Escherichia coli biotin ligase (BirA) has been harnessed for numerous biotechnological applications, including protein labeling,1-3 purification,4,5 and immobilization.6 BirA catalyzes the ATP-dependent covalent ligation of biotin to a lysine sidechain of a 15-amino acid recognition sequence called the "acceptor peptide" (AP).7 Previously, we showed that BirA could be applied to site-specific protein labeling on the surface of living cells by making use of a ketone isostere of biotin that could be ligated to AP fusion proteins, then derivatized with hydrazide or hydroxylamine probes.8 Since that work, we have focused on expanding the small-molecule specificity of BirA to incorporate other useful functional groups, such as azides and alkynes, which can be derivatized with even more chemoselective reactions than the ketone.9 Because we found that BirA does not ligate diverse structural analogs of biotin to the AP, in this study, we explored the activities of biotin ligases from nine other organisms. We discovered that yeast biotin ligase accepts an alkyne derivative of biotin, while Pyrococcus horikoshii biotin ligase utilizes both alkyne and azide biotin analogs. These new ligation reactions demonstrate the differential substrate specificities of ligases from different species, and open the door to novel protein labeling applications.Due to our interest in novel methodologies for targeting chemical probes to proteins in the cellular context, and the previous work of our lab in exploiting E. coli BirA for this purpose, 1,8 we synthesized or purchased each of the biotin analogs shown in Figure 1. Desthiobiotin azide (DTB-Az) 2 and propargyl biotin (PB) 3, whose syntheses are shown in Figure 2, contain unique functional group handles. Azides are naturally absent from cells and can be selectively derivatized with strained alkynes or phosphines under physiological conditions,9 while alkynes, also absent from cells, can be selectively derivatized with azides via [3+2] cycloaddition in the presence of a copper catalyst.10 Iminobiotin 4 and diaminobiotin 5 exhibit pH-dependent binding to streptavidin and can be used for protein purification.11 Nitrobenzoxadiazole gamma-amino butyric acid 6 is a fluorophore, and iodouracil and thiouracil valeric acid probes (7 and 8) are photoactivatable crosslinkers.12We found that wild-type BirA does not ligate analogs 2-8 to the AP (data not shown). Because precedent exists for differential substrate specificity among homologous enzymes from different species,13,14 we decided to clone, express, and evaluate biotin ligases from nine other species. All organisms express one or two biotin ligases,15,16 which attach biotin to protein domains involved in the transfer of carboxyl groups. In some organisms, the ligase plays an additional role in repressing transcription at the biotin biosynthetic operon or other biotin-sensitive genes.17 To select our panel of novel biotin ligases, we first noted that biochemical and/or structural data were available for the human,18 S. cerevisiae (yeast),...