title: Effects of salt on the structure, stability, and function of a halophilic dihydrofolate reductase from a hyperhalophilic archaeon, Haloarcula japonica strain TR-1.
abstract: The effects of salt on the structure, stability, and enzymatic function of a novel dihydrofolate reductase (HjDHFR P1) from a hyperhalophilic archaeon, Haloarcula japonica strain TR-1 living in a Japanese saltern, were studied using ultraviolet absorption, circular dichroism (CD), and fluorescence spectroscopy. HjDHFR P1 had a partial structure at pH 8.0 in the absence of NaCl, and the addition of NaCl (0-500 mM concentration) induced significant structural formation to HjDHFR P1. The addition of NADPH, which is a coenzyme for its catalytic reaction, and lowering the pH from 8 to 6 also induced the same CD change, indicating the formation of the NADPH-binding site in HjDHFR P1. The NaCl dependence of thermal and urea-induced unfolding measurements suggested that protein stability increased depending on NaCl concentration regardless of structural formation, and HjDHFR P1 achieved the same stability as Escherichia coli DHFR at 750 mM NaCl. Halophilic characteristics were also observed for enzymatic function, although its structure had already formed under the conditions that enzymatic activity was measured at due to the presence of NADPH. These results suggest that the halophilic mechanism on structural stability and function was caused by factors other than structural formation, which are suggested to be the contributions of preferential interactions between the protein and salt ions and the specific binding of salt ions.
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima, 739-8526, Japan.
authors.forename: Yurina
authors.initials: Y
authors.lastname: Miyashita
authors.forename: Eiji
authors.initials: E
authors.lastname: Ohmae
authors.forename: Kaoru
authors.initials: K
authors.lastname: Nakasone
authors.forename: Katsuo
authors.initials: K
authors.lastname: Katayanagi
mesh_terms: D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry* / Q000378:metabolism; D004795:Enzyme Stability; D019613:Haloarcula / Q000201:enzymology*; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D011489:Protein Denaturation; D012965:Sodium Chloride / Q000737:chemistry; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism; D014508:Urea / Q000737:chemistry
chemical_list: D019843:Archaeal Proteins; D012965:Sodium Chloride; D009249:NADP; D014508:Urea; D013762:Tetrahydrofolate Dehydrogenase