MoChia1 is a family 18 glycoside hydrolase from the rice blast fungus, Magnaporthe oryzae, that releases reducing-end GlcNAc from chitin oligosaccharides

Abstract

MoChia1, a family 18 glycoside hydrolase from the rice blast fungus Magnaporthe oryzae, was identified as an enzyme that binds chitin oligosaccharide elicitor to suppress the chitin-induced rice immune response. It hydrolyzed chitin oligosaccharides with a degree of polymerization of at least 3 [(GlcNAc)n (n[≥]3)] in an exo-splitting manner with anomeric retention. HPLC analysis of (GlcNAc)n cleavage by MoChia1 revealed that it specifically recognizes the {beta}-anomer of the substrate at the + 1 subsite and cleaves the terminal glycosidic linkage at the reducing end. It also hydrolyzed the partially deacetylated chitotrisaccharide GlcN-(GlcNAc)2 to GlcN-GlcNAc and GlcNAc, but not (GlcN)2-GlcNAc, indicating the requirement of the N-acetyl group at the -1 subsite for the hydrolytic reaction. MoChia1 released p-nitrophenol from pNP-(GlcNAc)2, p-nitrophenyl di-N-acetyl-{beta}-chitotrioside. Furthermore, it hydrolyzed the chitotriose derivatives with a modified GlcNAc residue on the reducing end, (GlcNAc)2-G, 4-O-{beta}-di-N-acetylchitobiosyl-2-acetamido-2-deoxy-2,3-anhydro-glucopyranose and (GlcNAc)2-L, 4-O-{beta}-di-N-acetylchitobiosyl-2-acetamido-2-deoxy-2,3-anhydro-glucono-{delta}-lactone, to (GlcNAc)2 and the modified GlcNAc, respectively. However, it did not hydrolyze 4-O-{beta}-di-N-acetylchitobiosyl moranoline [(GlcNAc)2-M], reduced chitotriose (chitotriitol), and -(GlcNAc)2 fluoride [(-(GlcNAc)2-F]. MoChia1 did not bind to chitin and barely hydrolyzed the polymeric substrate, glycol chitin. Taken together, we concluded that MoChia1 is a GH18 reducing end GlcNAc-releasing exo-oligochitinase with the {beta}-anomer selectivity. Allosamidin, a potent inhibitor of GH18 chitinases, was found to bind to MoChia1 and inhibit its hydrolytic activity with an IC50 of 54.4 {+/-} 6.91 M, indicating that allosamidin may be a potential candidate for a pesticide to prevent rice blast infection by inhibiting the chitinase activity of MoChia1.