title: Cooperative effect of two surface amino acid mutations (Q252L and E170K) in glucose dehydrogenase from Bacillus megaterium IWG3 on stabilization of its oligomeric state.
abstract: 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-angstroms 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.
authors.affiliation: Marine Biotechnology Institute, Heita Kamaishi, Iwate 026-0001, Japan. baik-sangho@nite.go.jp
authors.forename: Sang-Ho
authors.initials: SH
authors.lastname: Baik
authors.forename: Fabrice
authors.initials: F
authors.lastname: Michel
authors.forename: Nushin
authors.initials: N
authors.lastname: Aghajari
authors.forename: Richard
authors.initials: R
authors.lastname: Haser
authors.forename: Shigeaki
authors.initials: S
authors.lastname: Harayama
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001410:Bacillus megaterium / Q000201:enzymology* / Q000235:genetics; D003460:Crystallization; D018360:Crystallography, X-Ray; D019281:Dimerization; D004795:Enzyme Stability; D005948:Glucose Dehydrogenases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D016415:Sequence Alignment
chemical_list: D005948:Glucose Dehydrogenases