[P001] Poster

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

[P01-011] The reaction mechanism of tetrathionate hydrolysis based on the crystal structure analysis of tetrathionate hydrolase from the iron- and sulfur-oxidizing bacterium, Acidithiobacillus ferrooxidans.

○Akitaka Nishikawa¹, Michiko Nemoto¹, Takashi Tamura¹, Taro Tamada², Tadayoshi Kanao¹ (1. Okayama Univ. (Japan), 2. National Institutes for Quantum and Radiological Science and Technology (QST) (Japan))

Keywords:

Tetrathionate hydrolase,Enzymology,Crystal structure,Sulfur metabolism,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. Tetrathionate hydrolase (TTH) is a unique enzyme distributed among acidophilic sulfur-oxidizing microorganisms (bacteria and archaea) and plays an important role in dissimilatory sulfur-oxidation pathway which called S4 intermediate (S4I) pathway together with thiosulfate:quinone oxidoreductase to generate energy and reducing power. 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. Although heterologous expression of Af-tth in Escherichia coli resulted in the formation of the recombinant protein (Af-Tth) as inactive inclusion bodies, TTH activity was recovered by acidic refolding treatment. We have successfully obtained the crystals of recombinant Af-Tth and performed X-ray diffraction analysis. The diffraction dataset of recombinant Af-Tth was collected at a resolution of 1.95 Å, and all regions except for the N- and C-termini and several loop regions were successfully modeled. Af-Tth adopts a dimeric structure, and each monomer is rich in β-sheet structures. The monomer contains 33 β-strands and a β-propeller motif composed of eight blades. In each monomer, the L1 loop forms a cavity within the β-propeller region, where a large electron density was observed. Asp325, the only acidic residue located within 4 Å of this electron density, is conserved between TTHs from Acidithiobacillus species and the thermoacidophilic archaeon Acidianus ambivalens. The Af-Tth D325N mutant, in which Asp325 was substituted with asparagine, exhibited no detectable enzymatic activity, suggesting that the Asp325 residue is essential for tetrathionate hydrolysis. Af-Tth catalyzes a cysteine-independent reaction mechanism that does not involve a protein-bound intermediate. Structural and mutational analyses revealed that Asp325 plays a critical role in the initial step of tetrathionate hydrolysis. Although aspartate residues generally function as either acids or bases in catalytic reactions, Af-Tth exhibited activity maximum at pH 3.0 and no detectable activity above pH 6.0, indicating that Asp325 most likely acts as an acid during the reaction. The detailed reaction mechanism of the first step of tetrathionate hydrolysis concerning Asp325 was discussed in this presentation.