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Investigating the chemistry of lipoyl synthase

Investigating the chemistry of lipoyl synthase
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.
Douglas, Paul
a6304b96-3482-4468-9a45-bc05153ca270
Douglas, Paul
a6304b96-3482-4468-9a45-bc05153ca270
Roach, Peter
ca94060c-4443-482b-af3e-979243488ba9

Douglas, Paul (2008) Investigating the chemistry of lipoyl synthase. University of Southampton, School of Chemistry, Doctoral Thesis, 159pp.

Record type: Thesis (Doctoral)

Abstract

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.

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Published date: September 2008
Organisations: University of Southampton

Identifiers

Local EPrints ID: 67197
URI: http://eprints.soton.ac.uk/id/eprint/67197
PURE UUID: 715bd6f3-0d9e-4def-9b5c-f1d9de7b186a
ORCID for Peter Roach: ORCID iD orcid.org/0000-0001-9880-2877

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Date deposited: 07 Aug 2009
Last modified: 30 Jan 2020 01:30

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