Glycobiology Advance Access published online on July 28, 2006
Glycobiology, doi:10.1093/glycob/cwl026
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1 Department of Advanced Bioscience, Kinki University, 3327-204 Nakamachi, Nara, 631-8505 Japan
* To whom correspondence should be addressed. Catalytic residues and the mode of action of the exo-
Received May 24, 2006
Revised July 20, 2006
Accepted July 21, 2006
Article
Exo-
Tamo Fukamizo 1 *, Alain Fleury 2, Nathalie Côté 3, Masaru Mitsutomi 4, and Ryszard Brzezinski 2
-D-glucosaminidase from Amycolatopsis orientalis: catalytic residues, sugar recognition specificity, kinetics, and synergism
2 Centre d’Étude et de Valorisation de la Diversité Microbienne, Département de Biologie, Faculté des Sciences, Université de Sherbrooke, Sherbrooke (Québec) J1K 2R1 Canada
3 Centre d’Étude et de Valorisation de la Diversité Microbienne, Département de Biologie, Faculté des Sciences, Université de Sherbrooke, Sherbrooke (Québec) J1K 2R1 Canada; Present address: ISM Biopolymer Inc., Granby, Qc, Canada
4 Department of Applied Biological Sciences, Saga University, Saga 840-8502, Japan
Tamo Fukamizo, E-mail: fukamizo{at}nara.kindai.ac.jp
Abstract 
-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) which 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, 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 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)(+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.-glucosaminidase/catalytic residue/specificity/subsites/synergism.
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