MoChia1 is a GH18 Reducing-End GlcNAc–Releasing Chitin Oligosaccharide Hydrolase from the Rice Blast Fungus Magnaporthe oryzae

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 (n3) in an exo-splitting manner with anomeric retention.HPLC analysis of (GlcNAc)n cleavage by MoChia1 revealed that it specifically recognizes the -anomer of the substrate at the +1 subsite and cleaves the terminal glycosidic linkage at the reducing end.It also hydrolyzed the hetero-chitotriose GlcN-GlcNAc-GlcNAc, producing GlcN-GlcNAc and GlcNAc, but not GlcN-GlcN-GlcNAc, indicating the requirement of the N-acetyl group at the -1 subsite for the hydrolytic reaction.MoChia1 released p-nitrophenol from pNP-Furthermore, it hydrolyzed the chitotriose derivatives with a modified GlcNAc residue on the reducing end, (GlcNAc)2-G, 4-O--di-N-acetylchitobiosyl-2-acetamido-2-deoxy-2,3-anhydroglucopyranose and (GlcNAc)2-L, 4-O--di-N-acetylchitobiosyl-2-acetamido-2-deoxy-2,3-anhydro-glucono--lactone, to (GlcNAc)2 and the modified GlcNAc, respectively.However, it did not hydrolyze 4-O--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 chitin oligosaccharide hydrolase with the -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 J o u r n a l P r e -p r o o f activity of MoChia1.Abbreviations -(GlcNAc)2-F, -(GlcNAc)2 fluoride; BSA, bovine serum albumin; (GlcNAc)n, -1,4linked oligosaccharide of GlcNAc with a polymerization degree of n; GlcN, Dglucosamine; GlcNAc, N-acetyl-D-glucosamine; GH, glycoside hydrolase; (GlcNAc)2-G, 4-O--di-N-acetylchitobiosyl-2-acetamido-2-deoxy-2,3-anhydro-glucopyranose; (GlcNAc)2-L, 4-O--di-N-acetylchitobiosyl-2-acetamido-2-deoxy-2,3-anhydro-glucono-lactone; (GlcNAc)2-M,