title: Increase of salt dependence of halophilic nucleoside diphosphate kinase caused by a single amino acid substitution.
abstract: Nucleoside diphosphate kinase (HsNDK) from an extremely halophilic archaea, Halobacterium salinarum, is composed of a homo hexamer, assembled as a trimer of basic dimeric units. It requires >2 M NaCl for refolding, although it does not require NaCl for stability or enzymatic activity below 30 °C. A HisN111L mutant with an N-terminal extension sequence containing hexa-His tag, in which Asn111 was replaced with Leu, was designed to be less stable between basic dimeric units. This mutant can lose between 6 and 12 hydrogen bonds between basic dimeric units in the hexamer structure. The HisN111L mutant had enhanced salt requirements for enzymatic activity and refolding even though the secondary structure of the HisN111L mutant was confirmed to be similar to the control, HisNDK, in low and high salt solutions using circular dichroism. We reported previously that G114R and D148C mutants, which had enhanced interactions between basic dimeric units, showed facilitated refolding and stabilization in low salt solution. The results of this study help to elucidate the process for engineering industrial enzymes by controlling subunit-subunit interactions through mutations.
authors.affiliation: Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan. matu@chem.agri.kagoshima-u.ac.jp
authors.forename: Matsujiro
authors.initials: M
authors.lastname: Ishibashi
authors.forename: Tomoe
authors.initials: T
authors.lastname: Hayashi
authors.forename: Chiho
authors.initials: C
authors.lastname: Yoshida
authors.forename: Masao
authors.initials: M
authors.lastname: Tokunaga
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability; D006217:Halobacterium / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D009701:Nucleoside-Diphosphate Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011489:Protein Denaturation; D055503:Protein Multimerization; D012965:Sodium Chloride / Q000737:chemistry*; D013696:Temperature
chemical_list: D019843:Archaeal Proteins; D012965:Sodium Chloride; D009701:Nucleoside-Diphosphate Kinase