Glycobiology Advance Access originally published online on July 28, 2006
Glycobiology 2006 16(11):1064-1072; doi:10.1093/glycob/cwl026
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Exo-ß-D-glucosaminidase from Amycolatopsis orientalis: catalytic residues, sugar recognition specificity, kinetics, and synergism
3 Department of Advanced Bioscience, Kinki University, 3327-204 Nakamachi, Nara 631-8505, Japan; 4 Centre d’Étude et de Valorisation de la Diversité Microbienne, Département de Biologie, Faculté des Sciences, Université de Sherbrooke, Sherbrooke (Québec) J1K 2R1, Canada; and 5 Department of Applied Biological Sciences, Saga University, Saga 840-8502, Japan
1 To whom correspondence should be addressed; e-mail: fukamizo{at}nara.kindai.ac.jp
2 Present address: ISM Biopolymer Inc., Granby, QC, Canada
Received on May 24, 2006; revised on July 20, 2006; accepted on July 21, 2006
Catalytic residues and the mode of action of the exo-ß-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.
Key words: catalytic residue / exo-ß-d-glucosaminidase / specificity / subsites / synergism