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Xi Chen - One of the best experts on this subject based on the ideXlab platform.

  • interaction of neisseria meningitidis group x n acetylglucosamine 1 phosphotransferase with its Donor Substrate
    Glycobiology, 2018
    Co-Authors: Shonoi Ming, Yi Chen, Xi Chen, Ebony Cottmanthomas, Natalee C Black, Vamsee Veeramachineni, Dwight Peterson, Lauren M Tedaldi, Gerd K Wagner, Chao Cai
    Abstract:

    Neisseria meningitidis Group X is an emerging cause of bacterial meningitis in Sub-Saharan Africa. The capsular polysaccharide of Group X is a homopolymer of N-acetylglucosamine α(1-4) phosphate and is a vaccine target for prevention of disease associated with this meningococcal serogroup. We have demonstrated previously that the formation of the polymer is catalyzed by a phosphotransferase which transfers N-acetylglucosamine-1-phosphate from UDP-N-acetylglucosamine to the 4-hydroxyl of the N-acetylglucosamine on the nonreducing end of the growing chain. In this study, we use Substrate analogs of UDP-GlcNAc to define the enzyme/Donor Substrate interactions critical for catalysis. Our kinetic analysis of the phosphotransferase reaction is consistent with a sequential mechanism of Substrate addition and product release. The use of novel uracil modified analogs designed by Wagner et al. enabled us to assess whether the CsxA-catalyzed reaction is consistent with a Donor dependent conformational change. As expected with this model for glycosyltransferases, UDP-GlcNAc analogs with bulky uracil modifications are not Substrates but are inhibitors. An analog with a smaller iodo uracil substitution is a Substrate and a less potent inhibitor. Moreover, our survey of analogs with modifications on the N-acetylglucosamine residue of the sugar nucleotide Donor highlights the importance of substituents at C2 and C4 of the sugar residue. The hydroxyl group at C4 and the structure of the acyl group at C2 are very important for specificity and Substrate interactions during the polymerization reaction. While most analogs modified at C2 were inhibitors, acetamido analogs were also Substrates suggesting the importance of the carbonyl group.

  • Donor Substrate promiscuity of bacterial β1 3 n acetylglucosaminyltransferases and acceptor Substrate flexibility of β1 4 galactosyltransferases
    Bioorganic & Medicinal Chemistry, 2016
    Co-Authors: Mengyang Xue, Xue Sheng, Jie Zeng, Vireak Thon, Yi Chen, Musleh M Muthana, Peng George Wang, Xi Chen
    Abstract:

    β1-3-N-Acetylglucosaminyltransferases (β3GlcNAcTs) and β1-4-galactosyltransferases (β4GalTs) have been broadly used in enzymatic synthesis of N-acetyllactosamine (LacNAc)-containing oligosaccharides and glycoconjugates including poly-LacNAc, and lacto-N-neotetraose (LNnT) found in the milk of human and other mammals. In order to explore oligosaccharides and derivatives that can be synthesized by the combination of β3GlcNAcTs and β4GalTs, Donor Substrate specificity studies of two bacterial β3GlcNAcTs from Helicobacter pylori (Hpβ3GlcNAcT) and Neisseria meningitidis (NmLgtA), respectively, using a library of 39 sugar nucleotides were carried out. The two β3GlcNAcTs have complementary Donor Substrate promiscuity and 13 different trisaccharides were produced. They were used to investigate the acceptor Substrate specificities of three β4GalTs from Neisseria meningitidis (NmLgtB), Helicobacter pylori (Hpβ4GalT), and bovine (Bβ4GalT), respectively. Ten of the 13 trisaccharides were shown to be tolerable acceptors for at least one of these β4GalTs. The application of NmLgtA in one-pot multienzyme (OPME) synthesis of two trisaccharides including GalNAcβ1-3Galβ1-4GlcβProN3 and Galβ1-3Galβ1-4Glc was demonstrated. The study provides important information for using these glycosyltransferases as powerful catalysts in enzymatic and chemoenzymatic syntheses of oligosaccharides and derivatives which can be useful probes and reagents.

