Structure and Function of Exo-.BETA.-glucosaminidase from Amycolatopsis orientalis

• Fukamizo Tamo

  Department of Advanced Bioscience, Kinki University

• Brzezinski Ryszard

  Centre d’Étude et de Valorisation de la Diversité Microbienne, Département de Biologie, Faculté des Sciences,Université de Sherbrooke

Bibliographic Information

Other Title

• <i>Amycolatopsis orientalis</i>由来エキソ-β-グルコサミニダーゼの構造と機能
• Structure and function of exo-β-glucosamninidase from Amycolatopsis orientalis
• Structure and function of exo v glucosamninidase from Amycolatopsis orientalis

Abstract

We cloned and sequenced the gene encoding exo-β-glucosaminidase (GlcNase) from Amycolatopsis orientalis, and found that the gene has an open reading frame of 1032 residues with a calculated molecular mass of 110,557. The GlcNase has been classified as a member of family GH-2. Sequence alignments identified a group of GlcNase-related protein sequences forming a distinct subclass of family GH-2. When mono-N-acetylated chitotetraose [(GlcN)₃-GlcNAc] was hydrolyzed by the enzyme, the GlcN unit was produced from the nonreducing end together with the transglycosylation products. ¹H-NMR spectroscopy revealed that the enzyme is a retaining glycoside hydrolase. The rate of hydrolysis of the disaccharide, GlcN-GlcNAc, was somewhat lower than that of (GlcN)₂, suggesting that the N-acetyl group of the sugar residue located at (+1) site partly interferes with the catalytic reaction. Based on the time-course of the enzymatic hydrolysis of the completely deacetylated chitotetraose [(GlcN)₄], we obtained 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). Synergism resulting from mixing the A. orientalis GlcNase with Streptomyces sp. N174 endochitosanase was also observed when chitosan polysaccharide was used as the substrate. To identify the catalytic residue, 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 from sequence alignment of enzymes belonging to GH-2 family.

Journal

• Journal of Applied Glycoscience

  Journal of Applied Glycoscience 54 (2), 133-138, 2007

  The Japanese Society of Applied Glycoscience

Keywords

• <i>Amycolatopsis orientalis</i>
• exo-β-glucosaminidase
• chitosanase
• subsites
• chitooligosaccharides

Details

• CRID: 1390001206293730944
• NII Article ID: 10018617656
• NII Book ID: AN10453916
• DOI: 10.5458/jag.54.133
• ISSN: 18807291
• ISSN: 13447882
• NDL BIB ID: 8828061
• Web Site: https://ndlsearch.ndl.go.jp/books/R000000004-I8828061
• Web Site: http://www.jstage.jst.go.jp/article/jag/54/2/54_2_133/_pdf
• Text Lang: en
• Data Source:
  • JaLC
  • NDL
  • Crossref
  • CiNii Articles
  • KAKEN
• Abstract License Flag: Disallowed