Investigations into lipoic acid biosynthesis

The protein lipoyl synthase (LipA) is essential for lipoic acid biosynthesis via sulfur insertions at the C6 and C8 positions of a protein-bound octanoyl group. LipA from Escherichia coli binds two [4Fe-4S]1+2/2+ and is a member of the radical SAM superfamily of proteins. To facilitate mechanistic investigations into LipA dependent lipoyl synthesis, a novel in vitro assay has been developed which makes use of synthetic peptide substrates. These peptides contain an N(ε)-octanoyl lysine residue, corresponding in sequence to the lipoyl binding domain of the E2 subunit of pyruvate dehydrogenase. The activity LipA from Sulfolobus solfataricus was measured using these substrates analogues. The optimal temperature for the S. solfataricus LipA dependent formation of the lipoyl group was found to be 60 oC. Time dependent activity of LipA has been investigated over a wide range of temperatures (23-60 oC) and rate constants approximated for the observed rates of loss of octanoyl starting material and lipoyl formation at each temperature. This has allowed calculation of rate constants for the overall rate, kinetic analysis over a range of temperatures has allowed the activation energy for overall lipoyl formation (47.2 ± 5.4 kJ/mol) and for sulfur insertion at C6 (38.3 ± 4.9 kJ/mol) and C8 (46.9 ± 5.7 kJ/mol) to be determined. In all experiments using of S. solfataricus LipA, reconstitution with exogenous iron and sulphide was found to be essential for activity and spectroscopic studies have been carried out which show that S. solfataricus LipA can bind two [4Fe-4S] 1+/2+ and therefore closely resembles E. coli LipA. The mechanistic roles of these clusters have been investigated.