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Understanding the role of electron donors in the reaction catalyzed by Tsrm, a cobalamin-dependent radical S-adenosylmethionine methylase.
J Biol Inorg Chem 2019; 24(6):831-839JB

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.

Authors+Show Affiliations

Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA, 16802, USA.Department of Chemistry, The Pennsylvania State University, University Park, PA, 16802, USA.Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA, 16802, USA. squire@psu.edu. Department of Chemistry, The Pennsylvania State University, University Park, PA, 16802, USA. squire@psu.edu. The Howard Hughes Medical Institute, The Pennsylvania State University, University Park, PA, 16802, USA. squire@psu.edu.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

31350635

Citation

Blaszczyk, Anthony J., et al. "Understanding the Role of Electron Donors in the Reaction Catalyzed By Tsrm, a Cobalamin-dependent Radical S-adenosylmethionine Methylase." Journal of Biological Inorganic Chemistry : JBIC : a Publication of the Society of Biological Inorganic Chemistry, vol. 24, no. 6, 2019, pp. 831-839.
Blaszczyk AJ, Knox HL, Booker SJ. Understanding the role of electron donors in the reaction catalyzed by Tsrm, a cobalamin-dependent radical S-adenosylmethionine methylase. J Biol Inorg Chem. 2019;24(6):831-839.
Blaszczyk, A. J., Knox, H. L., & Booker, S. J. (2019). Understanding the role of electron donors in the reaction catalyzed by Tsrm, a cobalamin-dependent radical S-adenosylmethionine methylase. Journal of Biological Inorganic Chemistry : JBIC : a Publication of the Society of Biological Inorganic Chemistry, 24(6), pp. 831-839. doi:10.1007/s00775-019-01689-8.
Blaszczyk AJ, Knox HL, Booker SJ. Understanding the Role of Electron Donors in the Reaction Catalyzed By Tsrm, a Cobalamin-dependent Radical S-adenosylmethionine Methylase. J Biol Inorg Chem. 2019;24(6):831-839. PubMed PMID: 31350635.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Understanding the role of electron donors in the reaction catalyzed by Tsrm, a cobalamin-dependent radical S-adenosylmethionine methylase. AU - Blaszczyk,Anthony J, AU - Knox,Hayley L, AU - Booker,Squire J, Y1 - 2019/07/26/ PY - 2019/05/18/received PY - 2019/07/11/accepted PY - 2019/7/28/pubmed PY - 2019/7/28/medline PY - 2019/7/28/entrez KW - Cobalamin KW - Enzyme catalysis KW - Iron–sulfur clusters KW - Methylation KW - S-adenosylmethionine KW - Tryptophan SP - 831 EP - 839 JF - Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry JO - J. Biol. Inorg. Chem. VL - 24 IS - 6 N2 - 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. SN - 1432-1327 UR - https://www.unboundmedicine.com/medline/citation/31350635/Understanding_the_role_of_electron_donors_in_the_reaction_catalyzed_by_Tsrm,_a_cobalamin-dependent_radical_S-adenosylmethionine_methylase L2 - https://dx.doi.org/10.1007/s00775-019-01689-8 DB - PRIME DP - Unbound Medicine ER -