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Applied Biochemistry and Biotechnology

, Volume 77, Issue 1–3, pp 159–168 | Cite as

Site-directed mutagenesis study on the thermal stability of a chemiric PQQ glucose dehydrogenase and its structural interpretation

  • Arief Budi Witarto
  • Takafumi Ohtera
  • Koji SodeEmail author
Article

Abstract

We have previously reported that a chimeric pyrroloquinoline quinone (PQQ) glucose dehydrogenase (GDH), E97A3, which was made up of 97% of Escherichia coli PQQGDH sequence and 3% of Acinetobacter calcoaceticus PQQGDH, showed increased thermal stability compared with both parental enzymes. Site-directed mutagenesis studies were carried out in order to investigate the role of amino-acid substitution at the C-terminal region, Ser 771, of a chimeric PQQGDHs on their thermal stability. A series of Ser 771 substitutions of a chimeric PQQGDH, E99A1, confirmed that hydrophobic interaction governs the thermal stability of the chimeric enzymes. Comparison of the thermal denaturation of E. coli PQQGDH and E97A3 followed by far-ultraviolet (UV) circular dichroism (CD) spectroscopy revealed that E97 A3 acquired stability at the first step of denaturation, which is reversible, and where no significant secondary structure change was observed. These results suggested that the interaction between C-terminal and N-terminal regions may play a crucial role in maintaining the overall structure of β-propeller proteins.

Index Entries

PQQ glucose dehydrogenase β-propeller protein site-directed mutagenesis CD spectroscopy denaturation pathway 

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Copyright information

© Humana Press Inc. 1999

Authors and Affiliations

  • Arief Budi Witarto
    • 1
  • Takafumi Ohtera
    • 1
  • Koji Sode
    • 1
    Email author
  1. 1.Department of BiotechnologyTokyo University of Agriculture and TechnologyTokyoJapan

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