title: Exo-beta-D-glucosaminidase from Amycolatopsis orientalis: catalytic residues, sugar recognition specificity, kinetics, and synergism.
abstract: Catalytic residues and the mode of action of the exo-beta-D-glucosaminidase (GlcNase) from Amycolatopsis orientalis were investigated using the wild-type and mutated enzymes. Mutations were introduced into the putative catalytic residues resulting in five mutated enzymes (D469A, D469E, E541D, E541Q, and S468N/D469E) that were successfully produced. The four single mutants were devoid of enzymatic activity, indicating that Asp469 and Glu541 are essential for catalysis as predicted by sequence alignments of enzymes belonging to GH-2 family. When mono-N-acetylated chitotetraose [(GlcN)3-GlcNAc] was hydrolyzed by the enzyme, the nonreducing-end glucosamine unit was produced together with the transglycosylation products. The rate of hydrolysis of the disaccharide, 2-amino-2-deoxy-D-glucopyranosyl 2-acetamido-2-deoxy-D-glucopyranose (GlcN-GlcNAc), was slightly lower than that of (GlcN)2, suggesting that N-acetyl group of the sugar residue located at (+1) site partly interferes with the catalytic reaction. The time-course of the enzymatic hydrolysis of the completely deacetylated chitotetraose [(GlcN)4] was quantitatively determined by high-performance liquid chromatography (HPLC) and used for in silico modeling of the enzymatic hydrolysis. The modeling study provided the values of binding free energy changes of +7.0, -2.9, -1.8, -0.9, -1.0, and -0.5 kcal/mol corresponding, respectively, to subsites (-2), (-1), (+1), (+2), (+3), and (+4). When chitosan polysaccharide was hydrolyzed by a binary enzyme system consisting of A. orientalis GlcNase and Streptomyces sp. N174 endochitosanase, the highest synergy in the rate of product formation was observed at the molar ratio 2:1. Thus, GlcNase would be an efficient tool for industrial production of glucosamine monosaccharide.
authors.affiliation: Department of Advanced Bioscience, Kinki University, 3327-204 Nakamichi, Nara 631-8505, Japan.
authors.forename: Tamo
authors.initials: T
authors.lastname: Fukamizo
authors.forename: Alain
authors.initials: A
authors.lastname: Fleury
authors.forename: Nathalie
authors.initials: N
authors.lastname: Côté
authors.forename: Masaru
authors.initials: M
authors.lastname: Mitsutomi
authors.forename: Ryszard
authors.initials: R
authors.lastname: Brzezinski
mesh_terms: D000191:Actinomycetaceae / Q000201:enzymology*; D020134:Catalytic Domain; D002688:Chitinases / Q000737:chemistry; D048271:Chitosan / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D004187:Disaccharides / Q000737:chemistry; D006031:Glycosylation; D006596:Hexosaminidases / Q000737:chemistry* / Q000235:genetics; D006868:Hydrolysis; D007700:Kinetics; D008956:Models, Chemical; D009154:Mutation; D009844:Oligosaccharides / Q000737:chemistry; D012440:Saccharomyces / Q000201:enzymology; D013379:Substrate Specificity
chemical_list: C517973:2-amino-2-deoxyglucopyranosyl 2-acetamido-2-deoxyglucopyranose; D004187:Disaccharides; D009844:Oligosaccharides; C012238:chitotetrose; D048271:Chitosan; D006596:Hexosaminidases; C064499:exo-beta-D-glucosaminidase; D002688:Chitinases