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

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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.

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Authors and Affiliations

  1. Department of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16 Naka-machi, Koganei, 184-8588, Tokyo, Japan

    Arief Budi Witarto, Takafumi Ohtera & Koji Sode

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  1. Arief Budi Witarto
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  2. Takafumi Ohtera
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  3. Koji Sode
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Correspondence to Koji Sode.

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Witarto, A.B., Ohtera, T. & Sode, K. Site-directed mutagenesis study on the thermal stability of a chemiric PQQ glucose dehydrogenase and its structural interpretation. Appl Biochem Biotechnol 77, 159–168 (1999). https://doi.org/10.1385/ABAB:77:1-3:159

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  • DOI: https://doi.org/10.1385/ABAB:77:1-3:159

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