Structure and substrate specificity of a eukaryotic fucosidase from Fusarium graminearum.

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.