Biochemical Approaches for Understanding Iron–Sulfur Cluster Regeneration in Escherichia coli Lipoyl Synthase During Catalysis

Erin L. McCarthy, Squire J. Booker

Research output: Chapter in Book/Report/Conference proceedingChapter

1 Citation (Scopus)

Abstract

Lipoyl synthase (LipA in bacteria) is a radical S-adenosylmethionine (SAM) enzyme that catalyzes the second step of the de novo biosynthesis of the lipoyl cofactor: the insertion of sulfur at C6 and C8 of a pendant octanoyl chain. In addition to the [4Fe–4S] cluster that is characteristic of the radical SAM (RS) enzymes, LipA contains a second [4Fe–4S] cluster that, though controversial, has been proposed to be degraded during turnover to supply the inserted sulfur atoms. A consequence of this proposed role is that the destruction of its iron–sulfur cluster renders the enzyme in an inactive state. Recently, it was shown that Escherichia coli proteins NfuA or IscU can confer catalytic properties to E. coli LipA in vitro. In this chapter, we present methods for characterizing LipA and analyzing its activity in vitro, and provide strategies to monitor the pathway for the regeneration of LipA's auxiliary cluster by E. coli iron–sulfur carrier protein NfuA.

Original languageEnglish (US)
Title of host publicationMethods in Enzymology
EditorsVahe Bandarian
PublisherAcademic Press Inc.
Pages217-239
Number of pages23
ISBN (Print)9780128127940
DOIs
StatePublished - Jan 1 2018

Publication series

NameMethods in Enzymology
Volume606
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Fingerprint

Catalysis
Escherichia coli
Regeneration
S-Adenosylmethionine
Sulfur
Enzymes
Escherichia coli Proteins
Biosynthesis
Bacteria
Carrier Proteins
Atoms
In Vitro Techniques

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

Cite this

McCarthy, E. L., & Booker, S. J. (2018). Biochemical Approaches for Understanding Iron–Sulfur Cluster Regeneration in Escherichia coli Lipoyl Synthase During Catalysis. In V. Bandarian (Ed.), Methods in Enzymology (pp. 217-239). (Methods in Enzymology; Vol. 606). Academic Press Inc.. https://doi.org/10.1016/bs.mie.2018.06.006
McCarthy, Erin L. ; Booker, Squire J. / Biochemical Approaches for Understanding Iron–Sulfur Cluster Regeneration in Escherichia coli Lipoyl Synthase During Catalysis. Methods in Enzymology. editor / Vahe Bandarian. Academic Press Inc., 2018. pp. 217-239 (Methods in Enzymology).
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McCarthy, EL & Booker, SJ 2018, Biochemical Approaches for Understanding Iron–Sulfur Cluster Regeneration in Escherichia coli Lipoyl Synthase During Catalysis. in V Bandarian (ed.), Methods in Enzymology. Methods in Enzymology, vol. 606, Academic Press Inc., pp. 217-239. https://doi.org/10.1016/bs.mie.2018.06.006

Biochemical Approaches for Understanding Iron–Sulfur Cluster Regeneration in Escherichia coli Lipoyl Synthase During Catalysis. / McCarthy, Erin L.; Booker, Squire J.

Methods in Enzymology. ed. / Vahe Bandarian. Academic Press Inc., 2018. p. 217-239 (Methods in Enzymology; Vol. 606).

Research output: Chapter in Book/Report/Conference proceedingChapter

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McCarthy EL, Booker SJ. Biochemical Approaches for Understanding Iron–Sulfur Cluster Regeneration in Escherichia coli Lipoyl Synthase During Catalysis. In Bandarian V, editor, Methods in Enzymology. Academic Press Inc. 2018. p. 217-239. (Methods in Enzymology). https://doi.org/10.1016/bs.mie.2018.06.006