title: The C-terminal domain of biotin protein ligase from E. coli is required for catalytic activity.
abstract: Biotin protein ligase of Escherichia coli, the BirA protein, catalyses the covalent attachment of the biotin prosthetic group to a specific lysine of the biotin carboxyl carrier protein (BCCP) subunit of acetyl-CoA carboxylase. BirA also functions to repress the biotin biosynthetic operon and synthesizes its own corepressor, biotinyl-5'-AMP, the catalytic intermediate in the biotinylation reaction. We have previously identified two charge substitution mutants in BCCP, E119K, and E147K that are poorly biotinylated by BirA. Here we used site-directed mutagenesis to investigate residues in BirA that may interact with E119 or E147 in BCCP. None of the complementary charge substitution mutations at selected residues in BirA restored activity to wild-type levels when assayed with our BCCP mutant substrates. However, a BirA variant, in which K277 of the C-terminal domain was substituted with Glu, had significantly higher activity with E119K BCCP than did wild-type BirA. No function has been identified previously for the BirA C-terminal domain, which is distinct from the central domain thought to contain the ATP binding site and is known to contain the biotin binding site. Kinetic analysis of several purified mutant enzymes indicated that a single amino acid substitution within the C-terminal domain (R317E) and located some distance from the presumptive ATP binding site resulted in a 25-fold decrease in the affinity for ATP. Our data indicate that the C-terminal domain of BirA is essential for the catalytic activity of the enzyme and contributes to the interaction with ATP and the protein substrate, the BCCP biotin domain.
authors.affiliation: Department of Molecular Biosciences, University of Adelaide, South Australia 5005, Australia. anne.chapmansmith@adelaide.edu.au
authors.forename: A
authors.initials: A
authors.lastname: Chapman-Smith
authors.forename: T D
authors.initials: TD
authors.lastname: Mulhern
authors.forename: F
authors.initials: F
authors.lastname: Whelan
authors.forename: J E
authors.initials: JE
authors.lastname: Cronan
authors.forename: J C
authors.initials: JC
authors.lastname: Wallace
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D001710:Biotin / Q000737:chemistry*; D019921:Biotinylation; D019731:Carbon-Nitrogen Ligases / Q000737:chemistry* / Q000235:genetics; D002384:Catalysis; D020134:Catalytic Domain; D004252:DNA Mutational Analysis; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D014157:Transcription Factors
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D001710:Biotin; D000255:Adenosine Triphosphate; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli