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Title: BioI : the P450 implicated in biotin biosynthesis
Author: Green, Amanda Jane
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2001
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The three genes believed to encode the redox system for cytochrome P450 BioI have been cloned (bioI, orf2 and fer). Protein overexpression of both BioI and the ferredoxin has been achieved to high levels and purification protocols determined for both. Characterisation of BioI using a variety of spectroscopic methods has proved that the enzyme is indeed a P450, showing typical spectral shifts on binding of exogenous ligands such as CO and NO. Ligand binding studies have indicated a high affinity of the protein for long-chain saturated fatty acids, with the optimal ligand being myristate, C14 Crystals of BioI have also been obtained, but solution of the structure has so far proved elusive. The Bacillus subtilis ferredoxin appeared to be the only candidate electron donor to BioI and sequence similarities suggested it contained a 4Fe-4S cluster. Characterisation of the ferredoxin by UV-visible spectroscopy, CD and EPR (in the reduced form) confirmed this. ICP-AES and the oxidised EPR data seemed to suggest the presence of at least some 3De-4S cluster, which appears to suggest oxidative degradation of the cluster. Using E. coli flavodoxin reductase (FLDR) as a redox partner alongside the ferredoxin, electron transfer to BioI has been demonstrated, albeit very slowly. Electron transfer from the FLDR to the ferredoxin was shown to be very rapid, indicating the slow step to be electron transfer from ferredoxin to BioI or steps after this. The ability of this system to hydroxylate fatty acids has also been demonstrated using the fatty acid chromophore, p-nitrophenoxydodecanoic acid (Schwaneberg et al., 1999).
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available