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https://hdl.handle.net/2440/13421
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Type: | Journal article |
Title: | Mutated Barley (1,3)-beta-D-Glucan Endohydrolases Synthesize Crystalline (1,3)-beta-D-Glucans |
Author: | Hrmova, M. Imai, T. Rutten, S. Fairweather, J. Pelosi, L. Bulone, V. Driguez, H. Fincher, G. |
Citation: | Journal of Biological Chemistry, 2002; 277(33):30102-30111 |
Publisher: | Amer Soc Biochemistry Molecular Biology Inc |
Issue Date: | 2002 |
ISSN: | 0021-9258 1083-351X |
Statement of Responsibility: | Maria Hrmova, Tomoya Imai, Simon J. Rutten, Jon K. Fairweather, Ludovic Pelosi, Vincent Bulone, Hugues Driguez, and Geoffrey B. Fincher |
Abstract: | Barley (1,3)-beta-D-glucan endohydrolases (EC ), inactivated by site-directed mutagenesis of their catalytic nucleophiles, show autocondensation glucosynthetic activity with alpha-laminaribiosyl fluoride and heterocondensation glycosynthetic activity with alpha-laminaribiosyl fluoride and 4'-nitrophenyl beta-D-glucopyranoside. The native enzyme is a retaining endohydrolase of the family 17 group and catalyzes glycosyl transfer reactions at high substrate concentrations. Catalytic efficiencies (k(cat) K(m)(-1)) of mutants E231G, E231S, and E231A as glycosynthases are 28.9, 0.9, and 0.5 x 10(-4) m(-1) s(-1), respectively. Glycosynthase reactions appear to be processive and proceed with pH optima of 6-8 and yields of up to 75%. Insoluble products formed during the glycosynthase reaction appear as lamellar, hexagonal crystals when observed by electron microscopy. Methylation, NMR, and matrix-assisted laser desorption ionization time-of-flight analyses show that the reaction products are linear (1,3)-beta-D-glucans with a degree of polymerization of 30-34, whereas electron and x-ray diffraction patterns indicate that these (1,3)-beta-D-glucan chains adopt a parallel, triple helical conformation. The (1,3)-beta-D-glucan triple helices are orientated perpendicularly to the plane of the lamellar crystals. The barley (1,3)-beta-D-glucan glycosynthases have considerable potential for tailored and high efficiency synthesis of (1,3)-beta-D-linked oligo- and polysaccharides, some of which could have immunomodulating activity, or for the coupling of (1,3)-beta-D-linked glucosyl residues onto other oligosaccharides or glycoproteins. |
Keywords: | Hordeum Glucans Glycoside Hydrolases beta-Glucans DNA Primers Microscopy, Electron Crystallization Magnetic Resonance Spectroscopy Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization Mutagenesis, Site-Directed Base Sequence Carbohydrate Sequence Catalysis |
Description: | Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc. |
DOI: | 10.1074/jbc.M203971200 |
Published version: | http://dx.doi.org/10.1074/jbc.m203971200 |
Appears in Collections: | Agriculture, Food and Wine publications Aurora harvest 7 |
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