Purification and properties of the lipoate protein ligase of Escherichia coli

Biochem J. 1995 Aug 1;309 ( Pt 3)(Pt 3):853-62. doi: 10.1042/bj3090853.


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 Mg(2+)- 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 degrees C, but slowly oxidizes to an inactive form containing at least one intramolecular disulphide bond at 4 degrees 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 Km values for ATP, Mg2+ and DL-lipoate were determined. Selenolipoate 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 lplA mutants lacking LPL activity indicated that E. coli possesses another distinct lipoylation system, although no such activity could be detected in vitro.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Cations
  • Chromatography, Affinity
  • Chromatography, Gel
  • Electrophoresis, Polyacrylamide Gel
  • Enzyme Activation
  • Enzyme Stability
  • Escherichia coli / enzymology*
  • Gene Amplification
  • Hydrogen-Ion Concentration
  • Hydrolysis
  • Kinetics
  • Molecular Sequence Data
  • Peptide Synthases / antagonists & inhibitors
  • Peptide Synthases / chemistry
  • Peptide Synthases / isolation & purification*
  • Substrate Specificity


  • Cations
  • Peptide Synthases
  • lipoate-protein ligase