EFFICIENT PRODUCTION OF URIDINE 5′-DIPHOSPHO-<i>N</i>-ACETYLGLUCOSAMINE BY THE COMBINATION OF THREE RECOMBINANT ENZYMES AND YEAST CELLS

Zhou, Jiewen; Fan, Limei; Wei, Peilian; Huang, Lei; Cai, Jin; Xu, Zhinan

doi:10.1080/10826068.2010.488518

Cited by 4 publications

(1 citation statement)

References 16 publications

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“…The enzymatic method for preparing UDP-GlcNAc described here offers marked benefits over previously described methods with respect to isotope labeling for NMR and MS-based studies. Other enzymatic methods for synthesizing UDP-GlcNAc that start from GlcNAc or GlcN have been described. − However, isotope-labeled GlcNAc and GlcN can be cost prohibitive and are available with only limited labeling patterns, unlike the scheme presented here that can be used to produce a wide array of custom labeling patterns starting with inexpensive starting materials, including glucose and glutamine. Numerous chemical methods are also available and can be adapted for the synthesis of GlcNAc analogues; however, these methods are less efficient than the one-pot enzymatic method presented here (not limited to refs − ).…”

Section: Resultsmentioning

confidence: 99%

Intramolecular N-Glycan/Polypeptide Interactions Observed at Multiple N-Glycan Remodeling Steps through [¹³C,¹⁵N]-N-Acetylglucosamine Labeling of Immunoglobulin G1

Barb

2014

Biochemistry

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Asparagine-linked (N) glycosylation is a common eukaryotic protein modification that affects protein folding, function, and stability through intramolecular interactions between N-glycan and polypeptide residues. Attempts to characterize the structure–activity relationship of each N-glycan are hindered by inherent properties of the glycoprotein, including glycan conformational and compositional heterogeneity. These limitations can be addressed by using a combination of nuclear magnetic resonance techniques following enzymatic glycan remodeling to simultaneously generate homogeneous glycoforms. However, widely applicable methods do not yet exist. To address this technological gap, immature glycoforms of the immunoglobulin G1 fragment crystallizable (Fc) were isolated in a homogeneous state and enzymatically remodeled with [13C,15N]-N-acetylglucosamine (GlcNAc). UDP-[13C,15N]GlcNAc was synthesized enzymatically in a one-pot reaction from [13C]glucose and [15N-amido]glutamine. Modifying Fc with recombinantly expressed glycosyltransferases (Gnt1 and Gnt2) and UDP-[13C,15N]GlcNAc resulted in complete glycoform conversion as judged by mass spectrometry. Two-dimensional heteronuclear single-quantum coherence spectra of the Gnt1 product, containing a single [13C,15N]GlcNAc residue on each N-glycan, showed that the N-glycan is stabilized through interactions with polypeptide residues. Similar spectra of homogeneous glycoforms, halted at different points along the N-glycan remodeling pathway, revealed the presence of an increased level of interaction between the N-glycan and polypeptide at each step, including mannose trimming, as the N-glycan was converted to a complex-type, biantennary form. Thus, conformational restriction increases as Fc N-glycan maturation proceeds. Gnt1 and Gnt2 catalyze fundamental reactions in the synthesis of every glycoprotein with a complex-type N-glycan; thus, the strategies presented herein can be applied to a broad range of glycoprotein studies.

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Section: Resultsmentioning

confidence: 99%

Intramolecular N-Glycan/Polypeptide Interactions Observed at Multiple N-Glycan Remodeling Steps through [¹³C,¹⁵N]-N-Acetylglucosamine Labeling of Immunoglobulin G1

Barb

2014

Biochemistry

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Reconstitution of the peptidoglycan cytoplasmic precursor biosynthetic pathway in cell-free system and rapid screening of antisense oligonucleotides for Mur enzymes

