A Bioorthogonal Precision Tool for human N-acetylglucosaminyltransferase V

Correct elaboration of N-linked glycans in the secretory pathway of human cells is essential in physiology. Early N-glycan biosynthesis follows an assembly line principle before undergoing crucial elaboration points that feature the sequential incorporation of the sugar N-acetylglucosamine (GlcNAc). The activity of GlcNAc transferase V (MGAT5) primes the biosynthesis of an N-glycan antenna that is heavily upregulated in cancer. Yet, the functional relevance and substrate choice of MGAT5 are ill-defined. Here, we employ a tactic termed bump-and-hole engineering to develop a bioorthogonal substrate analog for the activity of MGAT5. Chemoenzymatic synthesis is used to produce a collection of nucleotide-sugar analogs with bulky, bioorthogonal acylamide side chains. Protein engineering yields an MGAT5 variant that specifically uses one such substrate instead of the native nucleotide-sugar. We validate the enzymatic bump-and-hole system and show that the orthogonal enzyme-substrate pair is suitable to bioorthogonally tag glycoproteins. Through X-ray crystallography and molecular dynamic simulations, we establish the structural basis of bump-and-hole engineering, informing the design rules for bioorthogonal precision chemical tools.