Structural and functional studies of biotin protein ligase and its bacterial substrate acetyl-CoA carboxylase

• Main Author: Tron, Cecile M. V.
• Published: University of Edinburgh 2008
• Subjects: 572.7
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.663049

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 <i>Aquifex aeolicus </i>were overexpressed in <i>E. coli</i> and the recombinant protein <i>Aa</i>BPL was purified to homogeneity. <i>Aa</i>BPL was purified to homogeneity.  <i>Aa</i>BPL 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 <i>Aa</i>BPL, 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 <i>via </i>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 <i>A. aeolicus </i>by chemical cross-linking and mutational studies have identified a salt bridge between <i>Aa</i>BPL and BCCPΔ67 which is important for heterodimerisation. The structures at 2.4 Å resolution of <i>Aa</i>BPL 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-<i>Aa</i>BPL 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.