Structure and substrate specificity of a eukaryotic fucosidase from Fusarium graminearum [Protein Structure and Folding]

August 1st, 2014 by Cao, H., Walton, J. D., Brumm, P., Phillips, G. N.

The secreted glycoside hydrolase family 29 (GH29) α-L-fucosidase from plant pathogenic fungus Fusarium graminearum (FgFCO1) actively releases fucose from xyloglucan fragment. We solved crystal structures of two active-site conformations, ie., open and closed, of apo FgFCO1 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 pNP-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 highest specificty with two natural substrates harboring Fucα1-2Gal glycosidic linkage, a xyloglucan-derived nonasaccharide and 2(prime)-fucosyllactose. Furthermore, FgFCO1 hydrolyzes H-disaccharide (lacking a +2 subsite sugar) at a rate 103-fold slower than 2(prime)-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.