title: Structure and substrate specificity of a eukaryotic fucosidase from Fusarium graminearum.
abstract: The secreted glycoside hydrolase family 29 (GH29) α-L-fucosidase from plant pathogenic fungus Fusarium graminearum (FgFCO1) actively releases fucose from the xyloglucan fragment. We solved crystal structures of two active-site conformations, i.e. open and closed, of apoFgFCO1 and an open complex with product fucose at atomic resolution. The closed conformation supports catalysis by orienting the conserved general acid/base Glu-288 nearest the predicted glycosidic position, whereas the open conformation possibly represents an unreactive state with Glu-288 positioned away from the catalytic center. A flexible loop near the substrate binding site containing a non-conserved GGSFT sequence is ordered in the closed but not the open form. We also identified a novel C-terminal βγ-crystallin domain in FgFCO1 devoid of calcium binding motif whose homologous sequences are present in various glycoside hydrolase families. N-Glycosylated FgFCO1 adopts a monomeric state as verified by solution small angle x-ray scattering in contrast to reported multimeric fucosidases. Steady-state kinetics shows that FgFCO1 prefers α1,2 over α1,3/4 linkages and displays minimal activity with p-nitrophenyl fucoside with an acidic pH optimum of 4.6. Despite a retaining GH29 family fold, the overall specificity of FgFCO1 most closely resembles inverting GH95 α-fucosidase, which displays the highest specificity with two natural substrates harboring the Fucα1-2Gal glycosidic linkage, a xyloglucan-derived nonasaccharide, and 2'-fucosyllactose. Furthermore, FgFCO1 hydrolyzes H-disaccharide (lacking a +2 subsite sugar) at a rate 10(3)-fold slower than 2'-fucosyllactose. We demonstrated the structurally dynamic active site of FgFCO1 with flexible general acid/base Glu, a common feature shared by several bacterial GH29 fucosidases to various extents.
authors.affiliation: From Rice University, Houston Texas 77005, Great Lakes Bioenergy Research Center, University of Wisconsin, Madison, Wisconsin 53706.
authors.forename: Hongnan
authors.initials: H
authors.lastname: Cao
authors.affiliation: Great Lakes Bioenergy Research Center, Michigan State University, East Lansing, Michigan 48824 and.
authors.forename: Jonathan D
authors.initials: JD
authors.lastname: Walton
authors.affiliation: C5-6 Technologies Corp., Middleton, Wisconsin 53562.
authors.forename: Phil
authors.initials: P
authors.lastname: Brumm
authors.affiliation: From Rice University, Houston Texas 77005, Great Lakes Bioenergy Research Center, University of Wisconsin, Madison, Wisconsin 53706, georgep@rice.edu.
authors.forename: George N
authors.initials: GN
authors.lastname: Phillips
mesh_terms: D000595:Amino Acid Sequence; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics; D018360:Crystallography, X-Ray; D005643:Fucose / Q000737:chemistry*; D005670:Fusarium / Q000737:chemistry*; D007700:Kinetics; D008968:Molecular Conformation; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D005644:alpha-L-Fucosidase / Q000737:chemistry* / Q000378:metabolism
chemical_list: D005643:Fucose; D005644:alpha-L-Fucosidase
keywords: Crystal Structure; Crystallin; Fucosidase; Glycoprotein; Molecular Dynamics; Substrate Specificity
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