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Applied and Environmental Microbiology, June 2005, p. 3285-3293, Vol. 71, No. 6
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.6.3285-3293.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Cooperative Effect of Two Surface Amino Acid Mutations (Q252L and E170K) in Glucose Dehydrogenase from Bacillus megaterium IWG3 on Stabilization of Its Oligomeric State

Sang-Ho Baik,1,3* Fabrice Michel,2 Nushin Aghajari,2 Richard Haser,2 and Shigeaki Harayama1,3

Marine Biotechnology Institute, 3-75-1 Heita Kamaishi, Iwate 026-0001, Japan,1 Institut de Biologie et Chimie des Protéines, UMR5086-CNRS, Laboratoire de BioCristallographie, IFR128 BioSciences Lyon-Gerland, 7 Passage du Vercors, 69367 Lyon cedex 07, France,2 Biotechnology Development Center, National Institute of Technology and Evaluation, 2-5-8 Kazusa-Kamatari, Kisarazu-shi, Chiba 292-0818, Japan3

Received 11 August 2004/ Accepted 21 December 2004

A thermostable glucose dehydrogenase (GlcDH) mutant of Bacillus megaterium IWG3 harboring the Q252L substitution (Y. Makino, S. Negoro, I. Urabe, and H. Okada, J. Biol. Chem. 264:6381-6385, 1989) is stable at pH values above 9, but only in the presence of 2 M NaCl. Another GlcDH mutant exhibiting increased stability at an alkaline pH in the absence of NaCl has been isolated previously (S.-H. Baik, T. Ide, H. Yoshida, O. Kagami, and S. Harayama, Appl. Microbiol. Biotechnol. 61:329-335, 2003). This mutant had two amino acid substitutions, Q252L and E170K. In the present study, we characterized three GlcDH mutants harboring the substitutions Q252L, E170K, and Q252L/E170K under low-salt conditions. The GlcDH mutant harboring two substitutions, Q252L/E170K, was stable, but mutants harboring a single substitution, either Q252L or E170K, were unstable at an alkaline pH. Gel filtration chromatography analyses demonstrated that the oligomeric state of the Q252/E170K enzyme was stable, while the tetramers of the enzymes harboring a single substitution (Q252L or E170K) dissociated into dimers at an alkaline pH. These results indicated that the Q252L and E170K substitutions synergistically strengthened the interaction at the dimer-dimer interface. The crystal structure of the E170K/Q252L mutant, determined at 2.0-Å resolution, showed that residues 170 and 252 are located in a hydrophobic cavity at the subunit-subunit interface. We concluded that these residues in the wild-type enzyme have thermodynamically unfavorable effects, while the Q252L and E170K substitutions increase the subunit-subunit interactions by stabilizing the hydrophobic cavity.

* Corresponding author. Mailing address: Biotechnology Development Center, National Institute of Technology and Evaluation, 2-5-8 Kazusa-Kamatari, Kisarazu-shi, Chiba 292-0818, Japan. Phone: 81-438-20-5908. Fax: 81-438-20-5766. E-mail: baik-sangho{at}nite.go.jp.

Applied and Environmental Microbiology, June 2005, p. 3285-3293, Vol. 71, No. 6
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.6.3285-3293.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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