title: Synthesis of cyclic beta-glucan using laminarinase 16A glycosynthase mutant from the basidiomycete Phanerochaete chrysosporium.
abstract: Glycosynthases are precise molecular instruments for making specifically linked oligosaccharides. X-ray crystallography screening of ligands bound to the 1,3(4)-beta-D-glucanase nucleophile mutant E115S of Phanerochaete chrysosporium Laminarinase 16A (Lam16A) showed that laminariheptaose (L7) bound in an arch with the reducing and nonreducing ends occupying either side of the catalytic cleft of the enzyme. The X-ray structure of Lam16A E115S in complex with alpha-laminariheptaosyl fluoride (alphaL7F) revealed how alphaL7F could make a nucleophilic attack upon itself. Indeed, when Lam16A E115S was allowed to react with alphaL7F the major product was a cyclic beta-1,3-heptaglucan, as shown by mass spectrometry. NMR confirmed uniquely beta-1,3-linkages and no reducing end. Molecular dynamics simulations indicate that the cyclic laminariheptaose molecule is not completely planar and that torsion angles at the glycosidic linkages fluctuate between two energy minima. This is the first report of a glycosynthase that joins the reducing and nonreducing ends of a single oligosaccharide and the first reported synthesis of cyclic beta-glucan.
authors.affiliation: Department of Molecular Biology, Swedish University of Agricultural Sciences, POB 590, SE-754 21 Uppsala, Sweden. jonas.vasur@gmail.com
authors.forename: Jonas
authors.initials: J
authors.lastname: Vasur
authors.forename: Rie
authors.initials: R
authors.lastname: Kawai
authors.forename: K Hanna M
authors.initials: KH
authors.lastname: Jonsson
authors.forename: Göran
authors.initials: G
authors.lastname: Widmalm
authors.forename: Ake
authors.initials: A
authors.lastname: Engström
authors.forename: Martin
authors.initials: M
authors.lastname: Frank
authors.forename: Evalena
authors.initials: E
authors.lastname: Andersson
authors.forename: Henrik
authors.initials: H
authors.lastname: Hansson
authors.forename: Zarah
authors.initials: Z
authors.lastname: Forsberg
authors.forename: Kiyohiko
authors.initials: K
authors.lastname: Igarashi
authors.forename: Masahiro
authors.initials: M
authors.lastname: Samejima
authors.forename: Mats
authors.initials: M
authors.lastname: Sandgren
authors.forename: Jerry
authors.initials: J
authors.lastname: Ståhlberg
mesh_terms: D020134:Catalytic Domain; D044602:Cellulases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray; D008024:Ligands; D056004:Molecular Dynamics Simulation; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020075:Phanerochaete / Q000201:enzymology*; D047071:beta-Glucans / Q000737:chemistry / Q000378:metabolism*
chemical_list: D008024:Ligands; D050505:Mutant Proteins; D047071:beta-Glucans; D044602:Cellulases