RlmN and AtsB as models for the overproduction and characterization of radical SAM proteins

Nicholas D. Lanz, Tyler L. Grove, Camelia Baleanu Gogonea, Kyung Hoon Lee, Carsten Krebs, Squire J. Booker

Research output: Chapter in Book/Report/Conference proceedingChapter

67 Scopus citations

Abstract

An explosion of remarkable chemical transformations has been witnessed in the past decade as a result of the radical S-adenosyl-l-methionine (SAM) (RS) superfamily of proteins. These proteins share the ability to cleave SAM reductively to l-methionine and a 5′-deoxyadenosyl 5′-radical (5′-dA ). The 5′-dA initiates > 40 distinct reaction types by abstracting target hydrogen atoms on small-molecule and macromolecular substrates. All RS enzymes contain a [4Fe-4S] cluster coordinated by SAM that supplies the electron for SAM cleavage. A subset of RS enzymes contains additional iron-sulfur (Fe/S) clusters that serve alternative purposes, many remaining to be defined. The oxygen lability of their [4Fe-4S] clusters causes RS enzymes to be more tedious to purify, characterize, and study. Moreover, the type(s) and stoichiometry of Fe/S clusters in RS enzymes has often been a source of debate. Herein, we use RlmN and AtsB as models to highlight methods for purifying and characterizing RS enzymes, focusing on using Mössbauer spectroscopy in concert with methods for quantifying iron and acid-labile sulfide to assign cluster content accurately.

Original languageEnglish (US)
Title of host publicationMethods in Enzymology
PublisherAcademic Press Inc.
Pages125-152
Number of pages28
DOIs
StatePublished - 2012

Publication series

NameMethods in Enzymology
Volume516
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

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    Lanz, N. D., Grove, T. L., Gogonea, C. B., Lee, K. H., Krebs, C., & Booker, S. J. (2012). RlmN and AtsB as models for the overproduction and characterization of radical SAM proteins. In Methods in Enzymology (pp. 125-152). (Methods in Enzymology; Vol. 516). Academic Press Inc.. https://doi.org/10.1016/B978-0-12-394291-3.00030-7