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Title: Structural and functional studies of biotin protein ligase and its bacterial substrate acetyl-CoA carboxylase
Author: Tron, Cecile M. V.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2008
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Biotin protein ligase (BPL) catalyzes the formation of biotinyl-5’-AMP from biotin and ATP and the succeeding biotinylation of the biotin carboxyl carrier protein (BCCP). The genes encoding the BPL from the hyperthermophyle Aquifex aeolicus were overexpressed in E. coli and the recombinant protein AaBPL was purified to homogeneity. AaBPL was purified to homogeneity.  AaBPL was proven to be catalytically active and to biotinylate specifically the C-terminal biotinyl domain of BCCP (BCCPΔ67). It was determined by isothermal titration calorimetry experiments that in the class I AaBPL, the presence of biotin is not required for ATP binding in absence of Mg2+ ions and the binding of biotin and ATP has been determined to occur via a random but cooperative process. In the second step of the enzymatic reaction, BPL has been suggested to form a BPL:BCCP complex. This complex was characterized in A. aeolicus by chemical cross-linking and mutational studies have identified a salt bridge between AaBPL and BCCPΔ67 which is important for heterodimerisation. The structures at 2.4 Å resolution of AaBPL in the apo-form and in complex with biotin and ATP were determined. These are the first crystal structures of a BPL complex with biotin and ATP and also of an ATP-bound BPL. The adenylate binding loop is ordered in the structure of apo-AaBPL and the ATP binding pocket is well defined. The solvent-exposed β- and γ-phosphates of ATP are located in the inter-subunit cavity formed by the N- and C-terminal domains. The Arg40 residue from the conserved GXGRXG motif is shown to interact with the carboxyl group of biotin and to stabilise the α- and β- phosphates of the nucleotide.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available