The activity of a thermostable lipoyl synthase from Sulfolobus solfataricus with a synthetic octanoyl substrate.
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.
Bryant
Penny
P
School of Chemistry, University of Southampton, Highfield, Southampton SO17 1BJ, UK.
Kriek
Marco
M
Wood
Robert J
RJ
Roach
Peter L
PL
D001483:Base Sequence; D002210:Caprylates / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D008025:Ligases / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D013379:Substrate Specificity; D048229:Sulfolobus solfataricus / Q000201:enzymology*; D013696:Temperature
D002210:Caprylates; D017931:DNA Primers; D008025:Ligases; C031492:octanoic acid