TsrM as a Model for Purifying and Characterizing Cobalamin-Dependent Radical S-Adenosylmethionine Methylases

Anthony J. Blaszczyk, Roy X. Wang, Squire J. Booker

Research output: Chapter in Book/Report/Conference proceedingChapter

7 Citations (Scopus)

Abstract

Cobalamin-dependent radical S-adenosylmethionine (SAM) methylases play vital roles in the de novo biosynthesis of many antibiotics, cofactors, and other important natural products, yet remain an understudied subclass of radical SAM enzymes. In addition to a [4Fe–4S] cluster that is ligated by three cysteine residues, these enzymes also contain an N-terminal cobalamin-binding domain. In vitro studies of these enzymes have been severely limited because many are insoluble or sparingly soluble upon their overproduction in Escherichia coli. This solubility issue has led a number of groups either to purify the protein from inclusion bodies or to purify soluble protein that often lacks proper cofactor incorporation. Herein, we use TsrM as a model to describe methods that we have used to generate soluble protein that is purified in an active form with both cobalamin and [4Fe–4S] cluster cofactors bound. Additionally, we highlight the methods that we developed to characterize the enzyme following purification.

Original languageEnglish (US)
Title of host publicationMethods in Enzymology
PublisherAcademic Press Inc.
Pages303-329
Number of pages27
DOIs
StatePublished - Jan 1 2017

Publication series

NameMethods in Enzymology
Volume595
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Fingerprint

S-Adenosylmethionine
Vitamin B 12
Enzymes
Proteins
Biosynthesis
Inclusion Bodies
Biological Products
Solubility
Escherichia coli
Purification
Cysteine
Anti-Bacterial Agents

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

Cite this

Blaszczyk, A. J., Wang, R. X., & Booker, S. J. (2017). TsrM as a Model for Purifying and Characterizing Cobalamin-Dependent Radical S-Adenosylmethionine Methylases. In Methods in Enzymology (pp. 303-329). (Methods in Enzymology; Vol. 595). Academic Press Inc.. https://doi.org/10.1016/bs.mie.2017.07.007
Blaszczyk, Anthony J. ; Wang, Roy X. ; Booker, Squire J. / TsrM as a Model for Purifying and Characterizing Cobalamin-Dependent Radical S-Adenosylmethionine Methylases. Methods in Enzymology. Academic Press Inc., 2017. pp. 303-329 (Methods in Enzymology).
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Blaszczyk, AJ, Wang, RX & Booker, SJ 2017, TsrM as a Model for Purifying and Characterizing Cobalamin-Dependent Radical S-Adenosylmethionine Methylases. in Methods in Enzymology. Methods in Enzymology, vol. 595, Academic Press Inc., pp. 303-329. https://doi.org/10.1016/bs.mie.2017.07.007

TsrM as a Model for Purifying and Characterizing Cobalamin-Dependent Radical S-Adenosylmethionine Methylases. / Blaszczyk, Anthony J.; Wang, Roy X.; Booker, Squire J.

Methods in Enzymology. Academic Press Inc., 2017. p. 303-329 (Methods in Enzymology; Vol. 595).

Research output: Chapter in Book/Report/Conference proceedingChapter

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Blaszczyk AJ, Wang RX, Booker SJ. TsrM as a Model for Purifying and Characterizing Cobalamin-Dependent Radical S-Adenosylmethionine Methylases. In Methods in Enzymology. Academic Press Inc. 2017. p. 303-329. (Methods in Enzymology). https://doi.org/10.1016/bs.mie.2017.07.007