Spectroscopic and Electrochemical Characterization of the Iron–Sulfur and Cobalamin Cofactors of TsrM, an Unusual Radical S-Adenosylmethionine Methylase
| Autor: | Sean Elliott, Squire J. Booker, Nicholas D. Lanz, Stephanie J. Maiocco, Alexey Silakov, Bo Zhang, Wendy L. Kelly, Anthony J. Blaszczyk, Carsten Krebs |
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| Rok vydání: | 2016 |
| Předmět: |
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 |
| Zdroj: | Journal of the American Chemical Society. 138:3416-3426 |
| ISSN: | 1520-5126 0002-7863 |
| DOI: | 10.1021/jacs.5b12592 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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