[P001] Poster

12:10 PM - 1:00 PM JST(3:10 AM - 4:00 AM UTC)

[P01-013] Crystallization of recombinant tetrathionate hydrolase from Acidithiobacillus ferrooxidans refolded under acidic conditions.

○Reina Kubo¹, Michiko Nemoto¹, Takashi Tamura¹, Taro Tamada², Tadayoshi Kanao¹ (1. Okayama University (Japan), 2. QST (Japan))

Keywords:

tetrathionate hydrolase,crystallization,sulfur methabolism,refolding,acidophile

The biological oxidation of reduced inorganic sulfur compounds (RISCs) is one of the most important processes in the environmental sulfur cycle. Especially, in acidic environments such as volcanic and geothermal areas and also in acid mine drainage, RISCs are abundant and suitable energy substrates for sulfur-oxidizing microorganisms. At least two major dissimilatory RISCs oxidation pathways, Sox system and S4 intermediate (S4I) pathway have been proposed, and various enzymes involved in RISCs oxidation metabolism have been identified and characterized. The multienzyme system required for thiosulfate oxidation in the Sox pathway consists of the SoxXA, SoxB, and SoxYZ complex. This system has been well studied in the neutrophilic sulfur-oxidizing bacteria. Conversely, in the S4I pathway, thiosulfate dehydrogenase (Tsd) and thiosulfate:quinone oxidoreductase (TQO) catalyze the oxidation of two molecules of thiosulfate to generate tetrathionate, which is subsequently hydrolyzed by tetrathionate hydrolase (TTH), a reaction that plays an important role in the pathway. TTH has been purified from the acidophilic iron- and sulfur-oxidizing bacterium, Acidithiobacillus ferrooxidans. We have previously identified the gene encoding TTH (Af-tth) in this bacterium. Heterologous expression of Af-tth in Escherichia coli resulted in the formation of the recombinant protein (Af-Tth) as inactive inclusion bodies. The protein solubilized from inclusion bodies with 6 M guanidine hydrochloride was successfully activated by in vitro refolding at 4℃ for 14 h in a buffer containing 10 mM beta-alanine, 2 mM dithiothreitol, 0.4 M ammonium sulfate, and 30% (v/v) glycerol, adjusted to pH 4.0 with sulfuric acid. Af-Tth exhibited activity when refolded under pH 2.0-6.0 conditions, with maximal activity observed at pH 4.0. These results indicate that exposure to an acidic environment during the protein-folding process is essential for activation of Af-Tth. This property reflects the physiological characteristics of Af-Tth, which is localized in the outer membrane of the acidophilic bacterium A. ferrooxidans, and strongly supports the notion that it folds within the periplasmic space. The optimal glycerol concentration for refolding of Af-Tth was 30%. Although high initial protein concentrations promoted protein aggregation, the highest yield of 4THase was obtained when refolding was performed at an initial protein concentration of 0.5 mg mL^-1. Despite exhibiting sequence similarity to members of the pyrroloquinoline quinone (PQQ) protein family, Af-Tth did not require cofactors such as PQQ during the refolding process. The recombinant protein was crystallized by the hanging-drop vapor diffusion method using 20 mM glycine buffer (pH 10) containing 50 mM sodium chloride and 33% (v/v) PEG 1000. The resulting crystals were hexagonal cylinder with dimensions of 0.2 × 0.05 × 0.05 mm. X-ray crystallographic analysis revealed that the crystals diffracted to a resolution of 2.15 Å and belonged to space group P3₁ or P3₂, with unit-cell parameters a = b = 92.1 Å and c = 232.6 Å.