Biotinylation in the hyperthermophile Aquifex aeolicus.
Biotin protein ligase (BPL) catalyses the biotinylation of the biotin carboxyl carrier protein (BCCP) subunit of acetyl CoA carboxylase and this post-translational modification of a single lysine residue is exceptionally specific. The exact details of the protein-protein interactions involved are unclear as a BPL:BCCP complex has not yet been isolated. Moreover, detailed information is lacking on the composition, biosynthesis and role of fatty acids in hyperthermophilic organisms. We have cloned, overexpressed and purified recombinant BPL and the biotinyl domain of BCCP (BCCP Delta 67) from the extreme hyperthermophile Aquifex aeolicus. In vitro assays have demonstrated that BPL catalyses biotinylation of lysine 117 on BCCP Delta 67 at temperatures of up to 70 degrees C. Limited proteolysis of BPL with trypsin and chymotrypsin revealed a single protease-sensitive site located 44 residues from the N-terminus. This site is adjacent to the predicted substrate-binding site and proteolysis of BPL is significantly reduced in the presence of MgATP and biotin. Chemical crosslinking with 1-ethyl-3-(dimethylamino-propyl)-carbodiimide (EDC) allowed the isolation of a BPL:apo-BCCP Delta 67 complex. Furthermore, this complex was also formed between BPL and a BCCP Delta 67 mutant lacking the lysine residue (BCCP Delta 67 K117L) however, complex formation was considerably reduced using holo-BCCP Delta 67. These observations provide evidence that addition of the biotin prosthetic group reduces the ability of BCCP Delta 67 to heterodimerize with BPL, and emphasizes that a network of interactions between residues on both proteins mediates protein recognition.
Clarke
David J
DJ
School of Chemistry, University of Edinburgh, UK.
Coulson
Joseph
J
Baillie
Ranald
R
Campopiano
Dominic J
DJ
D000103:Acetyl-CoA Carboxylase / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology*; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001710:Biotin / Q000378:metabolism; D019921:Biotinylation; D019731:Carbon-Nitrogen Ligases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D054889:Fatty Acid Synthase, Type II; D008969:Molecular Sequence Data; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016415:Sequence Alignment; D014157:Transcription Factors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D001426:Bacterial Proteins; D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D001710:Biotin; D054889:Fatty Acid Synthase, Type II; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli; D000103:Acetyl-CoA Carboxylase; C079633:biotin carboxyl carrier protein