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Understanding the role of electron donors in the reaction catalyzed by Tsrm, a cobalamin-dependent radical S-adenosylmethionine methylase

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Abstract

The cobalamin-dependent radical S-adenosylmethionine (SAM) enzyme TsrM catalyzes the methylation of C2 of l-tryptophan to form 2-methyltryptophan during the biosynthesis of thiostrepton A. Although TsrM is a member of the radical SAM superfamily, unlike all other annotated members, it does not catalyze a reductive cleavage of SAM to a 5′-deoxyadenosyl 5′-radical intermediate. In fact, it has been proposed that TsrM catalyzes its reaction through two polar nucleophilic displacements, with its cobalamin cofactor cycling directly between methylcobalamin (MeCbl) and cob(I)alamin. Nevertheless, the enzyme has been stated to require the action of a reductant, which can be satisfied by dithiothreitol. By contrast, all other annotated RS enzymes require a reductant that exhibits a much lower reduction potential, which is necessary for the reductive cleavage of SAM. Herein, we show that TsrM can catalyze multiple turnovers in the absence of any reducing agent, but only when it is pre-loaded with MeCbl. When hydroxocobalamin (OHCbl) or cob(II)alamin is bound to TsrM, a reductant is required to convert it to cob(I)alamin, which can acquire a methyl group directly from SAM. Our studies suggest that TsrM uses an external reductant to prime its reaction by converting bound OHCbl or cob(II)alamin to MeCbl, and to regenerate the MeCbl form of the cofactor upon adventitious oxidation of the cob(I)alamin intermediate to cob(II)alamin.

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Abbreviations

5′-dA:

5′-deoxyadenosyl radical

β:

Mercaptoethanol

DTT:

Dithiothreitol

flr:

Flavodoxin reductase

flv:

Flavodoxin

LC–MS/MS:

Liquid chromatography tandem mass spectrometry

MeCbl:

Methylcobalamin

MetH:

Methionine synthase

MeTrp:

2-methyltryptophan

RS:

Radical S-adenosylmethionine

SAM:

S-adenosylmethionine

TCEP:

Tris (2-carboxyethyl) phosphine

Trp:

Tryptophan

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Funding

This work was supported an NIH grant (GM-122595) and the Eberly Family Distinguished Chair in Science to S. J. B, who is also an investigator of the Howard Hughes Medical Institute.

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Author notes

  1. Anthony J. Blaszczyk and Hayley L. Knox contributed equally.

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA, 16802, USA

    Anthony J. Blaszczyk & Squire J. Booker

  2. Department of Chemistry, The Pennsylvania State University, University Park, PA, 16802, USA

    Hayley L. Knox & Squire J. Booker

  3. The Howard Hughes Medical Institute, The Pennsylvania State University, University Park, PA, 16802, USA

    Squire J. Booker

Authors
  1. Anthony J. Blaszczyk
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  2. Hayley L. Knox
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  3. Squire J. Booker
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Correspondence to Squire J. Booker.

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Blaszczyk, A.J., Knox, H.L. & Booker, S.J. Understanding the role of electron donors in the reaction catalyzed by Tsrm, a cobalamin-dependent radical S-adenosylmethionine methylase. J Biol Inorg Chem 24, 831–839 (2019). https://doi.org/10.1007/s00775-019-01689-8

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  • DOI: https://doi.org/10.1007/s00775-019-01689-8

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