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Henrik Vibe Scheller - One of the best experts on this subject based on the ideXlab platform.
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microscale thermophoresis as a powerful tool for screening Glycosyltransferases involved in cell wall biosynthesis
Plant Methods, 2020Co-Authors: Wanchen Shao, Rita Sharma, Mads Hartvig Clausen, Henrik Vibe SchellerAbstract:Identification and characterization of key enzymes associated with cell wall biosynthesis and modification is fundamental to gain insights into cell wall dynamics. However, it is a challenge that activity assays of Glycosyltransferases are very low throughput and acceptor substrates are generally not available. We optimized and validated microscale thermophoresis (MST) to achieve high throughput screening for Glycosyltransferase substrates. MST is a powerful method for the quantitative analysis of protein–ligand interactions with low sample consumption. The technique is based on the motion of molecules along local temperature gradients, measured by fluorescence changes. We expressed Glycosyltransferases as YFP-fusion proteins in tobacco and optimized the MST method to allow the determination of substrate binding affinity without purification of the target protein from the cell lysate. The application of this MST method to the β-1,4-galactosyltransferase AtGALS1 validated the capability to screen both nucleotide-sugar donor substrates and acceptor substrates. We also expanded the application to members of Glycosyltransferase family GT61 in sorghum for substrate screening and function prediction. This method is rapid and sensitive to allow determination of both donor and acceptor substrates of Glycosyltransferases. MST enables high throughput screening of Glycosyltransferases for likely substrates, which will narrow down their in vivo function and help to select candidates for further studies. Additionally, this method gives insight into biochemical mechanism of Glycosyltransferase function.
Tatsuo Yanagisawa - One of the best experts on this subject based on the ideXlab platform.
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Structural basis of protein arginine rhamnosylation by Glycosyltransferase EarP
Nature Chemical Biology, 2018Co-Authors: Toru Sengoku, Takehiro Suzuki, Naoshi Dohmae, Chiduru Watanabe, Teruki Honma, Yasushi Hikida, Yoshiki Yamaguchi, Hideyuki Takahashi, Shigeyuki Yokoyama, Tatsuo YanagisawaAbstract:The crystal structure of EarP, an inverting Glycosyltransferase that generates rhamnosyl-arginine modifications, suggests that the enzyme uses an S_N2 reaction mechanism that may involve perturbation of the donor sugar nucleotide conformation. Protein glycosylation regulates many cellular processes. Numerous Glycosyltransferases with broad substrate specificities have been structurally characterized. A novel inverting Glycosyltransferase, EarP, specifically transfers rhamnose from dTDP-β- l -rhamnose to Arg32 of bacterial translation elongation factor P (EF-P) to activate its function. Here we report a crystallographic study of Neisseria meningitidis EarP. The EarP structure contains two tandem Rossmann-fold domains, which classifies EarP in Glycosyltransferase superfamily B. In contrast to other structurally characterized protein Glycosyltransferases, EarP binds the entire β-sheet structure of EF-P domain I through numerous interactions that specifically recognize its conserved residues. Thus Arg32 is properly located at the active site, and causes structural change in a conserved dTDP-β- l -rhamnose-binding loop of EarP. Rhamnosylation by EarP should occur via an S_N2 reaction, with Asp20 as the general base. The Arg32 binding and accompanying structural change of EarP may induce a change in the rhamnose-ring conformation suitable for the reaction.
