title: Heteronuclear NMR studies of the specificity of the post-translational modification of biotinyl domains by biotinyl protein ligase.
abstract: The lipoyl domains of 2-oxo acid dehydrogenase multienzyme complexes and the biotinyl domains of biotin-dependent enzymes have homologous structures, but the target lysine residue in each domain is correctly selected for posttranslational modification by lipoyl protein ligase and biotinyl protein ligase, respectively. We have applied two-dimensional heteronuclear NMR spectroscopy to investigate the interaction between the apo form of the biotinyl domain of the biotin carboxyl carrier protein of acetyl-CoA carboxylase and the biotinyl protein ligase (BPL) from Escherichia coli. Heteronuclear multiple quantum coherence NMR spectra of the 15N-labelled biotinyl domain were recorded in the presence and absence of the ligase and backbone amide 1H and 15N chemical shifts were evaluated. Small, but significant, changes in chemical shift were found in two regions, including the tight beta-turn that houses the lysine residue targetted for biotinylation, and the beta-strand 2 and the loop that precedes it in the domain. When compared with the three-dimensional structure, sequence alignments of other biotinyl and lipoyl domains, and mutagenesis data, these results give a clear indication of how the biotinyl domain is both recognised by BPL and distinguished from the structurally related lipoyl domain to ensure correct posttranslational modification.
authors.affiliation: Cambridge Centre for Molecular Recognition, Department of Biochemistry, University of Cambridge, UK. reche@dnax.org
authors.forename: P A
authors.initials: PA
authors.lastname: Reche
authors.forename: M J
authors.initials: MJ
authors.lastname: Howard
authors.forename: R W
authors.initials: RW
authors.lastname: Broadhurst
authors.forename: R N
authors.initials: RN
authors.lastname: Perham
mesh_terms: D000103:Acetyl-CoA Carboxylase / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D019921:Biotinylation; D019731:Carbon-Nitrogen Ligases / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014157:Transcription Factors
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli; D000103:Acetyl-CoA Carboxylase