Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.662987
Title: Spectroscopic studies of the biotin biosynthase enzymes
Author: Tomczyk, Nick
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2006
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Abstract:
Biotin synthase (BS) catalyses the final sulfur incorporation step in the biosynthesis of biotin. Its chemical mechanism, primary amino acid sequence and tertiary place it in the Radical-SAM-dependent superfamily of >600 proteins. The gene encoding E. coli biotin synthase (bioB) has been expressed as a histidine-fusion protein (6HisBS) and the recombinant protein purified in a single step using immobilised metal-affinity chromatography (IMAC). In addition, a number of single and double amino acid mutations of the conserved cysteine residues within the ‘cys box’ were constructed and purified using similar methodology. Wild-type and mutant 6HisBS proteins were compared using mass spectrometry (LC-ESI-MS) and UV-visible spectroscopy to probe characteristics altered by the mutation. Biotin synthase was analysed for its ability to bind the cofactor pyridoxal 5’-phosphate (PLP). Two single point mutations (K490Q and K49R) of a putative PLP-binding residue, Lys 49, were isolated and compared with wild-type BS using a combination of biochemical and mass spectrometry techniques. Each protein was subjected to enzymatic proteolysis and peptide modification analysed by means of MALDI-ToF MS, ESI-QTOF MS and MS/MS. We identified a unique Lys49-containing fragment of the 6HisBS which corresponds to the PLP modified peptide. In contrast, this modified peptide is absent in the mutant proteins. This observation is further confirmed by MS/MS sequencing of this MS observed species which shows CID of both the peptide backbone and covalently attached PLP moiety. Biotin synthase was prepared in the presence of excess PLP and its interaction with cysteine monitored with UV-visible spectroscopy. We observed marked changes in the UV-vis profile of biotin synthase under these conditions which is consistent observations of PLP-dependent cysteine desulferase enzymes.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.662987  DOI: Not available
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