  • Donor Substrate promiscuity of the n acetylglucosaminyltransferase activities of pasteurella multocida heparosan synthase 2 pmhs2 and escherichia coli k5 kfia
    Applied Microbiology and Biotechnology, 2014
    Co-Authors: Yanhong Li, Vireak Thon, Yi Chen, Musleh M Muthana, Hai Yu, Jingyao Qu, Xi Chen
    Abstract:

    The biological activities of heparan sulfate (HS) and heparin (HP) are closely related to their molecular structures. Both Pasteurella multocida heparosan synthase 2 (PmHS2) and Escherichia coli K5 KfiA have been used for enzymatic and chemoenzymatic synthesis of HS and HP oligosaccharides and their derivatives. We show here that cloning using the pET15b vector and expressing PmHS2 as an N-His6-tagged fusion protein improve its expression level in E. coli. Investigation of the Donor Substrate specificity of the N-acetylglucosaminyltransferase activities of P. multocida heparosan synthase 2 (PmHS2) and E. coli K5 KfiA indicates the Substrate promiscuities of PmHS2 and KfiA. Overall, both PmHS2 and KfiA can use uridine 5'-diphosphate-N-acetylglucosamine (UDP-GlcNAc) and some of its C2'- and C6'-derivatives as Donor Substrates for their α1–4-GlcNAcT activities. Nevertheless, PmHS2 has a broader tolerance towards Substrate modifications. Other than the UDP-sugars that can be used by KfiA, additional C6'-derivatives of UDP-GlcNAc, UDP-glucose, and UDP-N-acetylgalactosamine (UDP-GalNAc) are tolerable Substrates for the α1–4-GlcNAcT activity of PmHS2. The Substrate promiscuities of PmHS2 and KfiA will allow efficient chemoenzymatic synthesis of diverse HS and HP oligosaccharide derivatives which may have improved or altered activities compared to their natural counterparts.

Juan M. Tomás - One of the best experts on this subject based on the ideXlab platform.

  • a udp hexnac polyprenol p galnac 1 p transferase wecp representing a new subgroup of the enzyme family
    Journal of Bacteriology, 2011
    Co-Authors: Susana Merino, Raquel Molero, Lamiaa Bouamama, Miguel Regue, Natalia Jimenez, Juan M. Tomás
    Abstract:

    The Aeromonas hydrophila AH-3 WecP represents a new class of UDP-HexNAc:polyprenol-P HexNAc-1-P transferases. These enzymes use a membrane-associated polyprenol phosphate acceptor (undecaprenyl phosphate [Und-P]) and a cytoplasmic UDP-d-N-acetylhexosamine sugar nucleotide as the Donor Substrate. Until now, all the WecA enzymes tested were able to transfer UDP-GlcNAc to the Und-P. In this study, we present in vitro and in vivo proofs that A. hydrophila AH-3 WecP transfers GalNAc to Und-P and is unable to transfer GlcNAc to the same enzyme Substrate. The molecular topology of WecP is more similar to that of WbaP (UDP-Gal polyprenol-P transferase) than to that of WecA (UDP-GlcNAc polyprenol-P transferase). WecP is the first UDP-HexNAc:polyprenol-P GalNAc-1-P transferase described.

Philippe Delaporte - One of the best experts on this subject based on the ideXlab platform.

  • laser printing of a semiconducting oligomer as active layer in organic thin film transistors impact of a protecting triazene layer
    Thin Solid Films, 2012
    Co-Authors: Ludovic Rapp, Anne Patricia Alloncle, Abdou Karim Diallo, Christine Videlotackermann, Frederic Fages, Sébastien Nénon, Matthias Nagel, Thomas Lippert, Philippe Delaporte
    Abstract:

    Abstract Organic thin-film transistor (OTFT) devices were achieved using the laser-induced forward transfer technique. As p-type organic semiconductor, distyryl-quaterthiophene (DS4T) was vacuum-deposited on a Donor Substrate and transferred with picosecond laser pulses on Si/SiO2-based receiver Substrates to form an organic active layer. To avoid laser damage of the organic thin film, a UV-sensitive aryltriazene polymer as a sacrificial layer was used. The polymer layer, deposited on the Donor Substrate prior to the organic layer deposition, has high absorption at the laser wavelength and does not contaminate the printed pixels. The DS4T pixels printed on receiver Substrates have well defined morphological properties as shown by atomic force microscopy and scanning electronic microscopy. OTFT devices were characterized in top- and bottom-contact configurations using thermally evaporated gold lines as source-drain electrodes. DS4T pixels printed as active layer for charge transport not only issue mobility values comparable to DS4T layers prepared by vacuum evaporation but also a relative electrical stability over time.

  • pulsed laser printing process for organic thin film transistors fabrication
    INTERNATIONAL SYMPOSIUM ON HIGH POWER LASER ABLATION 2010, 2010
    Co-Authors: Ludovic Rapp, Anne Patricia Alloncle, Abdou Karim Diallo, Christine Videlotackermann, Frederic Fages, Sébastien Nénon, Philippe Delaporte
    Abstract:

    This paper presents a Pulsed‐Laser Printing process applied to metals, liquid, polymers and oligomers with the goal to fabricate Organic Thin‐Film Transistors. The Laser‐Induced Forward Transfer (LIFT) technique has been used as a spatially‐resolved laser deposition method. All materials have been transferred from a Donor Substrate onto a receiver Substrate upon laser pulses in the picosecond regime. The broad nature of transferred patterns and the efficiency of the LIFT confirm the important potential of a laser printing technique in the development of the plastic microelectronics.

