Investigating the chemistry of lipoyl synthase

The radical SAM protein lipoyl synthase (LipA) is essential for lipoic acid biosynthesis via sulfur insertions into the unactivated C6 and C8 centres of a protein-bound octanoyl group. Using an in vitro assay which makes use of a small peptide mimic of the protein substrate, it has now been shown at which carbon centre sulfur insertion first occurs. LCMS analysis of reactions using labeled substrates and proton NMR characterization of an isolated monothiolated adduct have been used to show that sulfur insertion proceeds in a stepwise manner, with sulfur insertion occurring preferentially at the C6 centre. The associated kinetic isotope effects (KIE’s) for hydrogen atom abstraction from the C6 and C8 centres have been calculated and found to equal 2 and 15 respectively. The inhibition of LipA by methionine and AdoH, which are products from reactions involving radical SAM proteins, was investigated. Methionine offered no clear inhibition whilst AdoH had a slight inhibitionary effect (IC50 = 990 ± 83 ?M). When both methionine and AdoH were used together, a strong synergistic inhibition was present (IC50 = 327 ± 22 ?M). However, when an enzyme (Pfs) which cleaves the glycosidic bond in AdoH was added to the reaction, this inhibition was removed and a 1.4 fold increase in activity was observed.The ability of LipA to accept larger substrates was also tested using a nonanoyl peptide analogue. LCMS analysis of these reactions identified that as well as the expected single and double sulfur inserted products there were two further unexpected products formed in the reaction mixture. Proton NMR characterized these as a trans-alkene and a thietane. Mechanisms for their formations have been proposed.