Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.628752
Title: Investigations into lipoic acid biosynthesis
Author: Hiscox, Martyn
ISNI:       0000 0004 5346 938X
Awarding Body: University of Southampton
Current Institution: University of Southampton
Date of Award: 2014
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Abstract:
Lipoic acid is a ubiquitous biomolecule required for the metabolism of a-keto acids and glycine. The final step of lipoic acid biosynthesis is catalysed by lipoyl synthase (LipA), a member of the radical SAM enzyme superfamily. LipA catalyses the double sulfur insertion at the C6 and C8 positions of a protein bound octanoyl chain to form the lipoyl cofactor. The LipA mechanism has been probed with peptide substrate mimics. The previous synthetic routes to these mimics were time and resource costly. New methods have been developed for the rapid synthesis of modified Fmoc-lysine derivatives to prepare a range of substrate and product like peptides. Kinetic analysis of a LipA time course has demonstrated that the previously described 6-thio-octanoyl species, is a kinetically competent intermediate and therefore the formation of the lipoyl group proceeds via two sequential sulfur insertion steps. A bioinformatics and crystallisation study of LipA resulted in two structures of LipA-2 from Thermosynechocccus elongatus. These structures have shown a m-sulfide of the [4Fe-4S]Aux cluster positioned to donate a sulfur atom and reveal an novel and completely conserved serine ligand for the [4Fe-4S]Aux cluster. Mutagenesis of the serine to either an alanine or a cysteine resulted in a loss of activity in both enzymes. However the cysteine mutant was able to catalyse a single sulfur insertion at a very low level. EPR studies have suggested that the [4Fe-4S]Aux cluster is EPR active when reduced with sodium dithionite, pointing towards a co-operative role in cluster reduction. Additional spectra suggest that during the turnover of LipA the [4Fe-4S]Aux cluster of LipA is reduced from a 1+ state to a 0 state, analogous to biotin synthase whose [2Fe-2S] cluster is reduced upon sulfur insertion. The work presented in this thesis suggests that the [4Fe-4S]Aux cluster, ligated by a unique serine is the source of the sulfur atom inserted at the C6 position of the octanoyl chain.
Supervisor: Roach, Peter Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.628752  DOI: Not available
Keywords: QD Chemistry
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