9F5H

Crystal structure of MGAT5 bump-and-hole mutant in complex with UDP and M592

Summary for 9F5H

• Entry DOI: 10.2210/pdb9f5h/pdb
• Descriptor: Secreted alpha-1,6-mannosylglycoprotein 6-beta-N-acetylglucosaminyltransferase A, URIDINE-5'-DIPHOSPHATE, 2-acetamido-2-deoxy-beta-D-glucopyranose, ... (6 entities in total)
• Functional Keywords: bump and hole, bioorthogonal tag, transferase
• Biological source: Homo sapiens (human); More
• Total number of polymer chains: 2
• Total formula weight: 119420.74

Authors

Liu, Y.,Bineva-Todd, G.,Meek, R.,Mazo, L.,Piniello, B.,Moroz, O.V.,Begum, N.,Roustan, C.,Tomita, S.,Kjaer, S.,Rovira, C.,Davies, G.J.,Schumann, B. (deposition date: 2024-04-28, release date: 2024-10-02, Last modification date: 2024-10-16)

Primary citation

Liu, Y.,Bineva-Todd, G.,Meek, R.W.,Mazo, L.,Piniello, B.,Moroz, O.,Burnap, S.A.,Begum, N.,Ohara, A.,Roustan, C.,Tomita, S.,Kjaer, S.,Polizzi, K.,Struwe, W.B.,Rovira, C.,Davies, G.J.,Schumann, B.
A Bioorthogonal Precision Tool for Human N -Acetylglucosaminyltransferase V.
J.Am.Chem.Soc., 146:26707-26718, 2024

PubMed Abstract: 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 -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.

PubMed: 39287665
DOI: 10.1021/jacs.4c05955

Experimental method

X-RAY DIFFRACTION (1.97 Å)