Biotin ligase

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. 

All four carboxylases use bicarbonate as their one-carbon substrate and, in all, the biotin is covalently linked by an amide bond between the carboxyl of biotin and an epsilon amino group of a lysyl residue in the holocarboxylase synthase (= biotin ligase) that catalyzes the formation of the covalent bond. Biotinylation of histones is involved in regulation of gene transcription and may also play a role in packaging of deoxyribonucleic acid (DNA). Biotin has also been found to inhibit the generation of reactive oxygen species (ROS) by neutrophils in vitro. 

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]... 

Biotin acceptor peptides >15 Keto isostere of biotin Biotin ligase, ATP, hydrazide derivatives Covalent and reversible Cell surface Two-step labeling required [32]... 

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,... 

Fig. 10.3-16 Introduction of unnatural functional groups through posttranslational modification, (a) Ketones and azides can be introduced onto cell surfaces by feeding cells with unnatural sialic acid precursors, such as mannosamine derivatives 40b and c. These are incorporated into cell-surface glycans, which can be further elaborated using additional bioconjugation reactions, (b) Specific amino acid sequences can be modified using biotin ligase. Interestingly, ketobiotin" is also recognized as a substrate for the enzyme, allowing a...

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).

The AviTag (Avidity, Colorado, USA) is an in vitro synthesized 15 residue peptide that is specifically biotinylated by the E. coli biotin ligase (31). 

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

Holocarboxylase synthetase (EC 6.3.4.10) (HCS or biotin ligase) catalyzes biotin incorporation into various carboxylases which are essential for housekeeping cellular metabolism, as for example ACCase that realizes the first committed step in fatty acid biosynthesis. These carboxylases are synthesized at first as inactive apoproteins which are then modified into active holocarboxylases by post-translational addition of D-biotin to a specific Lysine residue in the apoprotein. This covalent reaction occurs via an amide linkage between the biotin carboxyl group and the e-amino group of a unique Lysine residue present in a specific sequence of all biotin-dependent carboxylases. This covalent attachment occurs in two steps (1) and (2) as follows ... 

Current efforts are directed at attempts to introduce the C. cryptica ACCase gene into yeast. Because the structures of yeast and C. cryptica ACCase are reasonably well conserved, it is anticipated that the endogenous biotin ligase of yeast will function more efficiently in biotinylating C. cryptica ACCase than was the case in E. coli. If so, the ftmctionality of the recombinant ACCase can readily be evaluated. 

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