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structural basis of protein arginine rhamnosylation by Glycosyltransferase earp
Nature Chemical Biology, 2018Co-Authors: Toru Sengoku, Takehiro Suzuki, Naoshi Dohmae, Chiduru Watanabe, Teruki Honma, Yasushi Hikida, Yoshiki Yamaguchi, Hideyuki Takahashi, Shigeyuki Yokoyama, Tatsuo YanagisawaAbstract:Protein glycosylation regulates many cellular processes. Numerous Glycosyltransferases with broad substrate specificities have been structurally characterized. A novel inverting Glycosyltransferase, EarP, specifically transfers rhamnose from dTDP-β-L-rhamnose to Arg32 of bacterial translation elongation factor P (EF-P) to activate its function. Here we report a crystallographic study of Neisseria meningitidis EarP. The EarP structure contains two tandem Rossmann-fold domains, which classifies EarP in Glycosyltransferase superfamily B. In contrast to other structurally characterized protein Glycosyltransferases, EarP binds the entire β-sheet structure of EF-P domain I through numerous interactions that specifically recognize its conserved residues. Thus Arg32 is properly located at the active site, and causes structural change in a conserved dTDP-β-L-rhamnose-binding loop of EarP. Rhamnosylation by EarP should occur via an SN2 reaction, with Asp20 as the general base. The Arg32 binding and accompanying structural change of EarP may induce a change in the rhamnose-ring conformation suitable for the reaction.
Wanchen Shao - One of the best experts on this subject based on the ideXlab platform.
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microscale thermophoresis as a powerful tool for screening Glycosyltransferases involved in cell wall biosynthesis
Plant Methods, 2020Co-Authors: Wanchen Shao, Rita Sharma, Mads Hartvig Clausen, Henrik Vibe SchellerAbstract:Identification and characterization of key enzymes associated with cell wall biosynthesis and modification is fundamental to gain insights into cell wall dynamics. However, it is a challenge that activity assays of Glycosyltransferases are very low throughput and acceptor substrates are generally not available. We optimized and validated microscale thermophoresis (MST) to achieve high throughput screening for Glycosyltransferase substrates. MST is a powerful method for the quantitative analysis of protein–ligand interactions with low sample consumption. The technique is based on the motion of molecules along local temperature gradients, measured by fluorescence changes. We expressed Glycosyltransferases as YFP-fusion proteins in tobacco and optimized the MST method to allow the determination of substrate binding affinity without purification of the target protein from the cell lysate. The application of this MST method to the β-1,4-galactosyltransferase AtGALS1 validated the capability to screen both nucleotide-sugar donor substrates and acceptor substrates. We also expanded the application to members of Glycosyltransferase family GT61 in sorghum for substrate screening and function prediction. This method is rapid and sensitive to allow determination of both donor and acceptor substrates of Glycosyltransferases. MST enables high throughput screening of Glycosyltransferases for likely substrates, which will narrow down their in vivo function and help to select candidates for further studies. Additionally, this method gives insight into biochemical mechanism of Glycosyltransferase function.
Toru Sengoku - One of the best experts on this subject based on the ideXlab platform.
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Structural basis of protein arginine rhamnosylation by Glycosyltransferase EarP
Nature Chemical Biology, 2018Co-Authors: Toru Sengoku, Takehiro Suzuki, Naoshi Dohmae, Chiduru Watanabe, Teruki Honma, Yasushi Hikida, Yoshiki Yamaguchi, Hideyuki Takahashi, Shigeyuki Yokoyama, Tatsuo YanagisawaAbstract:The crystal structure of EarP, an inverting Glycosyltransferase that generates rhamnosyl-arginine modifications, suggests that the enzyme uses an S_N2 reaction mechanism that may involve perturbation of the donor sugar nucleotide conformation. Protein glycosylation regulates many cellular processes. Numerous Glycosyltransferases with broad substrate specificities have been structurally characterized. A novel inverting Glycosyltransferase, EarP, specifically transfers rhamnose from dTDP-β- l -rhamnose to Arg32 of bacterial translation elongation factor P (EF-P) to activate its function. Here we report a crystallographic study of Neisseria meningitidis EarP. The EarP structure contains two tandem Rossmann-fold domains, which classifies EarP in Glycosyltransferase superfamily B. In contrast to other structurally characterized protein Glycosyltransferases, EarP binds the entire β-sheet structure of EF-P domain I through numerous interactions that specifically recognize its conserved residues. Thus Arg32 is properly located at the active site, and causes structural change in a conserved dTDP-β- l -rhamnose-binding loop of EarP. Rhamnosylation by EarP should occur via an S_N2 reaction, with Asp20 as the general base. The Arg32 binding and accompanying structural change of EarP may induce a change in the rhamnose-ring conformation suitable for the reaction.
