title: Mutations of Asp540 and the domain-connecting residues synergistically enhance Pyrococcus furiosus DNA ligase activity.
abstract: The structure of Pyrococcus furiosus DNA ligase (PfuLig), which architecturally resembles human DNA ligase I (hLigI), revealed that the C-terminal helix stabilizes the closed conformation through several ionic interactions between two domains (adenylylation domain (AdD) and C-terminal OB-fold domain (OBD)). This helix is oriented differently in DNA-bound hLigI, suggesting that the disruption of its interactions with AdD facilitates DNA binding. Previously, we demonstrated that the replacement of Asp540 with arginine improves the ligation activity. Here we report that the combination of the Asp540-replacement and the elimination of ionic residues in the helix, forming interactions with AdD, effectively enhanced the activity.
authors.affiliation: Central Research Laboratory, Hitachi Ltd., 1-280 Higashi-koigakubo, Kokubunji-shi, Tokyo 185-8601, Japan.
authors.forename: Maiko
authors.initials: M
authors.lastname: Tanabe
authors.affiliation: Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka-shi, Fukuoka 812-8581, Japan.
authors.forename: Sonoko
authors.initials: S
authors.lastname: Ishino
authors.affiliation: Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka-shi, Fukuoka 812-8581, Japan.
authors.forename: Yoshizumi
authors.initials: Y
authors.lastname: Ishino
authors.affiliation: Central Research Laboratory, Hitachi Ltd., 1-280 Higashi-koigakubo, Kokubunji-shi, Tokyo 185-8601, Japan. Electronic address: hirokazu.nishida.ab@hitachi.com.
authors.forename: Hirokazu
authors.initials: H
authors.lastname: Nishida
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001224:Aspartic Acid; D011088:DNA Ligases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D020027:Pyrococcus furiosus / Q000201:enzymology*
chemical_list: D001224:Aspartic Acid; D011088:DNA Ligases
keywords: Archaea; DNA ligase; Mutational analysis; Protein engineering; Thermostable enzyme