Recombinant tetrathionate hydrolase from Acidithiobacillus ferrooxidans requires exposure to acidic conditions for proper folding.
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 beta-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 degrees 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.
Kanao
Tadayoshi
T
Department of Instrumental Analysis, Advanced Science Research Center, Okayama University, Kita-ku, Okayama, Japan. tkanao@cc.okayama-u.ac.jp
Matsumoto
Chie
C
Shiraga
Kumiko
K
Yoshida
Kyoya
K
Takada
Jun
J
Kamimura
Kazuo
K
D042763:Acidithiobacillus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D000143:Acids / Q000737:chemistry; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006867:Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D000143:Acids; D001426:Bacterial Proteins; D011994:Recombinant Proteins; D006867:Hydrolases; C104756:tetrathionate hydrolase