Chimeric PQQ Glucose Dehydrogenase 159 Applied Biochemistry and Biotechnology Vol. 77–79, 1999 Copyright © 1999 by Humana Press Inc. All rights of any nature whatsoever reserved. 0273-2289/99/77–79/0159/$12.50 159 *Author to whom all correspondence and reprint requests should be addressed. Site-Directed Mutagenesis Study on the Thermal Stability of a Chimeric PQQ Glucose Dehydrogenase and Its Structural Interpretation ARIEF BUDI WITARTO, TAKAFUMI OHTERA, AND KOJI SODE* Department of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16 Naka-machi, Koganei, Tokyo 184-8588, Japan, E-mail: sode@cc.tuat.ac.jp 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, Ser771, of a chimeric PQQGDHs on their thermal stability. A series of Ser771 substitutions of a chimeric PQQGDH, E99A1, confirmed that hydrophobic interaction governs the thermal stability of the chimeric enzymes. Compari- son of the thermal denaturation of E. coli PQQGDH and E97A3 followed by far-ultraviolet (UV) circular dichroism (CD) spectroscopy revealed that E97A3 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-propel- ler proteins. Index Entries: PQQ glucose dehydrogenase; β-propeller protein; site- directed mutagenesis; CD spectroscopy; denaturation pathway. Introduction Many Gram-negative bacteria possess membrane-bound pyrrolo- quinoline quinone (PQQ) glucose dehydrogenase (GDH), which catalyze the oxidation of glucose into glucono-δ-lactone in the periplasm (1). This enzyme is monomer with molecular weight of approx 87 kDa. Amino-acid