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Title: Studies on the protein components of biotin synthase
Author: McIver, Lisa
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2000
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The gene encoding the E. coli flavodoxin NADP+ oxidoreductase (FLDR) and flavodoxin (FLD) have been overexpressed in E. coli and the gene products purified to homogeneity. Physical characterisation of both proteins was carried out using steady-step and stopped flow kinetics, circular dichroism and fluorimetric studies. The molecular masses of the apoproteins were determined as 27648Da and 19606Da and the isoelectric points as 4.8 and 3.5, respectively. The midpoint reduction potentials of the oxidised/semiquinone and semiquinone/hydroquinone couples of both FLDR (-308mV and -268mV) and FLD (-254mV and -433mV) were measured using redox potentiometry. This confirms the electron-transfer route as NADPH (r) FLDR (r) FLD. Binding of 2' adenosine monophosphate increases the midpoint reduction potentials for both FLDR couples. These data highlight the strong stabilisation of the flavodoxin semiquinone with respect to the hydroquinone state and indicate that FLD must act as a single electron shuttle from the semiquinone form in its support of cellular functions. FLDR and FLD were covalently crosslinked using 1-ethyl-3(dimethylamino-propyl) carodiimide. A single species was formed with an apparent molecular weight of 47kD. This complex was catalytically active in that it could reduce cytochrome c and potassium ferricyanide almost as efficiently as the individual proteins. The gene encoding E. coli biotin synthase (bioB) has been expressed as a histidine-fusion protein and the protein purified in a single step using IMAC. The His6-tagged protein was fully functional in in vitro and in vivo biotin production assays. Analysis of all the published bioB sequences identified a number of conserved residues. Single point mutations, to either serine or threonine, were carried out on the four conserved (Cys-53, Cys-57), Cys-60 and Cys-188) and on non-conserved (Cys-288) cysteine residues and the purified mutant proteins tested both for ability to reconstitute the [2Fe-2S] clusters of the native (oxidised) dimer and enzymatic activity.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available