Expanding the Substrate Tolerance of Biotin Ligase through Exploration of Enzymes from Diverse Species

Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Journal of the American Chemical Society (Impact Factor: 12.11). 02/2008; 130(4):1160-2. DOI: 10.1021/ja076655i
Source: PubMed


Technologies that enable the site-specific conjugation of chemical probes onto proteins are extremely useful for applications in cell biology and proteomics. We cloned, expressed, and purified biotin ligases from nine different species and screened them for the ability to ligate unnatural analogues of biotin onto the human p67 biotin acceptor domain. We discovered that the biotin ligases of Saccharomyces cerevisiae (yeast) and Pyrococcus horikoshii could accept alkyne and azide derivatives of biotin, respectively. HPLC, gel-shift, and mass spectrometry assays confirmed that these ligation reactions were ATP-and enzyme-dependent, as well as site-specific. We used the P. horikoshii-catalyzed azide ligation reaction to site-specifically introduce a phosphine probe onto p67 using the Staudinger ligation. These new ligation reactions demonstrate the differential substrate specificities of biotin ligases from different organisms and open the door to novel protein labeling applications.

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Available from: Sarah A. Slavoff
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    • "However, yAP has a low catalytic value and the catalytic efficiency of the yAP-yBL pair is about 800 fold lower than the BirA-AP pair. To expand the substrate range of biotin ligase, Slavoff et al. examined the biotinylation reaction with biotin ligases from nine species and eight biotin isosters, using p67, a domain from one of the human biotin acceptor proteins (Slavoff et al., 2008). It was found that biotin ligases from S. cerevisiae and P. horikoshii accept alkyne and azide biotin analogues. "
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    • "In another study, FLAG phosphine (FLAG is a peptide epitope for a useful well-characterized mAb) was used to label azido-bearing biotin. The FLAG biotin was then used to tag an endogenous biotin acceptor protein (Slavoff et al., 2008). Despite the susceptibility of phosphines used for the Staudinger ligation to air oxidation (Agard et al., 2004) and their substoichiometric modification of azF in recombinant proteins (Huber et al., 2013; Yanagisawa et al., 2008) and azido sugars (Agard et al., 2006), their compatibility to cell-based applications (Prescher et al., 2004) makes them attractive candidates for further study. "
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    • "While some analogs such as desthiobiotin azide and cispropargyl biotin can be used as substrates by P. horikoshii and yeast BPLs for the site-specific introduction of azides and alkynes onto peptides the reactions are relatively slow (Slavoff et al., 2008). Our selection system can now potentially be used "
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