroscopies suggests that SAM does not bind to the unique iron site of the cluster in the same manner as in other radical SAM (RS) enzymes, yet its binding still perturbs the electronic configuration of both the Fe/S cluster and the cob(II)alamin cofactors. These biophysical studies suggest that TsrM is an atypical RS enzyme, consistent with its reported inability to catalyze formation of a 5'-deoxyadenosyl 5'-radical.
- Subjects :
- Iron-Sulfur Proteins
0301 basic medicine
S-Adenosylmethionine
Stereochemistry
Coenzymes
chemistry.chemical_element
010402 general chemistry
01 natural sciences
Biochemistry
Cobalamin
Catalysis
Cofactor
law.invention
Spectroscopy, Mossbauer
03 medical and health sciences
chemistry.chemical_compound
Colloid and Surface Chemistry
law
Electrochemistry
Moiety
Organic chemistry
Electron paramagnetic resonance
Indole test
biology
Electron Spin Resonance Spectroscopy
Methyltransferases
General Chemistry
Methylation
Sulfur
0104 chemical sciences
Vitamin B 12
030104 developmental biology
chemistry
biology.protein
Methyl group
Subjects
Details
- ISSN :
- 15205126 and 00027863
- Volume :
- 138
- Database :
- OpenAIRE
- Journal :
- Journal of the American Chemical Society
- Accession number :
- edsair.doi.dedup.....507cf81f67f8dcacc6b78380ea065c76