Phylogeny-based design of a B-subunit of DNA gyrase and its ATPase domain using a small set of homologous amino acid sequences

Satoshi Akanuma, Shoko Iwami, Tamaki Yokoi, Nana Nakamura, Hideaki Watanabe, Shin Ichi Yokobori, Akihiko Yamagishi

Research output: Contribution to journalArticle

14 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)212-225
Number of pages14
JournalJournal of Molecular Biology
Volume412
Issue number2
DOIs
Publication statusPublished - 2011 Sep 16
Externally publishedYes

Fingerprint

DNA Gyrase
Amino Acid Sequence Homology
Phylogeny
Adenosine Triphosphatases
Thermus thermophilus
Hot Temperature
Proteins
Escherichia coli
Amino Acids
Polymerase Chain Reaction
Genes

Keywords

  • ATP hydrolysis
  • consensus design
  • DNA gyrase
  • phylogenetic tree
  • temperature-induced unfolding

ASJC Scopus subject areas

  • Molecular Biology

Cite this

Phylogeny-based design of a B-subunit of DNA gyrase and its ATPase domain using a small set of homologous amino acid sequences. / Akanuma, Satoshi; Iwami, Shoko; Yokoi, Tamaki; Nakamura, Nana; Watanabe, Hideaki; Yokobori, Shin Ichi; Yamagishi, Akihiko.

In: Journal of Molecular Biology, Vol. 412, No. 2, 16.09.2011, p. 212-225.

Research output: Contribution to journalArticle

Akanuma, Satoshi ; Iwami, Shoko ; Yokoi, Tamaki ; Nakamura, Nana ; Watanabe, Hideaki ; Yokobori, Shin Ichi ; Yamagishi, Akihiko. / Phylogeny-based design of a B-subunit of DNA gyrase and its ATPase domain using a small set of homologous amino acid sequences. In: Journal of Molecular Biology. 2011 ; Vol. 412, No. 2. pp. 212-225.
@article{b20e0b8831c74e83967f3797a3739997,
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.",
keywords = "ATP hydrolysis, consensus design, DNA gyrase, phylogenetic tree, temperature-induced unfolding",
author = "Satoshi Akanuma and Shoko Iwami and Tamaki Yokoi and Nana Nakamura and Hideaki Watanabe and Yokobori, {Shin Ichi} and Akihiko Yamagishi",
year = "2011",
month = "9",
day = "16",
doi = "10.1016/j.jmb.2011.07.042",
language = "English",
volume = "412",
pages = "212--225",
journal = "Journal of Molecular Biology",
issn = "0022-2836",
publisher = "Academic Press Inc.",
number = "2",

}

TY - JOUR

T1 - Phylogeny-based design of a B-subunit of DNA gyrase and its ATPase domain using a small set of homologous amino acid sequences

AU - Akanuma, Satoshi

AU - Iwami, Shoko

AU - Yokoi, Tamaki

AU - Nakamura, Nana

AU - Watanabe, Hideaki

AU - Yokobori, Shin Ichi

AU - Yamagishi, Akihiko

PY - 2011/9/16

Y1 - 2011/9/16

N2 - 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.

AB - 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.

KW - ATP hydrolysis

KW - consensus design

KW - DNA gyrase

KW - phylogenetic tree

KW - temperature-induced unfolding

UR - http://www.scopus.com/inward/record.url?scp=80052024237&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=80052024237&partnerID=8YFLogxK

U2 - 10.1016/j.jmb.2011.07.042

DO - 10.1016/j.jmb.2011.07.042

M3 - Article

C2 - 21819994

AN - SCOPUS:80052024237

VL - 412

SP - 212

EP - 225

JO - Journal of Molecular Biology

JF - Journal of Molecular Biology

SN - 0022-2836

IS - 2

ER -