title: Characterization of the low-temperature activity of Sulfolobus tokodaii glucose-1-dehydrogenase mutants.
abstract: Thermophilic enzymes are potentially useful for industrial processes because they are generally more stable than are mesophilic or psychrophilic enzymes. However, a crucial drawback for their use in such processes is that most thermophilic enzymes are nearly inactive at moderate and low temperatures. We have previously proposed that modulation of the coenzyme-binding pocket of thermophilic dehydrogenases can produce mutated proteins with enhanced low-temperature activities. In the current study, we produced and characterized mutants of an NADP-dependent glucose-1-dehydrogenase from the hyperthermophile Sulfolobus tokodaii in which a predicted coenzyme-binding, non-polar residue was replaced by another non-polar residue. Detailed analyses of the kinetic properties of the wild-type enzyme and its mutants showed that one of the mutants (V254I) had improved kcat and kcat/Km values at both 25°C and 80°C. Temperature-induced unfolding experiments showed that the thermal stability of the mutant enzyme was comparable to that of the wild-type enzyme. Calculation of the energetic contribution of the V254I mutation for the dehydrogenase reaction revealed that the mutation destabilizes the enzyme-NADP(+)-glucose ternary complex and reduces the transition-state energy, thus enhancing catalysis.
authors.affiliation: Department of Applied Life Sciences, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.
authors.forename: Taisuke
authors.initials: T
authors.lastname: Sugii
authors.affiliation: Department of Applied Life Sciences, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan. Electronic address: akanuma@toyaku.ac.jp.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Akanuma
authors.affiliation: Department of Applied Life Sciences, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.
authors.forename: Sota
authors.initials: S
authors.lastname: Yagi
authors.affiliation: Department of Applied Life Sciences, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.
authors.forename: Kazuki
authors.initials: K
authors.lastname: Yagyu
authors.affiliation: Department of Applied Life Sciences, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Shimoda
authors.affiliation: Department of Applied Life Sciences, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.
authors.forename: Akihiko
authors.initials: A
authors.lastname: Yamagishi
mesh_terms: D019943:Amino Acid Substitution; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055162:Biocatalysis; D003080:Cold Temperature; D004795:Enzyme Stability; D005947:Glucose / Q000737:chemistry / Q000378:metabolism; D042843:Glucose 1-Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006358:Hot Temperature; D007532:Isoleucine / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D009154:Mutation; D009249:NADP / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D040681:Structural Homology, Protein; D016944:Sulfolobus / Q000737:chemistry* / Q000201:enzymology; D013816:Thermodynamics; D014633:Valine / Q000737:chemistry* / Q000378:metabolism
chemical_list: D019843:Archaeal Proteins; D011994:Recombinant Proteins; D007532:Isoleucine; D009249:NADP; D042843:Glucose 1-Dehydrogenase; D014633:Valine; D005947:Glucose
keywords: Circular dichroism; Enzyme activity; Glucose-1-dehydrogenase; Low-temperature activity; Site-directed mutagenesis; Steady-state kinetics; Thermal stability; Thermophilic enzyme