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  • Title: Purification and characterization of intact and truncated forms of the Escherichia coli biotin carboxyl carrier subunit of acetyl-CoA carboxylase.
    Author: Nenortas E, Beckett D.
    Journal: J Biol Chem; 1996 Mar 29; 271(13):7559-67. PubMed ID: 8631788.
    Abstract:
    Biotin biosynthesis and retention in Escherichia coli is regulated by the multifunctional protein, BirA. The protein acts as both the transcriptional repressor of the biotin biosynthetic operon and as a ligase for covalent attachment of biotin to a unique lysine residue of the acetyl-CoA carboxylase. Biotinyl-5'-AMP is the activated intermediate for the ligase reaction and the allosteric effector for DNA binding. We have purified and characterized apoBCCP and a truncated form containing the COOH-terminal 87 residues (apoBCCP87). Molecular masses of the proteins measured using matrix-assisted laser desorption ionization time-of-flight mass spectrometry conformed to the expected values. The assembly states of apoBCCP and apoBCCP87 were determined using sedimentation equilibrium ultracentrifugation. Nearly quantitative enzymatic transfer of biotin from BirA-biotinyl-5'-AMP to the apoBCCP forms was assessed using two methods, mass spectrometric analysis of acceptor proteins after incubation with BirA-bio-5'-AMP and a steady state fluorescence assay. The BirA catalyzed rates of transfer of biotin from bio-5'-AMP to apoBCCP and apoBCCP87 were measured by stopped-flow fluorescence. Kinetic parameters estimated from these measurements indicate that the intact and truncated forms of the acceptor protein are functionally identical.
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