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dc.contributor.authorHrmova, M.en
dc.contributor.authorImai, T.en
dc.contributor.authorRutten, S.en
dc.contributor.authorFairweather, J.en
dc.contributor.authorPelosi, L.en
dc.contributor.authorBulone, V.en
dc.contributor.authorDriguez, H.en
dc.contributor.authorFincher, G.en
dc.identifier.citationJournal of Biological Chemistry, 2002; 277(33):30102-30111en
dc.descriptionCopyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.en
dc.description.abstractBarley (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.en
dc.description.statementofresponsibilityMaria Hrmova, Tomoya Imai, Simon J. Rutten, Jon K. Fairweather, Ludovic Pelosi, Vincent Bulone, Hugues Driguez, and Geoffrey B. Fincheren
dc.publisherAmer Soc Biochemistry Molecular Biology Incen
dc.subjectHordeum; 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; Catalysisen
dc.titleMutated Barley (1,3)-beta-D-Glucan Endohydrolases Synthesize Crystalline (1,3)-beta-D-Glucansen
dc.typeJournal articleen
pubs.library.collectionAgriculture, Food and Wine publicationsen
dc.identifier.orcidHrmova, M. [0000-0002-3545-0605]en
dc.identifier.orcidBulone, V. [0000-0002-9742-4701]en
Appears in Collections:Agriculture, Food and Wine publications

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