Spectroscopic and Electrochemical Characterization of a Unique Radical S‐Adenosylmethionine Methylase

Le résumé fourni par la source

TsrM 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 S‐adenosylmethionine (SAM); however, the enzyme also requires cobalamin and iron‐sulfur cluster cofactors for turnover. In this work we demonstrate the ability to overproduce and purify TsrM and characterize its metallocofactors by UV‐visible, electron paramagnetic resonance, hyperfine sublevel correlation (HYSCORE), and Mössbauer spectroscopies, in addition to protein film electrochemistry. The enzyme contains one equivalent 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 is replaced by a ligand lacking nuclear spin, likely a water molecule. Electrochemical analysis of the protein by protein‐film electrochemistry indicates a one‐electron redox wave that exhibits a midpoint potential of ‐550 mV, 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 ion of the cluster in the same manner as other radical SAM enzymes, yet still disturbs the electronic configuration of the Fe/S cluster. These biophysical studies suggest that TsrM is an atypical radical SAM enzyme, consistent with its reported inability to catalyze formation of a 5′‐deoxyadenosyl 5′‐radical. Support or Funding Information National Institutes of Health

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