Biotin protein ligase

Protein biotinylation is catalyzed by biotin protein ligase (BPL). In the active site of the enzyme, biotin is activated at the expense of ATP to form AMP-biotin the activated biotin can then react with a nucleophile on the targeted protein. BPL transfers the biotin to a special lysine on biotin carboxyl carrier protein (BCCP), a subunit of AcCoA carboxylase (Scheme 21). Biotinylation of BCCP is very important in fatty acid biosynthesis, starting the growth of the fatty acid with AcCoA carboxylase to generate malonyl-CoA. Recently the crystal structures of mutated BPL and BCCP have been solved together with biotin and ATP to get a better idea of how the transfer fiinctions. ... 

Figure 11.1. Metabolism of biotin. Holocarboxylase synthetase (biotin protein ligase), EC 6.3.4.10 and biotinidase (biotinamide amidohydrolase), EC 3.5.1.12. Relative molecular mass (Mr) biotin, 244.3 and biocytin, 372.5.

A single holocarboxylase synthetase (biotin protein ligase, EC 6.3.4.10) acts on the apoenzymes of acetyl CoA, pyruvate, propionyl CoA, and methylcrotonyl CoA carboxylases. Acetyl CoA carboxylase is a cytosolic enzyme, whereas the other three enzymes are mitochondrial. Although holocarboxylase synthetase is found in both the cytosol and mitochondria, it is not clear whether biotin is incorporated into the mitochondrial enzymes before or after they are translocated into the mitochondria. 

Peters-Wendisch, P., Stansen, K.C., Gotker, S., and Wendisch, V.F. (2012) Biotin protein ligase from Corynebacterium glutamicum role for growth and L —lysine production. Appl. Microbiol Biotechnol, 93 (6), 2493-2502. 

Pimer HM and Stolz J. Biotin sensing in Saccharomyces cerevisiae is mediated by a conserved DNA element and requires the activity of biotin-protein ligase. J. Biol. Chem. 281 12381-12389(2006). 

Each ACC half-reaction is catalyzed by a different protein sub-complex. The vitamin biotin is covalently coupled through an amide bond to a lysine residue on biotin carboxyl carrier protein (BCCP, a homodimer of 16.7-kDa monomers encoded by accB) by a specific enzyme, biotin-apoprotein ligase (encoded by birA), and is essential to activity. The crystal and solution structures of the biotinyl domain of BCCP have been determined, and reveal a unique thumb required for activity (J. Cronan, 2001). Carboxylation of biotin is catalyzed by biotin carboxylase (encoded by accC), a homodimeric enzyme composed of 55-kDa subunits that is copurified complexed with BCCP. The accB and accC genes form an operon. The three-dimensional structure of the biotin carboxylase subunit has been solved by X-ray diffraction revealing an ATP-grasp motif for nucleotide binding. The mechanism of biotin carboxylation involves the reaction of ATP and CO2 to form the shortlived carboxyphosphate, which then interacts with biotin on BCCP for CO2 transfer to the I -nitrogen. 

Biotin ligase acceptor protein (AP) [ketone analog 15 1.7 lys +... 

Chen, I., Choi, Y. A. and Ting, A. Y. (2007). Phage display evolution of a peptide substrate for yeast biotin ligase and application to two-color quantum dot labeling of cell surface proteins. J. Am. Chem. Soc. 129, 6619-25. 

Howarth, M., Takao, K., Hayashi, Y. and Ting, A. Y. (2005). Targeting quantum dots to surface proteins in living cells with biotin ligase. Proc. Natl. Acad. Sci. USA 102, 7583-8. 

We observed that addition of free biotin to the growth medium increases the extent of biotinylation of the recombinant BCCP fusion protein. Strains overexpressing the E. coli biotin ligase are also available from Avidity (www.avidity.com) if desired, although we have not found this necessary when using the BCCP tag. The wash step prior to cell lysis is needed to remove free biotin before array fabrication if the protein is purified before array fabrication, this is not necessary. 

Chen I, Howarth M, Lin W, Ting AY. Site-specific labeling of cell surface proteins with biophysical probes using biotin ligase. Nat. Methods 2005 2 99-104. 

The question of the in vivo situation was of course open. As most organisms require only tiny amounts of biotin, the hypothesis that BS could also be noncatalytic in vivo had to, and has been considered. It looks, however, more reasonable to expect a catalytic function. It is now well established that in vivo synthesis of [Fe-S] centers makes use of a very complex machinery, namely, the Isc or Suf systems in E. colt They include chaperone proteins, which may be necessary for repairing the cluster, or for the regeneration of a native empty [2Fe-2S] site. A first answer to this puzzling question has been given in a recent paper that describes in vivo experiments. The amount of biotin produced from DTB was determined by coexpression in E. coli of BS, biotin ligase, and a fragment of acetyl-CoA carboxylase to trap the biotin produced, followed by quantification of the biotinylated protein. A turnover of 20-60 equivalents of biotin has been observed, but a quantitative evaluation is difficult due to the fact that turnover renders the protein susceptible to proteolysis. 

Biotin acceptor peptides >15 Biotin Biotin ligase, ATP Covalent and irreversible Intracellular, cell surface Allows use of derivatized streptavidins to label cell surface proteins [31]... 

Another general approach for the labeling of fusion proteins, which conceptually resembles the labeling of CP-fusion proteins is the biotinylation of so-called acceptor peptides by biotin ligase [31, 58]. The biotinylation of fusion proteins by itself is a valuable modification, as numerous streptavidin-and avidin-based probes and materials are commercially available. However,... 

As many posttranslational modification enzymes display exquisite specificity for a particular amino acid sequence, they are uniquely effective tools for labeling one particular protein present in a complex mixture. A recent report has capitalized on this feature by using biotin ligase to install biotin groups 41a on a single lysine residue embedded in a specific 15 amino acid sequence,... 

Fig. 1. Structure of the coli biotin carboxyl carrier protein (BCCP) domain. Residues 77-156 are drawn (coordinate file Ibdo), showing the N- and C-termini and the single biotin moiety that is attached to lysine 122 in vivo by biotin ligase. Representation produced using SwissPDBViewer (23).

Figure 1.20 Enzymes for site-specific protein modification (a) Biotin ligase/ (b) lipoic acid ligase/ and (c) protein farnesyltransferase. ...

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