Sheng

Huang

Zhu

et al. 2014

Appl Microbiol Biotechnol

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Bacterial peptidoglycan is the cell wall component responsible for various biological activities. Its cytoplasmic precursor UDP-N-acetylmuramyl pentapeptide is biosynthesized by the first six enzymes of peptidoglycan synthetic pathways (Mur enzymes), which are all proved to be important targets for antibiotic screening. In our present work, the genes encoding Mur enzymes from Escherichia coli were co-expressed in the cell-free protein synthesis (CFPS) system, and the activities of Mur enzymes derived from CFPS system were validated by the synthesis of the final product UDP-N-acetylmuramyl pentapeptide. Then this in vitro reconstituted Mur biosynthetic pathway was used to screen a panel of specific antisense oligonucleotides for MurA and MurB. The selected oligonucleotides were proved to eliminate the expression of Mur enzymes, and thus inhibit the Mur biosynthetic pathway. The present work not only developed a rapid method to reconstruct and regulate a biosynthetic pathway in vitro, but also may provide insight into the development of novel antibiotics targeting on peptidoglycan biosynthetic pathway.

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Quantifying Carbohydrate Motions Through Solution Measurements: Applications to Immunoglobulin G Fc

Barb

2017

NMR in Glycoscience and Glycotechnology

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This chapter describes the preparation of homogenous N-glycoproteins using in vivo or in vitro manipulation with a special emphasis on incorporating stable isotope labels for inspection by solution NMR spectroscopy. This also includes an introduction to basic solution NMR techniques for characterizing glycoprotein motion. The power of these techniques is highlighted by an analysis of recent breakthroughs in the characterization of the structure/activity relationship of antibody N-glycosylation with a discussion of future opportunities in this area of structural biology.

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EFFICIENT PRODUCTION OF URIDINE 5′-DIPHOSPHO-N-ACETYLGLUCOSAMINE BY THE COMBINATION OF THREE RECOMBINANT ENZYMES AND YEAST CELLS

Cited by 4 publications

References 16 publications

Intramolecular N-Glycan/Polypeptide Interactions Observed at Multiple N-Glycan Remodeling Steps through [¹³C,¹⁵N]-N-Acetylglucosamine Labeling of Immunoglobulin G1

Intramolecular N-Glycan/Polypeptide Interactions Observed at Multiple N-Glycan Remodeling Steps through [¹³C,¹⁵N]-N-Acetylglucosamine Labeling of Immunoglobulin G1

Reconstitution of the peptidoglycan cytoplasmic precursor biosynthetic pathway in cell-free system and rapid screening of antisense oligonucleotides for Mur enzymes

Quantifying Carbohydrate Motions Through Solution Measurements: Applications to Immunoglobulin G Fc

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EFFICIENT PRODUCTION OF URIDINE 5′-DIPHOSPHO-N-ACETYLGLUCOSAMINE BY THE COMBINATION OF THREE RECOMBINANT ENZYMES AND YEAST CELLS

Cited by 4 publications

References 16 publications

Intramolecular N-Glycan/Polypeptide Interactions Observed at Multiple N-Glycan Remodeling Steps through [13C,15N]-N-Acetylglucosamine Labeling of Immunoglobulin G1

Intramolecular N-Glycan/Polypeptide Interactions Observed at Multiple N-Glycan Remodeling Steps through [13C,15N]-N-Acetylglucosamine Labeling of Immunoglobulin G1

Reconstitution of the peptidoglycan cytoplasmic precursor biosynthetic pathway in cell-free system and rapid screening of antisense oligonucleotides for Mur enzymes

Quantifying Carbohydrate Motions Through Solution Measurements: Applications to Immunoglobulin G Fc

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Product

Resources

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Intramolecular N-Glycan/Polypeptide Interactions Observed at Multiple N-Glycan Remodeling Steps through [¹³C,¹⁵N]-N-Acetylglucosamine Labeling of Immunoglobulin G1

Intramolecular N-Glycan/Polypeptide Interactions Observed at Multiple N-Glycan Remodeling Steps through [¹³C,¹⁵N]-N-Acetylglucosamine Labeling of Immunoglobulin G1