Synthesis of cyclic beta-glucan using laminarinase 16A glycosynthase mutant from the basidiomycete Phanerochaete chrysosporium.
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.
Vasur
Jonas
J
Department of Molecular Biology, Swedish University of Agricultural Sciences, POB 590, SE-754 21 Uppsala, Sweden. jonas.vasur@gmail.com
Kawai
Rie
R
Jonsson
K Hanna M
KH
Widmalm
Göran
G
Engström
Ake
A
Frank
Martin
M
Andersson
Evalena
E
Hansson
Henrik
H
Forsberg
Zarah
Z
Igarashi
Kiyohiko
K
Samejima
Masahiro
M
Sandgren
Mats
M
Ståhlberg
Jerry
J
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*
D008024:Ligands; D050505:Mutant Proteins; D047071:beta-Glucans; D044602:Cellulases