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|Title:||Mutated Barley (1,3)-beta-D-Glucan Endohydrolases Synthesize Crystalline (1,3)-beta-D-Glucans|
|Citation:||Journal of Biological Chemistry, 2002; 277(33):30102-30111|
|Publisher:||Amer Soc Biochemistry Molecular Biology Inc|
|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.|
|Appears in Collections:||Agriculture, Food and Wine publications|
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