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Investigations into lipoic acid biosynthesis

Investigations into lipoic acid biosynthesis
Investigations into lipoic acid biosynthesis
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.
University of Southampton
Hiscox, Martyn J.
8d6e0a60-70c9-4f73-a9c0-9ff27a3e1e49
Hiscox, Martyn J.
8d6e0a60-70c9-4f73-a9c0-9ff27a3e1e49
Roach, Peter
ca94060c-4443-482b-af3e-979243488ba9

Hiscox, Martyn J. (2014) Investigations into lipoic acid biosynthesis. University of Southampton, Chemistry, Doctoral Thesis, 255pp.

Record type: Thesis (Doctoral)

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.

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More information

Published date: 1 June 2014
Organisations: University of Southampton, Chemistry, CBDT

Identifiers

Local EPrints ID: 367261
URI: http://eprints.soton.ac.uk/id/eprint/367261
PURE UUID: f4925ab4-8def-4f9e-a6d2-193f336c197a
ORCID for Peter Roach: ORCID iD orcid.org/0000-0001-9880-2877

Catalogue record

Date deposited: 23 Oct 2014 10:52
Last modified: 07 Jun 2019 04:01

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Contributors

Author: Martyn J. Hiscox
Thesis advisor: Peter Roach ORCID iD

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