title: Purification and characterization of intact and truncated forms of the Escherichia coli biotin carboxyl carrier subunit of acetyl-CoA carboxylase.
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.
authors.affiliation: Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore, 21228, USA.
authors.forename: E
authors.initials: E
authors.lastname: Nenortas
authors.forename: D
authors.initials: D
authors.lastname: Beckett
mesh_terms: D000103:Acetyl-CoA Carboxylase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D001710:Biotin / Q000096:biosynthesis / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D054889:Fatty Acid Synthase, Type II; D007700:Kinetics; D046911:Macromolecular Substances; D013058:Mass Spectrometry; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011487:Protein Conformation; D017384:Sequence Deletion; D013050:Spectrometry, Fluorescence; D014461:Ultracentrifugation
chemical_list: D002352:Carrier Proteins; D046911:Macromolecular Substances; D010446:Peptide Fragments; D001710:Biotin; D054889:Fatty Acid Synthase, Type II; D000103:Acetyl-CoA Carboxylase; C079633:biotin carboxyl carrier protein