Purification and properties of the lipoate protein ligase of Escherichia coli

D. E. Green, T. W. Morris, J. Green, J. E. Cronan, J. R. Guest

Research output: Contribution to journalArticle

Abstract

Lipoate is an essential component of the 2-oxoacid dehydrogenase complexes and the glycine-cleavage system of Escherichia coli. It is attached to specific lysine residues in the lipoyl domains of the E2p (lipoate acetyltransferase) subunit of the pyruvate dehydrogenase complex by a Mg2+- and ATP-dependent lipoate protein ligase (LPL). LPL was purified from wild-type E. coli, where its abundance is extremely low (< 10 molecules per cell) and from a genetically amplified source. The purified enzyme is a monomeric protein (M(r) 38 000) which forms irregular clusters of needle-like crystals, It is stable at -20°C, but slowly oxidizes to an inactive form containing at least one intramolecular disulphide bond at 4°C. The inactive form could be re-activated by reducing agents or by an as-yet unidentified component (reactivation factor) which is resolved from LPL at the final stage of purification. The pI is 5.80, and the K(m) values for ATP, Mg2+ and DL-lipoate were determined. Sclenolipoate and 6-thio-octanoate were alternative but poorer substrates. Lipoylation was reversibly inhibited by the 6- and 8-seleno-octanoates and 8-thio-octanoate, which reacted with the six cysteine thiol groups of LPL. LPL was inactivated by Cu2+ ions in a process that involved the formation of inter- and intra-molecular disulphide bonds. Studies with IplA mutants lacking LPL activity indicated that E. coli possesses another distinct lipoylation system, although no such activity could be detected in vitro.

Original languageEnglish (US)
Pages (from-to)853-862
Number of pages10
JournalBiochemical Journal
Volume309
Issue number3
DOIs
StatePublished - Jan 1 1995
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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