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structural basis of protein arginine rhamnosylation by Glycosyltransferase earp
Nature Chemical Biology, 2018Co-Authors: Toru Sengoku, Takehiro Suzuki, Naoshi Dohmae, Chiduru Watanabe, Teruki Honma, Yasushi Hikida, Yoshiki Yamaguchi, Hideyuki Takahashi, Shigeyuki Yokoyama, Tatsuo YanagisawaAbstract:Protein glycosylation regulates many cellular processes. Numerous Glycosyltransferases with broad substrate specificities have been structurally characterized. A novel inverting Glycosyltransferase, EarP, specifically transfers rhamnose from dTDP-β-L-rhamnose to Arg32 of bacterial translation elongation factor P (EF-P) to activate its function. Here we report a crystallographic study of Neisseria meningitidis EarP. The EarP structure contains two tandem Rossmann-fold domains, which classifies EarP in Glycosyltransferase superfamily B. In contrast to other structurally characterized protein Glycosyltransferases, EarP binds the entire β-sheet structure of EF-P domain I through numerous interactions that specifically recognize its conserved residues. Thus Arg32 is properly located at the active site, and causes structural change in a conserved dTDP-β-L-rhamnose-binding loop of EarP. Rhamnosylation by EarP should occur via an SN2 reaction, with Asp20 as the general base. The Arg32 binding and accompanying structural change of EarP may induce a change in the rhamnose-ring conformation suitable for the reaction.
Carolyn R Bertozzi - One of the best experts on this subject based on the ideXlab platform.
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Probing Glycosyltransferase activities with the Staudinger ligation.
Journal of the American Chemical Society, 2004Co-Authors: Howard C. Hang, Matthew R. Pratt, Carolyn R BertozziAbstract:The development of rapid screening methods for probing Glycosyltransferase activities is essential for advancing the field of glycobiology. While assays for specific Glycosyltransferases exist, there is no generalizable method that can be applied across the enzyme superfamily. Herein we describe a novel Glycosyltransferase assay that exploits their unnatural substrate tolerance and the unique chemical reactivity of the azide. We applied this “azido-ELISA” to the family of polypeptide α-N-acetylgalactosaminyltransferases (ppGalNAcTs), all of which were able to transfer N-azidoacetylgalactosamine (GalNAz) from the unnatural nucleotide sugar donor UDP-GalNAz. The azide was detected and quantified by Staudinger ligation with a phosphine probe in a microtiter plate format. This approach should be applicable to any Glycosyltransferase or group-transfer enzyme that tolerates unnatural azido substrates.
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regulating cell surface glycosylation by small molecule control of enzyme localization
Chemistry & Biology, 2003Co-Authors: Jennifer J Kohler, Carolyn R BertozziAbstract:Cell surface carbohydrates mediate interactions between the cell and its environment. Glycosyltransferases responsible for synthesis of cell surface oligosaccharides are therefore essential administrators of cellular communication. These enzymes often comprise large families. Redundacy of related family members and embryonic lethality both complicate genetic methods for deconvoluting functions of Glycosyltransferases. We report a chemical method in which the activity of an individual Glycosyltransferase is controlled by a small molecule. The approach exploits the requirement of Golgi localization, a common feature of Glycosyltransferase superfamily members. In our approach, the Glycosyltransferase is separated into two domains, one that determines localization and one responsible for catalysis. Control of enzyme activity is achieved using a small molecule to regulate association of the two domains. We used this method to regulate production of sialyl Lewis x by α1,3-fucosyltransferase VII in living cells.