We report a new chemoenzymatic strategy for the rapid and sensitive detection of O-GlcNAc posttranslational modifications. The approach exploits the ability of an engineered mutant of beta-1,4-galactosyltransferase to selectively transfer an unnatural ketone functionality onto O-GlcNAc glycosylated proteins. Once transferred, the ketone moiety serves as a versatile handle for the attachment of biotin, thereby enabling chemiluminescent detection of the modified protein. Importantly, this approach permits the rapid visualization of proteins that are at the limits of detection using traditional methods. Moreover, it bypasses the need for radioactive precursors and captures the glycosylated species without perturbing metabolic pathways. We anticipate that this general chemoenzymatic strategy will have broad application to the study of posttranslational modifications.

␤1,4-Galactosyltransferase I (Gal-T1) normally transfers Gal from UDP-Gal to GlcNAc in the presence of Mn 2؉ ion. In the presence of ␣-lactalbumin (LA), the Gal acceptor specificity is altered from GlcNAc to Glc. Gal-T1 also transfers GalNAc from UDP-GalNAc to GlcNAc, but with only ϳ0.1% of Gal-T activity. To understand this low GalNAc-transferase activity, we have carried out the crystal structure analysis of the Gal-T1⅐LA complex with UDP-GalNAc at 2.1-Å resolution. The crystal structure reveals that the UDP-GalNAc binding to Gal-T1 is similar to the binding of UDP-Gal to Gal-T1, except for an additional hydrogen bond formed between the N-acetyl group of GalNAc moiety with the Tyr-289 side chain hydroxyl group. Elimination of this additional hydrogen bond by mutating Tyr-289 residue to Leu, Ile, or Asn enhances the GalNAc-transferase activity. Although all three mutants exhibit enhanced GalNAc-transferase activity, the mutant Y289L exhibits GalNAc-transferase activity that is nearly 100% of its Gal-T activity, even while completely retaining its Gal-T activity. The steady state kinetic analyses on the Leu-289 mutant indicate that the K m for GlcNAc has increased compared to the wild type. On the other hand, the catalytic constant (k cat ) in the Gal-T reaction is comparable with the wild type, whereas it is 3-5-fold higher in the GalNAc-T reaction. Interestingly, in the presence of LA, these mutants also transfer GalNAc to Glc instead of to GlcNAc. The present study demonstrates that, in the Gal-T family, the Tyr-289/Phe-289 residue largely determines the sugar donor specificity.

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