Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.659735
Title: Studies on the mesophilic and hyperthermostable protein 8-amino-7-oxononanoate synthase : a biotin biosynthetic enzyme
Author: Mullan, Lisa
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2000
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Abstract:
The biotin biosynthetic enzyme 8-amino-7-oxononanoate synthase (AONS) from Escherichia coli is an homodimeric enzyme and relies on a pyridoxal-5'-phosphate (PLP) cofactor for catalytic activity. Specific activity of the over-expressed enzyme is approximately 0.4U at 30°C. To investigate the role of residues at the active site on the catalytic mechanism of the enzyme, two separate mutants have been created by site directed mutagenesis. H133 lies in parallel with the pyridine ring of the cofactor, and was mutated to a phenylalanine residue, with subsequent loss of cofactor binding. Alteration of the lysine 236 residue crucial for forming an aldimine linkage with the cofactor, to alanine resulted in a reduction, but no loss, of cofactor binding. The specific activities of both mutants were reduced by approximately ten-fold. The corresponding bioF gene from an hyperthermophilic bacterium, Aquifex aeolicus, was cloned and AONS over-expressed in an E. coli host. The enzyme was characterised by a number of biophysical techniques. It is a dimer in solution, with a monomeric mass of 42.3 kDa and one active site per subunit. Specific activity of the purified enzyme was 0.7U at 30°C, and optimum activity occurred at 80°C. The heat stable enzyme displays a greater affinity for its PLP cofactor and the binding of the coenzyme on mutation of the active site histidine residue corresponding to H133 in E. coli, indicates that PLP is bound by a residue not involved in E. coli AONS-PLP binding. Crystals grew in wells containing 1.6M-2.5M ammonium sulphate at 60°C produced orthorhombic crystals, which diffracted to 2.94Å. Analysis of the diffraction data suggested a space group of P21212 and a large unit cell containing at least 6 subunits in the asymmetric unit. Phasing was achieved by molecular replacement using the structure of the Bacillus sphaericus AONS protein.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.659735  DOI: Not available
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