Journal
ANALYTICAL BIOCHEMISTRY
Volume 351, Issue 1, Pages 44-49Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2006.01.023
Keywords
lipoyl synthase; sulfur insertion; radical sam; cofactor biosynthesis; Sulfolobus solfataricus
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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. (c) 2006 Elsevier Inc. All rights reserved.
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