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Reaction mechanism of tetrathionate hydrolysis based on the crystal structure of tetrathionate hydrolase from Acidithiobacillus ferrooxidans.

ABSTRACT: Tetrathionate hydrolase (4THase) plays an important role in dissimilatory sulfur oxidation in the acidophilic iron- and sulfur-oxidizing bacterium Acidithiobacillus ferrooxidans. The structure of recombinant 4THase from A. ferrooxidans (Af-Tth) was determined by X-ray crystallography to a resolution of 1.95 Å. Af-Tth is a homodimer, and its monomer structure exhibits an eight-bladed β-propeller motif. Two insertion loops participate in dimerization, and one loop forms a cavity with the β-propeller region. We observed unexplained electron densities in this cavity of the substrate-soaked structure. The anomalous difference map generated using diffraction data collected at a wavelength of 1.9 Å indicated the presence of polymerized sulfur atoms. Asp325, a highly conserved residue among 4THases, was located near the polymerized sulfur atoms. 4THase activity was completely abolished in the site-specific Af-Tth D325N variant, suggesting that Asp325 plays a crucial role in the first step of tetrathionate hydrolysis. Considering that the Af-Tth reaction occurs only under acidic pH, Asp325 acts as an acid for the tetrathionate hydrolysis reaction. The polymerized sulfur atoms in the active site cavity may represent the intermediate product in the subsequent step.

SUBMITTER: Kanao T

PROVIDER: S-EPMC7784748 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Crystallization and preliminary X-ray diffraction analysis of tetrathionate hydrolase from Acidithiobacillus ferrooxidans.

Project description:Tetrathionate hydrolase (4THase) from the iron- and sulfur-oxidizing bacterium Acidithiobacillus ferrooxidans catalyses the disproportionate hydrolysis of tetrathionate to elemental sulfur, thiosulfate and sulfate. The gene encoding 4THase (Af-tth) was expressed as inclusion bodies in recombinant Escherichia coli. Recombinant Af-Tth was activated by refolding under acidic conditions and was then purified to homogeneity. The recombinant protein was crystallized in 20 mM glycine buffer pH 10 containing 50 mM sodium chloride and 33%(v/v) PEG 1000 using the hanging-drop vapour-diffusion method. The crystal was a hexagonal cylinder with dimensions of 0.2 × 0.05 × 0.05 mm. X-ray crystallographic analysis showed that the crystal diffracted to 2.15 Å resolution and belongs to space group P3(1) or P3(2), with unit-cell parameters a = b = 92.1, c = 232.6 Å.

| S-EPMC3668597 | biostudies-literature

Tetrathionate-forming thiosulfate dehydrogenase from the acidophilic, chemolithoautotrophic bacterium Acidithiobacillus ferrooxidans.

Project description:Thiosulfate dehydrogenase is known to play a significant role in thiosulfate oxidation in the acidophilic, obligately chemolithoautotroph, Acidithiobacillus ferrooxidans. Enzyme activity measured using ferricyanide as the electron acceptor was detected in cell extracts of A. ferrooxidans ATCC 23270 grown on tetrathionate or sulfur, but no activity was detected in ferrous iron-grown cells. The enzyme was enriched 63-fold from cell extracts of tetrathionate-grown cells. Maximum enzyme activity (13.8 U mg(-1)) was observed at pH 2.5 and 70°C. The end product of the enzyme reaction was tetrathionate. The enzyme reduced neither ubiquinone nor horse heart cytochrome c, which serves as an electron acceptor. A major protein with a molecular mass of ∼25 kDa was detected in the partially purified preparation. Heme was not detected in the preparation, according to the results of spectroscopic analysis and heme staining. The open reading frame of AFE_0042 was identified by BLAST by using the N-terminal amino acid sequence of the protein. The gene was found within a region that was previously noted for sulfur metabolism-related gene clustering. The recombinant protein produced in Escherichia coli had a molecular mass of ∼25 kDa and showed thiosulfate dehydrogenase activity, with maximum enzyme activity (6.5 U mg(-1)) observed at pH 2.5 and 50°C.