  • pulsed laser printing of organic thin film transistors
    Applied Physics Letters, 2009
    Co-Authors: Ludovic Rapp, Anne Patricia Alloncle, Abdou Karim Diallo, Christine Videlotackermann, Frederic Fages, Philippe Delaporte
    Abstract:

    Organic thin-film transistors have been fabricated using laser-induced forward transfer as spatially resolved laser-printing method. Using this technique, source and drain electrodes were deposited from silver nanoparticle ink and the copper phthalocyanine (CuPc) was used to form the active layer. Both kinds of materials were transferred from a Donor Substrate onto a receiver Substrate upon irradiation with laser pulses in the picosecond regime. The latter Substrate formed the gate and the dielectric of the transistor. Electrical characterizations showed that the transistors are fully operative, showing well-defined linear and saturation regimes in the I-V curves.

Bernd Nidetzky - One of the best experts on this subject based on the ideXlab platform.

  • glycosynthase reaction meets the flow continuous synthesis of lacto n triose ii by engineered β hexosaminidase immobilized on solid support
    Biotechnology and Bioengineering, 2020
    Co-Authors: Lucija Ruzic, Juan M Bolivar, Bernd Nidetzky
    Abstract:

    The D746E variant of Bifidobacterium bifidum β-N-acetyl-hexosaminidase is a promising glycosynthase (engineered glycosidase deficient in hydrolase activity) for the synthesis of lacto-N-triose II (LNT II), a core structural unit of human milk oligosaccharides. Here, we develop a flow process for the glycosynthase reaction, which is the regioselective β-1,3-glycosylation of lactose from a d-glucosamine 1,2-oxazoline Donor. Using the glycosynthase immobilized on agarose beads (∼30 mg/g) packed into a fixed bed (1 ml), we show stable continuous production of LNT II (145-200 mM) at quantitative yield from the Donor Substrate. The wild-type β-N-acetyl-hexosaminidase used under exactly comparable conditions gives primarily (∼85%) the hydrolysis product d-glucosamine. By enabling short residence times (2 min) that are challenging for mixed-vessel types of reactor to establish, the glycosynthase flow reactor succeeds in an effective uncoupling of the LNT II formation (∼80-100 mM/min) from the slower side reactions (decomposition of Donor Substrate, enzymatic hydrolysis of LNT II) to obtain optimum synthetic efficiency. Our study thus provides a strong case for the application of flow chemistry principles to glycosynthase reactions and by that, it reveals the important synergy between enzyme and reaction engineering for biocatalytic synthesis of oligosaccharides.

  • lacto n tetraose synthesis by wild type and glycosynthase variants of the β n hexosaminidase from bifidobacterium bifidum
    Organic and Biomolecular Chemistry, 2019
    Co-Authors: Katharina Schmolzer, Melanie Weingarten, Kai Baldenius, Bernd Nidetzky
    Abstract:

    Lacto-N-biose 1,2-oxazoline was prepared chemo-enzymatically and shown to be a Donor Substrate for β-1,3-glycosylation of lactose by the wild-type and glycosynthase variants (D320E, D320A, Y419F) of Bifidobacterium bifidum β-N-hexosaminidase. Lacto-N-tetraose, a core structure of human milk oligosaccharides, was formed in 20-60% yield of Donor Substrate (up to 8 mM product titre), depending on the degree of selectivity control by the enzyme used.

  • β‑Glucosyl Fluoride as Reverse Reaction Donor Substrate and Mechanistic Probe of Inverting Sugar Nucleotide-Dependent Glycosyltransferases
    2018
    Co-Authors: Alexander Lepak, Alexander Gutmann, Bernd Nidetzky
    Abstract:

    For a set of flavonoid O- and C-β-glycosyltransferases, we show that β-glucosyl fluoride can function as Substrate for an enzymatic reaction wherein uridine 5′-diphosphate (UDP) α-glucose is synthesized in the presence of UDP. In pH and mutagenesis studies of the C-glycosyltransferase from rice, we show that reaction with the β-glucosyl fluoride can serve to identify the acid–base catalytic residue of the enzyme (His24). We also show that β-glucosyl fluoride can rescue activity in an enzyme variant (Ile121Asp) strongly impaired in the canonical reaction wherein flavonoid acceptor is glucosylated from UDP-glucose. Coupling of this variant with the wildtype C-glycosyltransferase in a one-pot reaction enabled efficient 3′-β-C-glucosylation of phloretin from β-glucosyl fluoride in the presence of substochiometric amounts of UDP

