tailieunhanh - Báo cáo khoa học: Biotinylation in the hyperthermophile Aquifex aeolicus Isolation of a cross-linked BPL:BCCP complex

Biotin protein ligase (BPL) catalyses the biotinylation of the biotin carboxyl carrier protein (BCCP) subunit of acetyl CoAcarboxylase and this post-translational modificationof 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 composi-tion, biosynthesis and role of fatty acids in hyperthermo-philic organisms. | Eur. J. Biochem. 270 1277-1287 2003 FEBS 2003 doi Biotinylation in the hyperthermophile Aquifex aeolicus Isolation of a cross-linked BPL BCCP complex David J. Clarke Joseph Coulson Ranald Baillie and Dominic J. Campopiano School of Chemistry University of Edinburgh UK 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 hyperthermo-philic organisms. We have cloned overexpressed and purified recombinant BPL and the biotinyl domain of BCCP BCCPA67 from the extreme hyperthermophile Aquifex aeolicus. In vitro assays have demonstrated that BPL catalyses biotinylation of lysine 117 on BCCPA67 at temperatures of up to 70 OC. 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-BCCPA67 complex. Furthermore this complex was also formed between BPL and a BCCPA67 mutant lacking the lysine residue BCCPA67 K117L however complex formation was considerably reduced using holo-BCCPA67. These observations provide evidence that addition of the biotin prosthetic group reduces the ability of BCCPA67 to heterodimerize with BPL and emphasizes that a network of interactions between residues on both proteins mediates protein recognition. Keywords biotin protein ligase Aquifex aeolicus biotinylation protein recognition chemical