Recombinant tetrathionate hydrolase from Acidithiobacillus ferrooxidans requires exposure to acidic conditions for proper folding

Tadayoshi Kanao, Chie Matsumoto, Kumiko Shiraga, Kyoya Yoshida, Jun Takada, Kazuo Kamimura

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

Tetrathionate hydrolase (4THase) plays an important role in dissimilatory sulfur metabolism in the acidophilic chemolithoautotrophic iron- and sulfur-oxidizing bacterium Acidithiobacillus ferrooxidans. We have already identified the gene encoding 4THase (Af-tth) in this bacterium. The heterologous expression of Af-tth in Escherichia coli resulted in the formation of inclusion bodies of the protein in an inactive form. The recombinant protein (Af-Tth) was successfully activated after an in vitro refolding treatment. The specific activity of the refolded Af-Tth obtained was 21.0±9.4 U mg-1 when the protein solubilized from inclusion bodies by 6 M guanidine hydrochloride solution was refolded in a buffer containing 10 mM β-alanine, 2 mM dithiothreitol, 0.4 M ammonium sulfate, and 30% v/v glycerol with the pH adjusted to 4.0 by sulfuric acid for 14 h at 4 °C. The in vitro refolding experiments revealed that Af-Tth required exposure to an acidic environment during protein folding for activation. This property reflects a physiological characteristic of the Af-Tth localized in the outer membrane of the acidophilic A. ferrooxidans. No cofactor such as pyrroloquinoline quinone (PQQ) was required during the refolding process in spite of the similarity in the primary structure of Af-Tth to the PQQ family of proteins.

Original languageEnglish
Pages (from-to)43-47
Number of pages5
JournalFEMS Microbiology Letters
Volume309
Issue number1
DOIs
Publication statusPublished - 2010

Fingerprint

Acidithiobacillus
PQQ Cofactor
Inclusion Bodies
Sulfur
Bacteria
Proteins
Dithiothreitol
Protein Folding
Guanidine
Ammonium Sulfate
Recombinant Proteins
Alanine
Glycerol
Buffers
Iron
Escherichia coli
Membranes
Genes
tetrathionate hydrolase
In Vitro Techniques

Keywords

  • acidophilic bacteria
  • refolding
  • sulfur metabolism
  • tetrathionate hydrolase

ASJC Scopus subject areas

  • Microbiology
  • Genetics
  • Molecular Biology
  • Medicine(all)

Cite this

Recombinant tetrathionate hydrolase from Acidithiobacillus ferrooxidans requires exposure to acidic conditions for proper folding. / Kanao, Tadayoshi; Matsumoto, Chie; Shiraga, Kumiko; Yoshida, Kyoya; Takada, Jun; Kamimura, Kazuo.

In: FEMS Microbiology Letters, Vol. 309, No. 1, 2010, p. 43-47.

Research output: Contribution to journalArticle

@article{7aab5490084641f2bec227a7ff26a3c6,
title = "Recombinant tetrathionate hydrolase from Acidithiobacillus ferrooxidans requires exposure to acidic conditions for proper folding",
abstract = "Tetrathionate hydrolase (4THase) plays an important role in dissimilatory sulfur metabolism in the acidophilic chemolithoautotrophic iron- and sulfur-oxidizing bacterium Acidithiobacillus ferrooxidans. We have already identified the gene encoding 4THase (Af-tth) in this bacterium. The heterologous expression of Af-tth in Escherichia coli resulted in the formation of inclusion bodies of the protein in an inactive form. The recombinant protein (Af-Tth) was successfully activated after an in vitro refolding treatment. The specific activity of the refolded Af-Tth obtained was 21.0±9.4 U mg-1 when the protein solubilized from inclusion bodies by 6 M guanidine hydrochloride solution was refolded in a buffer containing 10 mM β-alanine, 2 mM dithiothreitol, 0.4 M ammonium sulfate, and 30{\%} v/v glycerol with the pH adjusted to 4.0 by sulfuric acid for 14 h at 4 °C. The in vitro refolding experiments revealed that Af-Tth required exposure to an acidic environment during protein folding for activation. This property reflects a physiological characteristic of the Af-Tth localized in the outer membrane of the acidophilic A. ferrooxidans. No cofactor such as pyrroloquinoline quinone (PQQ) was required during the refolding process in spite of the similarity in the primary structure of Af-Tth to the PQQ family of proteins.",
keywords = "acidophilic bacteria, refolding, sulfur metabolism, tetrathionate hydrolase",
author = "Tadayoshi Kanao and Chie Matsumoto and Kumiko Shiraga and Kyoya Yoshida and Jun Takada and Kazuo Kamimura",
year = "2010",
doi = "10.1111/j.1574-6968.2010.02019.x",
language = "English",
volume = "309",
pages = "43--47",
journal = "FEMS Microbiology Letters",
issn = "0378-1097",
publisher = "Wiley-Blackwell",
number = "1",

