Probing peptide substrate specificities of N-glycosyltranferase isoforms from different bacterial species

Meng, Qingyun; Li, Kun; Rong, Yongheng; Wu, Qizheng; Zhang, Xunlian; Kong, Yun; Chen, Min

doi:10.1016/j.carres.2018.12.016

Cited by 9 publications

(6 citation statements)

References 20 publications

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“…[38][39][40]44 However, no such acidic residue pair or metal ion can be found in the ApNGT active site, and ApNGT is known as a metalindependent glycosyltransferase. 21,45,46 Notably, in our constructed models, we observe that two conserved residues, Y222 and H371, are positioned to establish HBs with the acceptor Asn, implying their potential involvement in the catalytic reaction, e.g., activating the nucleophilic ability of the acceptor Asn. To further examine the functional roles of Y222 and H371, we substituted these two residues with Ala or Phe to abolish the HBs.…”

Section: Determining Functional Roles Of Y222 and H371mentioning

confidence: 78%

“…Our computational modeling does not support this tautomerization mechanism for NGT, as no such general base exists in the active site of ApNGT. Another proposed mechanism for yeast OST involves the carboxamide twisting to activate the amide nitrogen by D47 and E360 (corresponding to D56 and E319 in PglB, respectively), which are bridged by a metal ion. − , However, no such acidic residue pair or metal ion can be found in the ApNGT active site, and ApNGT is known as a metal-independent glycosyltransferase. ,, …”

Section: Resultsmentioning

confidence: 99%

“… 38 − 40 , 44 However, no such acidic residue pair or metal ion can be found in the ApNGT active site, and ApNGT is known as a metal-independent glycosyltransferase. 21 , 45 , 46 …”

Section: Resultsmentioning

confidence: 99%

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

Hao

Guo

Yan

et al. 2023

JACS Au

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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 ( 215 DVYM 218 ) 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 Nglycoproteins for exploring the biological importance of the N-glycosylation at a specific site.

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Section: Determining Functional Roles Of Y222 and H371mentioning

confidence: 78%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

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

Hao

Guo

Yan

et al. 2023

JACS Au

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“…138,193 122 The introduction of synthetic glycosylation pathways with NGTs as ppGTs results in the synthesis of diverse, minimal glycan motifs with applications in vaccines and therapeutics. characterization of the acceptor specificity of NGTs using glycoproteomics and in vitro as well as cell-free methods 49,80,168,171,176 has illuminated detailed rules for the prediction and design of sequons for various NGTs. So far, the NGT from Actinobacillus pleuropneumoniae (ApNGT) has been the most extensively characterized and most often used for glycoengineering efforts, 78 discussed below.…”

Section: ■ Motivation and Scopementioning

confidence: 99%

Synthetic Glycobiology: Parts, Systems, and Applications

et al. 2020

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Protein glycosylation, the attachment of sugars to amino acid side chains, can endow proteins with a wide variety of properties of great interest to the engineering biology community. However, natural glycosylation systems are limited in the diversity of glycoproteins they can synthesize, the scale at which they can be harnessed for biotechnology, and the homogeneity of glycoprotein structures they can produce. Here we provide an overview of the emerging field of synthetic glycobiology, the application of synthetic biology tools and design principles to better understand and engineer glycosylation. Specifically, we focus on how the biosynthetic and analytical tools of synthetic biology have been used to redesign glycosylation systems to obtain defined glycosylation structures on proteins for diverse applications in medicine, materials, and diagnostics. We review the key biological parts available to synthetic biologists interested in engineering glycoproteins to solve compelling problems in glycoscience, describe recent efforts to construct synthetic glycoprotein synthesis systems, and outline exemplary applications as well as new opportunities in this emerging space.

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“…9 In addition to the ENGase-catalyzed topdown strategy, a bottom-up approach has also been developed recently whereby a monosaccharide is directly transferred to the N-glycosylation site using N-glycosyltransferases (NGTs). [10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29] Particularly, the NGT from Actinobacillus pleuropneumoniae (ApNGT) can recognize certain UDP-or GDP-activated sugars (e.g., UDP-Glc, UDP-GlcN, UDP-Gal, UDP-Xyl, GDP-Glc, and GDP-Man) as sugardonor substrates (Fig. 1a and b).…”

Section: Introductionmentioning

confidence: 99%

A kinetic model reveals the critical gating motifs for donor-substrate loading into Actinobacillus pleuropneumoniae N-glycosyltransferase

Hao,

Guo,

Peng

et al. 2024

Phys. Chem. Chem. Phys.

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Probing peptide substrate specificities of N-glycosyltranferase isoforms from different bacterial species

Cited by 9 publications

References 20 publications

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

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

Synthetic Glycobiology: Parts, Systems, and Applications

A kinetic model reveals the critical gating motifs for donor-substrate loading into Actinobacillus pleuropneumoniae N-glycosyltransferase

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