title: Mutated barley (1,3)-beta-D-glucan endohydrolases synthesize crystalline (1,3)-beta-D-glucans.
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.
authors.affiliation: Department of Plant Science, University of Adelaide, Waite Campus, Glen Osmond, South Australia 5064, Australia.
authors.forename: Maria
authors.initials: M
authors.lastname: Hrmova
authors.forename: Tomoya
authors.initials: T
authors.lastname: Imai
authors.forename: Simon J
authors.initials: SJ
authors.lastname: Rutten
authors.forename: Jon K
authors.initials: JK
authors.lastname: Fairweather
authors.forename: Ludovic
authors.initials: L
authors.lastname: Pelosi
authors.forename: Vincent
authors.initials: V
authors.lastname: Bulone
authors.forename: Hugues
authors.initials: H
authors.lastname: Driguez
authors.forename: Geoffrey B
authors.initials: GB
authors.lastname: Fincher
mesh_terms: D001483:Base Sequence; D002240:Carbohydrate Sequence; D002384:Catalysis; D003460:Crystallization; D017931:DNA Primers; D005936:Glucans / Q000096:biosynthesis* / Q000737:chemistry; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism*; D001467:Hordeum / Q000201:enzymology*; D009682:Magnetic Resonance Spectroscopy; D008854:Microscopy, Electron; D016297:Mutagenesis, Site-Directed; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D047071:beta-Glucans
chemical_list: D017931:DNA Primers; D005936:Glucans; D047071:beta-Glucans; C033363:beta-1,3-glucan; D006026:Glycoside Hydrolases; C028020:licheninase