The activity of a thermostable lipoyl synthase from Sulfolobus solfataricus with a synthetic octanoyl substrate

Anal Biochem. 2006 Apr 1;351(1):44-9. doi: 10.1016/j.ab.2006.01.023. Epub 2006 Feb 3.

Abstract

The protein lipoyl synthase (LipA) is essential for lipoic acid biosynthesis via sulfur insertions into a protein-bound octanoyl group. We have developed an in vitro assay for LipA using a synthetic tetrapeptide substrate, containing an N(epsilon)-octanoyl lysine residue, corresponding in sequence to the lipoyl binding domain of the E2 subunit of pyruvate dehydrogenase. A putative LipA from the hypothermophilic archaea Sulfolobus solfataricus was expressed in Escherichia coli and purified, and the activity was measured using this novel assay. The optimal temperature for the S. solfataricus LipA-dependent formation of the lipoyl group was found to be 60 degrees C.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Caprylates / metabolism*
  • Chromatography, High Pressure Liquid
  • DNA Primers
  • Electrophoresis, Polyacrylamide Gel
  • Enzyme Stability
  • Ligases / metabolism*
  • Magnetic Resonance Spectroscopy
  • Mass Spectrometry
  • Substrate Specificity
  • Sulfolobus solfataricus / enzymology*
  • Temperature

Substances

  • Caprylates
  • DNA Primers
  • Ligases
  • octanoic acid