Identification of a Catalytic Residue of Clostridium paraputrificum N-Acetyl-.BETA.-D-glucosaminidase Nag3A by Site-Directed Mutagenesis

• LI Huazhong

  School of Medicine, Southern Yangtze University

• ZHAO Guangshan

  Faculty of Bioresources, Mie University

• MIYAKE Hideo

  Faculty of Bioresources, Mie University

• UMEKAWA Hayato

  Faculty of Bioresources, Mie University

• KIMURA Tetsuya

  Faculty of Bioresources, Mie University

• OHMIYA Kunio

  Faculty of Bioresources, Mie University

• SAKKA Kazuo

  Faculty of Bioresources, Mie University

Bibliographic Information

Other Title

• Identification of a Catalytic Residue of Clostridium paraputrificum N-Acetyl-β-D-glucosaminidase Nag3A by Site-Directed Mutagenesis
• Identification of a Catalytic Residue of Clostridium paraputrificum N Acetyl ベータ D glucosaminidase Nag3A by Site Directed Mutagenesis
• Identification of a Catalytic Residue of<i>Clostridium paraputrificum</i><i>N</i>-Acetyl-β-<scp>D</scp>-glucosaminidase Nag3A by Site-Directed Mutagenesis

Abstract

Clostridium paraputrificum M-21 β-N-acetylglucosaminidase 3A (Nag3A) is an enzyme classified in family 3 of the glycoside hydrolases. To identify catalytic residues of this enzyme, mutations were introduced into highly conserved Glu and Asp residues. Replacement of Asp175 with Ala abolished the catalytic activity without change in the circular dichroism spectrum, strongly suggesting that this residue is a catalytic residue, a nucleophile/base or a proton donor. Since the K_m values of mutant enzymes D119N, D229N, D229A and D274N increased 17 to 41 times as compared with that of wild-type enzyme, Asp119, Asp229, and Asp274 appear to be involved in substrate recognition and binding. Taking previous studies into consideration, we presume that Asp303 is the catalytic nucleophile and Asp175 is the proton donor of C. paraputrificum Nag3A.

Journal

• Bioscience, Biotechnology, and Biochemistry

  Bioscience, Biotechnology, and Biochemistry 70 (5), 1127-1133, 2006

  Japan Society for Bioscience, Biotechnology, and Agrochemistry

Keywords

• β-<I>N</I>-acetylglucosaminidase
• <I>Clostridium paraputrificum</I>
• catalytic residue
• site-directed mutagenesis

Details

• CRID: 1390282681449505024
• NII Article ID: 10018531103
• NII Book ID: AA10824164
• DOI: 10.1271/bbb.70.1127
• ISSN: 13476947
• ISSN: 09168451
• NDL BIB ID: 7926042
• Web Site: https://ndlsearch.ndl.go.jp/books/R000000004-I7926042
• Web Site: http://www.tandfonline.com/doi/pdf/10.1271/bbb.70.1127
• Text Lang: en
• Data Source:
  • JaLC
  • NDL
  • Crossref
  • CiNii Articles
• Abstract License Flag: Disallowed