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Project description:Oxidative dissolution of stibnite (Sb2S3), one of the most prevalent geochemical processes for antimony (Sb) release, can be promoted by Sb-oxidizing microbes, which were studied under alkaline and neutral conditions but rarely under acidic conditions. This work is dedicated to unraveling the enhancement mechanism of stibnite dissolution by typical acidophile Acidithiobacillus ferrooxidans under extremely acidic conditions. The results of solution behavior showed that the dissolution of Sb2S3 was significantly enhanced by A. ferrooxidans, with lower pH and higher redox potential values and higher [Sb(III)] and [Sb(V)] than the sterile control. The surface morphology results showed that the cells adsorbed onto the mineral surface and formed biofilms. Much more filamentous secondary minerals were formed for the case with A. ferrooxidans. Further mineral phase compositions and Sb/S speciation transformation analyses showed that more secondary products Sb2O3/SbO2-, Sb2O5/SbO3-, SO42-, as well as intermediates, such as S0, S2O32- were formed for the biotic case, indicating that the dissolution of Sb2S3 and the Sb/S speciation transformation was promoted by A. ferrooxidans. These results were further clarified by the comparative transcriptome analysis. This work demonstrated that through the interaction with Sb2S3, A. ferrooxidans promotes S/Sb oxidation, so as to enhance S/Sb transformation and thus the dissolution of Sb2S3.

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2020-11-23 | GSE154815 | GEO

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Three-dimensional structure of nylon hydrolase and mechanism of nylon-6 hydrolysis.

Project description:We performed x-ray crystallographic analyses of the 6-aminohexanoate oligomer hydrolase (NylC) from Agromyces sp. at 2.0 Å-resolution. This enzyme is a member of the N-terminal nucleophile hydrolase superfamily that is responsible for the degradation of the nylon-6 industry byproduct. We observed four identical heterodimers (27 kDa + 9 kDa), which resulted from the autoprocessing of the precursor protein (36 kDa) and which constitute the doughnut-shaped quaternary structure. The catalytic residue of NylC was identified as the N-terminal Thr-267 of the 9-kDa subunit. Furthermore, each heterodimer is folded into a single domain, generating a stacked ???? core structure. Amino acid mutations at subunit interfaces of the tetramer were observed to drastically alter the thermostability of the protein. In particular, four mutations (D122G/H130Y/D36A/E263Q) of wild-type NylC from Arthrobacter sp. (plasmid pOAD2-encoding enzyme), with a heat denaturation temperature of T(m) = 52 °C, enhanced the protein thermostability by 36 °C (T(m) = 88 °C), whereas a single mutation (G111S or L137A) decreased the stability by ?10 °C. We examined the enzymatic hydrolysis of nylon-6 by the thermostable NylC mutant. Argon cluster secondary ion mass spectrometry analyses of the reaction products revealed that the major peak of nylon-6 (m/z 10,000-25,000) shifted to a smaller range, producing a new peak corresponding to m/z 1500-3000 after the enzyme treatment at 60 °C. In addition, smaller fragments in the soluble fraction were successively hydrolyzed to dimers and monomers. Based on these data, we propose that NylC should be designated as nylon hydrolase (or nylonase). Three potential uses of NylC for industrial and environmental applications are also discussed.

| S-EPMC3281642 | biostudies-literature

Draft genome sequence of Acidithiobacillus ferrooxidans YQH-1.

Project description:Acidithiobacillus ferrooxidans YQH-1 is a moderate acidophilic bacterium isolated from a river in a volcano of Northeast China. Here, we describe the draft genome of strain YQH-1, which was assembled into 123 contigs containing 3,111,222 bp with a G + C content of 58.63%. A large number of genes related to carbon dioxide fixation, dinitrogen fixation, pH tolerance, heavy metal detoxification, and oxidative stress defense were detected. The genome sequence can be accessed at DDBJ/EMBL/GenBank under the accession no. LJBT00000000.

