Open AccessRecombinant tetrathionate hydrolase from Acidithiobacillus ferrooxidans requires exposure to acidic conditions for proper folding
Author(s) -
Kanao Tadayoshi,
Matsumoto Chie,
Shiraga Kumiko,
Yoshida Kyoya,
Takada Jun,
Kamimura Kazuo
Publication year - 2010
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.2010.02019.x
Subject(s) - chemistry , biochemistry , pyrroloquinoline quinone , escherichia coli , acidithiobacillus , tetrathionate , heterologous expression , inclusion bodies , sulfur metabolism , recombinant dna , cofactor , sulfur , enzyme , acidithiobacillus ferrooxidans , thiosulfate , gene , organic chemistry , bioleaching , copper
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.