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

Kanao, Tadayoshi; Nakayama, Hiromasa; Kato, Mizuki; Kamimura, Kazuo

doi:10.1080/09168451.2014.948374

Cited by 5 publications

(8 citation statements)

References 30 publications

(46 reference statements)

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“…However, the three-dimensional structure was not obtained until now. It was reported that the sole cysteine residue (Cys301) in Af-TetH was involved in neither the tetrathionate hydrolysis reaction nor the subunit assembly, indicating a novel cysteine-independent reaction mechanism for this enzyme (Kanao et al, 2014).…”

Section: Thiosulfate Oxidationmentioning

confidence: 99%

Sulfur Oxidation in the Acidophilic Autotrophic Acidithiobacillus spp.

et al. 2019

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Sulfur oxidation is an essential component of the earth’s sulfur cycle. Acidithiobacillus spp. can oxidize various reduced inorganic sulfur compounds (RISCs) with high efficiency to obtain electrons for their autotrophic growth. Strains in this genus have been widely applied in bioleaching and biological desulfurization. Diverse sulfur-metabolic pathways and corresponding regulatory systems have been discovered in these acidophilic sulfur-oxidizing bacteria. The sulfur-metabolic enzymes in Acidithiobacillus spp. can be categorized as elemental sulfur oxidation enzymes (sulfur dioxygenase, sulfur oxygenase reductase, and Hdr-like complex), enzymes in thiosulfate oxidation pathways (tetrathionate intermediate thiosulfate oxidation (S4I) pathway, the sulfur oxidizing enzyme (Sox) system and thiosulfate dehydrogenase), sulfide oxidation enzymes (sulfide:quinone oxidoreductase) and sulfite oxidation pathways/enzymes. The two-component systems (TCSs) are the typical regulation elements for periplasmic thiosulfate metabolism in these autotrophic sulfur-oxidizing bacteria. Examples are RsrS/RsrR responsible for S4I pathway regulation and TspS/TspR for Sox system regulation. The proposal of sulfur metabolic and regulatory models provide new insights and overall understanding of the sulfur-metabolic processes in Acidithiobacillus spp. The future research directions and existing barriers in the bacterial sulfur metabolism are also emphasized here and the breakthroughs in these areas will accelerate the research on the sulfur oxidation in Acidithiobacillus spp. and other sulfur oxidizers.

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Section: Thiosulfate Oxidationmentioning

confidence: 99%

Sulfur Oxidation in the Acidophilic Autotrophic Acidithiobacillus spp.

et al. 2019

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“…Some ordered water molecules were present at both edges of the cavities (two water molecules are highlighted by the black arrows in Figure 2b). Although cysteine residues are indispensable in the reactions of many enzymes of dissimilatory sulfur oxidation metabolism, we previously reported that the sole cysteine residue (Cys301) of Af ‐Tth is not involved in the hydrolysis reaction 22 . Cys301 was more than 25 Å from this cavity.…”

Section: Resultsmentioning

confidence: 95%

“…Recombinant Af ‐Tth was synthesized in E. coli as inclusion bodies in an inactive form. The active enzyme was refolded from inclusion bodies under acidic conditions and purified to homogeneity as a homodimer via gel‐permeation column chromatography (GPC) at pH 4.0 22 . The crystallization of Af ‐Tth was previously described in detail 23 .…”

Section: Resultsmentioning

confidence: 99%

“…Although cysteine residues are normally conserved in each enzyme among different sulfur‐oxidizing microorganisms, only one cysteine residue (Cys301) can be detected in the primary structure of Af ‐Tth, and it is not conserved among 4THases from Acidithiobacillus spp 22 . Furthermore, the site‐specific variant ( Af ‐Tth C301A) exhibited similar activity as wild‐type Af ‐Tth 22 . This result suggests that Af ‐Tth has a novel cysteine‐independent reaction mechanism.…”

Section: Introductionmentioning

confidence: 94%

“…Cysteine residues in the catalytic center play an important role in many other enzymes involved in dissimilatory sulfur oxidation such as sulfide:quinone oxidoreductase, 19 sulfur oxygenase reductase, 20 and thiosulfate dehydrogenase 21 . Although cysteine residues are normally conserved in each enzyme among different sulfur‐oxidizing microorganisms, only one cysteine residue (Cys301) can be detected in the primary structure of Af ‐Tth, and it is not conserved among 4THases from Acidithiobacillus spp 22 . Furthermore, the site‐specific variant ( Af ‐Tth C301A) exhibited similar activity as wild‐type Af ‐Tth 22 .…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2020

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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 sitespecific 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.

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A novel soxO gene, encoding a glutathione disulfide reductase, is essential for tetrathionate oxidation in Advenella kashmirensis

Pyne

Alam

Ghosh

2017

Microbiological Research

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Molecular mechanisms of chemolithotrophic tetrathionate oxidation are not clearly understood. Here we used transposon(Tn5-mob)-insertion mutagenesis to search for novel tetrathionate oxidation genes in the facultatively chemolithoautotrophic betaproteobacterium Advenella kashmirensis that not only oxidizes tetrathionate, but also produces the same as an intermediate during thiosulfate oxidation. Genome-wide random insertion of Tn5-mob occurred at a frequency of one per 10 donor E. coli cells. A library of 8000 transconjugants yielded five tetrathionate-oxidation-impaired mutants, of which, the one named Ak_Tn_16 was studied here in detail. When grown chemolithoautotrophically on thiosulfate, Ak_Tn_16 converted the total thiosulfate supplied to equivalent amount of tetrathionate, exactly in the same way as the wild type. It could not, however, oxidize the intermediary tetrathionate to sulfate; Ak_Tn_16 could not also oxidize tetrathionate when it was supplied as the starting chemolithotrophic substrate. In the Ak_Tn_16 genome, Tn5-mob was found to have transposed in a novel soxO gene, located just-upstream of soxB, within the sox gene cluster. SoxO was predicted, via iterative threading assembly simulation, to be a glutathione-disulfide (GSSG) reductase. When Ak_Tn_16 was grown in tetrathionate-based chemolithoautotrophic medium supplemented with reduced glutathione (GSH) its tetrathionate-oxidation deficiency, remarkably, was ameliorated. Implications for a key role of GSH in tetrathionate oxidation are discussed in the light of other molecular evidences available for A. kashmirensis.

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

Cited by 5 publications

References 30 publications

Sulfur Oxidation in the Acidophilic Autotrophic Acidithiobacillus spp.

Sulfur Oxidation in the Acidophilic Autotrophic Acidithiobacillus spp.

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

A novel soxO gene, encoding a glutathione disulfide reductase, is essential for tetrathionate oxidation in Advenella kashmirensis

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