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Project description:BACKGROUND: Acidithiobacillus ferrooxidans is a major participant in consortia of microorganisms used for the industrial recovery of copper (bioleaching or biomining). It is a chemolithoautrophic, gamma-proteobacterium using energy from the oxidation of iron- and sulfur-containing minerals for growth. It thrives at extremely low pH (pH 1-2) and fixes both carbon and nitrogen from the atmosphere. It solubilizes copper and other metals from rocks and plays an important role in nutrient and metal biogeochemical cycling in acid environments. The lack of a well-developed system for genetic manipulation has prevented thorough exploration of its physiology. Also, confusion has been caused by prior metabolic models constructed based upon the examination of multiple, and sometimes distantly related, strains of the microorganism. RESULTS: The genome of the type strain A. ferrooxidans ATCC 23270 was sequenced and annotated to identify general features and provide a framework for in silico metabolic reconstruction. Earlier models of iron and sulfur oxidation, biofilm formation, quorum sensing, inorganic ion uptake, and amino acid metabolism are confirmed and extended. Initial models are presented for central carbon metabolism, anaerobic metabolism (including sulfur reduction, hydrogen metabolism and nitrogen fixation), stress responses, DNA repair, and metal and toxic compound fluxes. CONCLUSION: Bioinformatics analysis provides a valuable platform for gene discovery and functional prediction that helps explain the activity of A. ferrooxidans in industrial bioleaching and its role as a primary producer in acidic environments. An analysis of the genome of the type strain provides a coherent view of its gene content and metabolic potential.

| S-EPMC2621215 | biostudies-literature

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Project description:The iron oxidation system from sulfur-grown Acidithiobacillus ferrooxidans ATCC 23270 cells was reconstituted in vitro. Purified rusticyanin, cytochrome c, and aa(3)-type cytochrome oxidase were essential for reconstitution. The iron-oxidizing activity of the reconstituted system was 3.3-fold higher than that of the cell extract from which these components were purified.

| S-EPMC2576702 | biostudies-literature

Metabolic reconstruction of sulfur assimilation in the extremophile Acidithiobacillus ferrooxidans based on genome analysis.

Project description:BACKGROUND: Acidithiobacillus ferrooxidans is a gamma-proteobacterium that lives at pH2 and obtains energy by the oxidation of sulfur and iron. It is used in the biomining industry for the recovery of metals and is one of the causative agents of acid mine drainage. Effective tools for the study of its genetics and physiology are not in widespread use and, despite considerable effort, an understanding of its unusual physiology remains at a rudimentary level. Nearly complete genome sequences of A. ferrooxidans are available from two public sources and we have exploited this information to reconstruct aspects of its sulfur metabolism. RESULTS: Two candidate mechanisms for sulfate uptake from the environment were detected but both belong to large paralogous families of membrane transporters and their identification remains tentative. Prospective genes, pathways and regulatory mechanisms were identified that are likely to be involved in the assimilation of sulfate into cysteine and in the formation of Fe-S centers. Genes and regulatory networks were also uncovered that may link sulfur assimilation with nitrogen fixation, hydrogen utilization and sulfur reduction. Potential pathways were identified for sulfation of extracellular metabolites that may possibly be involved in cellular attachment to pyrite, sulfur and other solid substrates. CONCLUSIONS: A bioinformatic analysis of the genome sequence of A. ferrooxidans has revealed candidate genes, metabolic process and control mechanisms potentially involved in aspects of sulfur metabolism. Metabolic modeling provides an important preliminary step in understanding the unusual physiology of this extremophile especially given the severe difficulties involved in its genetic manipulation and biochemical analysis.

| S-EPMC324559 | biostudies-literature

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