  • examining the role of phosphate in glycosyl transfer reactions of cellulomonas uda cellobiose phosphorylase using d glucal as Donor Substrate
    Carbohydrate Research, 2012
    Co-Authors: Patricia Wildberger, Lothar Brecker, Bernd Nidetzky
    Abstract:

    Abstract Cellobiose phosphorylase from Cellulomonas uda ( Cu CPase) is shown to utilize d -glucal as slow alternative Donor Substrate for stereospecific glycosyl transfer to inorganic phosphate, giving 2-deoxy-α- d -glucose 1-phosphate as the product. When performed in D 2 O, enzymatic phosphorolysis of d -glucal proceeds with incorporation of deuterium in equatorial position at C-2, implying a stereochemical course of reaction where Substrate becomes protonated from below its six-membered ring through stereoselective re side attack at C-2. The proposed catalytic mechanism, which is supported by results of docking studies, involves direct protonation of d -glucal by the enzyme-bound phosphate, which then performs nucleophilic attack on the reactive C-1 of Donor Substrate. When offered d -glucose next to d -glucal and phosphate, Cu CPase produces 2-deoxy-β- d -glucosyl-(1→4)- d -glucose and 2-deoxy-α- d -glucose 1-phosphate in a ratio governed by mass action of the two acceptor Substrates present. Enzymatic synthesis of 2-deoxy-β- d -glucosyl-(1→4)- d -glucose is effectively promoted by catalytic concentrations of phosphate, suggesting that catalytic reaction proceeds through a quaternary complex of Cu CPase, d -glucal, phosphate, and d -glucose. Conversion of d -glucal and phosphate presents a convenient single-step synthesis of 2-deoxy-α- d -glucose 1-phosphate that is difficult to prepare chemically.

Alan John - One of the best experts on this subject based on the ideXlab platform.

  • structural basis of Substrate recognition and catalysis by fucosyltransferase 8
    Journal of Biological Chemistry, 2020
    Co-Authors: Michael Jarva, Marija Dramicanin, James P Lingford, Runyu Mao, Alan John
    Abstract:

    Fucosylation of the innermost GlcNAc of N-glycans by fucosyltransferase 8 (FUT8) is an important step in the maturation of complex and hybrid N-glycans. This simple modification can dramatically affect the activities and half-lives of glycoproteins, effects that are relevant to understanding the invasiveness of some cancers, development of mAb therapeutics, and the etiology of a congenital glycosylation disorder. The acceptor Substrate preferences of FUT8 are well-characterized and provide a framework for understanding N-glycan maturation in the Golgi; however, the structural basis of these Substrate preferences and the mechanism through which catalysis is achieved remain unknown. Here we describe several structures of mouse and human FUT8 in the apo state and in complex with GDP, a mimic of the Donor Substrate, and with a glycopeptide acceptor Substrate at 1.80-2.50 A resolution. These structures provide insights into a unique conformational change associated with Donor Substrate binding, common strategies employed by fucosyltransferases to coordinate GDP, features that define acceptor Substrate preferences, and a likely mechanism for enzyme catalysis. Together with molecular dynamics simulations, the structures also revealed how FUT8 dimerization plays an important role in defining the acceptor Substrate-binding site. Collectively, this information significantly builds on our understanding of the core fucosylation process.

  • structural basis of Substrate recognition and catalysis by fucosyltransferase 8
    bioRxiv, 2020
    Co-Authors: Michael Jarva, Marija Dramicanin, James P Lingford, Runyu Mao, Alan John
    Abstract:

    Fucosylation of the inner-most N-acetyl-glucosamine (GlcNAc) of N-glycans by fucosyltransferase 8 (FUT8) is an important step in the maturation of complex and hybrid N-glycans. This simple modification can have a dramatic impact on the activity and half-life of glycoproteins. These effects are relevant to understanding the invasiveness of some cancers, the development of monoclonal antibody therapeutics, and to a congenital disorder of glycosylation. The acceptor Substrate preferences of FUT8 are well characterised and provide a framework for understanding N-glycan maturation in the Golgi, however the structural basis for these Substrate preferences and the mechanism through which catalysis is achieved remains unknown. Here, we describe several structures of mouse and human FUT8 in the apo state and in complex with guanosine diphosphate (GDP), a mimic of the Donor Substrate, and a glycopeptide acceptor Substrate. These structures provide insights into: a unique conformational change associated with Donor Substrate binding; common strategies employed by fucosyltransferases to coordinate GDP; features that define acceptor Substrate preferences; and a likely mechanism for enzyme catalysis. Together with molecular dynamics simulations, the structures also reveal how FUT8 dimerisation plays an important role in defining the acceptor Substrate binding site. Collectively, this information significantly builds on our understanding of the core-fucosylation process.