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1 Department of Molecular Biosciences, University of Adelaide, South Australia 5005, Australia
2 Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3052, Australia
3 Departments of Microbiology and Biochemistry, University of Illinois, Urbana, Illinois 61801, USA
Reprint requests to: Dr. Anne Chapman-Smith, Department of Molecular Biosciences, University of Adelaide, Adelaide, SA 5005 Australia; e-mail: anne.chapmansmith{at}adelaide.edu.au; fax: 61-8-8303-4348.
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.
Keywords: Biotin protein ligase; biotin holoenzyme synthetase; ATP binding; protein–protein interactions; posttranslational modification
Abbreviations: BCCP, biotin carboxyl carrier protein • IPTG, isopropyl-1-thio-ß-D-galactopyranoside • PAGE, polyacrylamide gel electrophoresis • S.D., standard deviation
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