The A-type domain in Escherichia coli NfuA is required for regenerating the auxiliary [4Fe– 4S] cluster in Escherichia coli lipoyl synthase

Erin L. McCarthy, Ananda N. Rankin, Zerick R. Dill, Squire J. Booker

Research output: Contribution to journalArticle

2 Scopus citations

Abstract

The lipoyl cofactor plays an integral role in several essential biological processes. The last step in its de novo biosynthetic pathway, the attachment of two sulfur atoms at C6 and C8 of an n-octanoyllysyl chain, is catalyzed by lipoyl synthase (LipA), a member of the radical SAM superfamily. In addition to the [4Fe– 4S] cluster common to all radical SAM enzymes, LipA contains a second [4Fe– 4S] auxiliary cluster, which is sacrificed during catalysis to supply the requisite sulfur atoms, rendering the protein inactive for further turnovers. Recently, it was shown that the Fe–S cluster carrier protein NfuA from Escherichia coli can regenerate the auxiliary cluster of E. coli LipA after each turnover, but the molecular mechanism is incompletely understood. Herein, using protein–protein interaction and kinetic assays as well as site-directed mutagenesis, we provide further insight into the mechanism of NfuA-mediated cluster regeneration. In particular, we show that the N-terminal A-type domain of E. coli NfuA is essential for its tight interaction with LipA. Further, we demonstrate that NfuA from Mycobacterium tuberculosis can also regenerate the auxiliary cluster of E. coli LipA. However, an Nfu protein from Staphylococcus aureus, which lacks the A-type domain, was severely diminished in facilitating cluster regeneration. Of note, addition of the N-terminal domain of E. coli NfuA to S. aureus Nfu, fully restored cluster-regenerating activity. These results expand our understanding of the newly discovered mechanism by which the auxiliary cluster of LipA is restored after each turnover.

Original languageEnglish (US)
Pages (from-to)1609-1617
Number of pages9
JournalJournal of Biological Chemistry
Volume294
Issue number5
DOIs
StatePublished - Feb 1 2019

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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