{"doi":"10.1021/jacs.4c05955","title":"A Bioorthogonal Precision Tool for Human <i>N</i> -Acetylglucosaminyltransferase V","abstract":"High Resolution Image Download MS PowerPoint Slide 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. Still, the functional relevance and substrate choice of MGAT5 are ill-defined. Here, we employ protein 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. We find that WT-MGAT5 displays considerable activity toward such substrate analogues. Protein engineering yields an MGAT5 variant that loses activity against the native nucleotide sugar and increases activity toward a 4-azidobutyramide-containing substrate analogue. By such restriction of substrate specificity, we show that the orthogonal enzyme–substrate pair is suitable to bioorthogonally tag glycoproteins. Through X-ray crystallography and molecular dynamics simulations, we establish the structural basis of MGAT5 engineering, informing the design rules for bioorthogonal precision chemical tools.","journal":"Journal of the American Chemical Society","year":2024,"id":428987,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":11,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9511,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2024-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":288032,"name":"Ganka Bineva‐Todd","orcid":"0000-0001-8926-9417","position":1,"is_corresponding":false},{"id":1230635,"name":"Richard W. Meek","orcid":"0000-0002-1370-0896","position":2,"is_corresponding":false},{"id":1231051,"name":"Laura Mazo","orcid":null,"position":3,"is_corresponding":false},{"id":1230636,"name":"Beatriz Piniello","orcid":"0000-0001-5557-3146","position":4,"is_corresponding":false},{"id":1230637,"name":"Olga V. Moroz","orcid":"0000-0002-0354-6119","position":5,"is_corresponding":false},{"id":1172876,"name":"Sean A. Burnap","orcid":"0000-0002-3408-8608","position":6,"is_corresponding":false},{"id":1231052,"name":"Nadima Begum","orcid":null,"position":7,"is_corresponding":false},{"id":1230638,"name":"André Ohara","orcid":"0000-0002-9792-284X","position":8,"is_corresponding":false},{"id":288038,"name":"Chloë Roustan","orcid":"0000-0002-8439-8997","position":9,"is_corresponding":false},{"id":1230639,"name":"Sara Tomita","orcid":"0000-0001-7952-2474","position":10,"is_corresponding":false},{"id":288039,"name":"Svend Kjær","orcid":"0000-0001-9767-8683","position":11,"is_corresponding":false},{"id":842485,"name":"Karen M. Polizzi","orcid":"0000-0001-5435-2667","position":12,"is_corresponding":false},{"id":241657,"name":"Weston B. Struwe","orcid":"0000-0003-0594-226X","position":13,"is_corresponding":false},{"id":1230640,"name":"Carme Rovira","orcid":"0000-0003-1477-5010","position":14,"is_corresponding":false},{"id":1230641,"name":"G.J. Davies","orcid":"0000-0002-7343-776X","position":15,"is_corresponding":false},{"id":268915,"name":"Benjamin Schumann","orcid":"0000-0001-5504-0147","position":16,"is_corresponding":false},{"id":1230634,"name":"Liu Yu","orcid":"0009-0009-0722-182X","position":0,"is_corresponding":true}],"reference_count":74,"raw_metadata":null,"created_at":"2026-07-19T01:59:02.165535Z","pmid":"39287665","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}