title: Phylogeny-based design of a B-subunit of DNA gyrase and its ATPase domain using a small set of homologous amino acid sequences.
abstract: We have developed a phylogeny-based design method that has been used to produce mutated proteins with enhanced thermal stabilities. We previously validated the predictive worth of the method by producing and characterizing mutants in which one original residue or a small number of the original residues had been replaced with the one or the ones found in the phylogenetically predicted "ancestral" sequence. For the current study, this method was used to design a sequence for the deepest nodal position of a phylogenic tree composed of 16 gyrase B-subunit sequences, which was then synthesized and characterized. The sequence was inferred from the sequences of 16 extant DNA gyrases and 3 extant type VI DNA topoisomerases. Genes encoding the inferred sequence and its N-terminal ATPase domain were PCR constructed and expressed in Escherichia coli. The full-length designed protein is slightly less thermally stable than is subunit B from the extant thermophilic Thermus thermophilus DNA gyrase, whereas the thermal stability of the designed ATPase domain is more similar to that of the T. thermophilus ATPase domain. Moreover, the designed ATPase domain has significant catalytic activity. Therefore, even a small set of homologous amino acid sequences contains sufficient information to design a thermally stable and functional protein. Because the isolated designed ATPase domain is more thermally stable and catalytically active than is the sequence containing the most frequently occurring amino acids among the 16 gyrases, the phylogenetic approach was superior (in this case, at least) to the consensus approach when the same data set was used to predict the two sequences.
authors.affiliation: Department of Molecular Biology, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Akanuma
authors.forename: Shoko
authors.initials: S
authors.lastname: Iwami
authors.forename: Tamaki
authors.initials: T
authors.lastname: Yokoi
authors.forename: Nana
authors.initials: N
authors.lastname: Nakamura
authors.forename: Hideaki
authors.initials: H
authors.lastname: Watanabe
authors.forename: Shin-ichi
authors.initials: S
authors.lastname: Yokobori
authors.forename: Akihiko
authors.initials: A
authors.lastname: Yamagishi
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry*; D000595:Amino Acid Sequence; D027081:DNA Gyrase / Q000737:chemistry*; D008969:Molecular Sequence Data; D010802:Phylogeny
chemical_list: D000251:Adenosine Triphosphatases; D027081:DNA Gyrase