}

TY - JOUR

T1 - Recombinant tetrathionate hydrolase from Acidithiobacillus ferrooxidans requires exposure to acidic conditions for proper folding

AU - Kanao, Tadayoshi

AU - Matsumoto, Chie

AU - Shiraga, Kumiko

AU - Yoshida, Kyoya

AU - Takada, Jun

AU - Kamimura, Kazuo

PY - 2010

Y1 - 2010

N2 - Tetrathionate hydrolase (4THase) plays an important role in dissimilatory sulfur metabolism in the acidophilic chemolithoautotrophic iron- and sulfur-oxidizing bacterium Acidithiobacillus ferrooxidans. We have already identified the gene encoding 4THase (Af-tth) in this bacterium. The heterologous expression of Af-tth in Escherichia coli resulted in the formation of inclusion bodies of the protein in an inactive form. The recombinant protein (Af-Tth) was successfully activated after an in vitro refolding treatment. The specific activity of the refolded Af-Tth obtained was 21.0±9.4 U mg-1 when the protein solubilized from inclusion bodies by 6 M guanidine hydrochloride solution was refolded in a buffer containing 10 mM β-alanine, 2 mM dithiothreitol, 0.4 M ammonium sulfate, and 30% v/v glycerol with the pH adjusted to 4.0 by sulfuric acid for 14 h at 4 °C. The in vitro refolding experiments revealed that Af-Tth required exposure to an acidic environment during protein folding for activation. This property reflects a physiological characteristic of the Af-Tth localized in the outer membrane of the acidophilic A. ferrooxidans. No cofactor such as pyrroloquinoline quinone (PQQ) was required during the refolding process in spite of the similarity in the primary structure of Af-Tth to the PQQ family of proteins.

AB - Tetrathionate hydrolase (4THase) plays an important role in dissimilatory sulfur metabolism in the acidophilic chemolithoautotrophic iron- and sulfur-oxidizing bacterium Acidithiobacillus ferrooxidans. We have already identified the gene encoding 4THase (Af-tth) in this bacterium. The heterologous expression of Af-tth in Escherichia coli resulted in the formation of inclusion bodies of the protein in an inactive form. The recombinant protein (Af-Tth) was successfully activated after an in vitro refolding treatment. The specific activity of the refolded Af-Tth obtained was 21.0±9.4 U mg-1 when the protein solubilized from inclusion bodies by 6 M guanidine hydrochloride solution was refolded in a buffer containing 10 mM β-alanine, 2 mM dithiothreitol, 0.4 M ammonium sulfate, and 30% v/v glycerol with the pH adjusted to 4.0 by sulfuric acid for 14 h at 4 °C. The in vitro refolding experiments revealed that Af-Tth required exposure to an acidic environment during protein folding for activation. This property reflects a physiological characteristic of the Af-Tth localized in the outer membrane of the acidophilic A. ferrooxidans. No cofactor such as pyrroloquinoline quinone (PQQ) was required during the refolding process in spite of the similarity in the primary structure of Af-Tth to the PQQ family of proteins.

KW - acidophilic bacteria

KW - refolding

KW - sulfur metabolism

KW - tetrathionate hydrolase

UR - http://www.scopus.com/inward/record.url?scp=77954365665&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=77954365665&partnerID=8YFLogxK

U2 - 10.1111/j.1574-6968.2010.02019.x

DO - 10.1111/j.1574-6968.2010.02019.x

M3 - Article

VL - 309

SP - 43

EP - 47

JO - FEMS Microbiology Letters

JF - FEMS Microbiology Letters

SN - 0378-1097

IS - 1

ER -