An Extracellular Tetrathionate Hydrolase from the Thermoacidophilic Archaeon Acidianus Ambivalens with an Activity Optimum at pH 1

Abstract

Background: The thermoacidophilic and chemolithotrophic archaeon Acidianus ambivalens is routinely grown with sulfur and CO2-enriched air. We had described a membrane-bound, tetrathionate (TT) forming thiosulfate:quinone oxidoreductase. Here we describe the first TT hydrolase (TTH) from Archaea. Results: A. ambivalens cells grown aerobically with TT as sole sulfur source showed doubling times of 9 h and final cell densities of up to 8 × 108/ml. TTH activity (≈0.28 U/mg protein) was found in cell-free extracts of TT-grown but not of sulfur-grown cells. Differential fractionation of freshly harvested cells involving a pH shock showed that about 92% of the TTH activity was located in the pseudo-periplasmic fraction associated with the surface layer, while 7.3% and 0.3% were present in the soluble and membrane fractions, respectively. The enzyme was enriched 54-fold from the cytoplasmic fraction and 2.1-fold from the pseudo-periplasmic fraction. The molecular mass of the single subunit was 54 kDa. The optimal activity was at or above 95°C at pH 1. Neither PQQ nor divalent cations had a significant effect on activity. The gene (tth1) was identified following N-terminal sequencing of the protein. Northern hybridization showed that tth1 was transcribed in TT-grown cells in contrast to a second paralogous tth2 gene. The deduced amino acid sequences showed similarity to the TTH from Acidithiobacillus and other proteins from the PQQ dehydrogenase superfamily. It displayed a β-propeller structure when being modeled, however, important residues from the PQQ-binding site were absent. Conclusion: The soluble, extracellular, and acidophilic TTH identified in TT-grown A. ambivalens cells is essential for TT metabolism during growth but not for the downstream processing of the TQO reaction products in S°-grown cells. The liberation of TTH by pH shock from otherwise intact cells strongly supports the pseudo-periplasm hypothesis of the S-layer of Archaea.

Gene Identification and Substrate Regulation Provide Insights into Sulfur Accumulation during Bioleaching with the Psychrotolerant Acidophile Acidithiobacillus ferrivorans

“…Type I Sqr has been functionally and structurally characterized in several species, including A. ferrooxidans (32), while no bacterial type V Sqr has been shown to oxidize sulfide. Similarly, TetH1 grouped with previously described tetrathionate-hydrolyzing enzymes, and TetH2 grouped with Acidianus ambivalens TetH2, previously shown not to have tetrathionate-hydrolyzing activity (33). These analyses suggested that sqr1 and tetH1 were more likely to be involved in ISC metabolism.…”

The sole cysteine residue (Cys301) of tetrathionate hydrolase from Acidithiobacillus ferrooxidans does not play a role in enzyme activity

“…ambivalens. 23,25,27) Fig. 1 shows the alignment of the primary structure of the three 4THases excluding their putative signal peptides.…”

“…4THase from Ad. ambivalens has been reported to exist in both dimer and monomer forms, and enzyme activity has been detected in both forms, 25) indicating that subunit association is not essential for enzyme activity. Although 12 cysteine residues have been identified in the 4THase from Ad.…”

“…ambivalens. 25) 4THase of At. ferrooxidans (Af-Tth) catalyzes tetrathionate hydrolysis to generate elemental sulfur, thiosulfate, and sulfate.…”

“…caldus and Ad. ambivalens, 22,25) Af-Tth is synthesized in both tetrathionateand sulfur-grown cells.…”