Investigation of the Catalytic Mechanism of a Soluble N-glycosyltransferase Allows Synthesis of N-glycans at Noncanonical Sequons

The soluble N-glycosyltransferase from Actinobacilluspleuropneumoniae (ApNGT) can establish an N-glycosidic bondat the asparagine residuein the Asn-Xaa-Ser/Thr consensus sequon and is one of the most promisingtools for N-glycoprotein production. Here, by integrating computationaland experimental strategies, we revealed the molecular mechanism ofthe substrate recognition and following catalysis of ApNGT. Thesefindings allowed us to pinpoint a key structural motif ((DVYM218)-D-215) in ApNGT responsible for the peptide substraterecognition. Moreover, Y222 and H371 of ApNGT were found to participatein activating the acceptor Asn. The constructed models were supportedby further crystallographic studies and the functional roles of theidentified residues were validated by measuring the glycosylationactivity of various mutants against a library of synthetic peptides.Intriguingly, with particular mutants, site-selective N-glycosylationof canonical or noncanonical sequons within natural polypeptides fromthe SARS-CoV-2 spike protein could be achieved, which were used toinvestigate the biological roles of the N-glycosylation in membranefusion during virus entry. Our study thus provides in-depth molecularmechanisms underlying the substrate recognition and catalysis forApNGT, leading to the synthesis of previously unknown chemically definedN-glycoproteins for exploring the biological importance of the N-glycosylationat a specific site.