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

Zhiqiang Hao; Qiang Guo; Yuanyuan Feng; Zihan Zhang; Tiantian Li; Zhixin Tian; Jianting Zheng; Lin-Tai Da; Wenjie Peng

doi:10.1021/jacsau.3c00214

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

JACS Au. 2023 Aug 7;3(8):2144-2155. doi: 10.1021/jacsau.3c00214. eCollection 2023 Aug 28.

Authors

Zhiqiang Hao¹, Qiang Guo¹, Yuanyuan Feng², Zihan Zhang³, Tiantian Li¹, Zhixin Tian³, Jianting Zheng², Lin-Tai Da¹, Wenjie Peng¹

Affiliations

¹ Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University, Shanghai 200240, China.
² State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.
³ Shanghai Key Laboratory of Chemical Assessment and Sustainability, School of Chemical Science & Engineering, Tongji University, Shanghai 200092, China.

Abstract

The soluble N-glycosyltransferase from Actinobacillus pleuropneumoniae (ApNGT) can establish an N-glycosidic bond at the asparagine residue in the Asn-Xaa-Ser/Thr consensus sequon and is one of the most promising tools for N-glycoprotein production. Here, by integrating computational and experimental strategies, we revealed the molecular mechanism of the substrate recognition and following catalysis of ApNGT. These findings allowed us to pinpoint a key structural motif (²¹⁵DVYM²¹⁸) in ApNGT responsible for the peptide substrate recognition. Moreover, Y222 and H371 of ApNGT were found to participate in activating the acceptor Asn. The constructed models were supported by further crystallographic studies and the functional roles of the identified residues were validated by measuring the glycosylation activity of various mutants against a library of synthetic peptides. Intriguingly, with particular mutants, site-selective N-glycosylation of canonical or noncanonical sequons within natural polypeptides from the SARS-CoV-2 spike protein could be achieved, which were used to investigate the biological roles of the N-glycosylation in membrane fusion during virus entry. Our study thus provides in-depth molecular mechanisms underlying the substrate recognition and catalysis for ApNGT, leading to the synthesis of previously unknown chemically defined N-glycoproteins for exploring the biological importance of the N-glycosylation at a specific site.