Search Results (24)

Several eutardigrade species can withstand high doses of potassium cyanide (KCN), a potent inhibitor of mitochondrial respiration, suggesting defense mechanisms that remain poorly understood. We surveyed the available tardigrade genomes in search for 11 protein families associated with cyanide detoxification and resistance in other organisms. We identified 28 sequences putatively encoding mitochondrial alternative oxidase (AOX), as well as proteins from the nitrilase superfamily and rhodanese superfamily in three eutardigrades, but found no evidence for other canonical cyanide-detoxifying enzymes such as β-cyanoalanine synthase. We also experimentally tested KCN resistance of the model tardigrade species Hypsibius exemplaris using a standard exposure protocol (100 mM KCN, 10 min) to determine if its poor anhydrobiotic ability reflects on its capacity for KCN resistance. All specimens survived KCN exposure, with a mean recovery time that did not differ significantly from other tardigrade species, previously tested under identical conditions, suggesting that KCN resistance may be a shared trait among eutardigrades. However, confirming whether this represents a conserved feature across the Eutardigrada class will require broader sampling. While these findings highlight potential pathways, they are limited by the lack of functional or expression validation for the identified genes. Full article

Widespread release of cyanide from industrial activities represents a significant environmental challenge due to its acute toxicity and adverse effects on biological systems. In response to this concern, this study focused on the production of rhodanese from Kocuria rhizophila under submerged fermentation conditions and the assessment of its relevance for cyanide detoxification applications. A soil-derived Gram-positive bacterium was isolated and identified as Kocuria rhizophila based on morphological traits, biochemical profile-based VITEK 2 analysis, and 16S rRNA gene sequencing. Preliminary screening confirmed rhodanese production with an activity of 0.968 RU/mL. Under cyanide-induced submerged fermentation, enzyme production followed a growth-associated pattern and reached maximal activity at 40 h under optimized conditions (35 °C, pH 8.0). Partial purification using sequential precipitation and chromatographic steps enhanced enzyme purity, and SDS–PAGE analysis of the final fraction revealed protein bands at approximately 40, 140, and 260 kDa. Biochemical characterization showed Km values of 33.9 mM for KCN and 19.7 mM for sodium thiosulfate, with a Vmax of ~5.6 µmol min⁻¹ mL⁻¹ for KCN and optimal activity at pH 7–8 and 35 °C. Functional assays demonstrated efficient cyanide detoxification, achieving >85% conversion of KCN, ~92% of NaCN, and 65–77% of Ca (CN)₂ within 60 min in vitro. Collectively, these findings demonstrate that Kocuria rhizophila represents a promising microbial source of rhodanese with efficient cyanide-detoxifying activity, highlighting its potential for biotechnological and environmental remediation applications. Full article

The aim of this research was to determine the response of white mustard (Sinapis alba L.) cultivated in 2021–2022 in Lucim (53°23′06″ N 17°50′08″ E) in Poland to the soil nitrogen doses: N₄₀—40 kg N·ha⁻¹, N₆₀—60 kg N·ha⁻¹, N₈₀—(40 + 40)-80 kg N·ha⁻¹, as well as the method of its foliar application (0%—no foliar fertilization, 50%—half of the nitrogen dose and 75%—⅔ of the nitrogen dose). The effect of mustard cultivation on the activity of soil dehydrogenase, arylsulfatase, and rhodanese was examined. The highest white mustard seed yield was obtained after application of 80 kg N·ha⁻¹ (1.577, 1.597 Mg·ha⁻¹) and after application of Multi-N_50% (1.490, 1.515 Mg·ha⁻¹). Total nitrogen (50 g·kg⁻¹) content was highest in seeds from treatments fertilized with 80 kg N·ha⁻¹. Similar total nitrogen content in seeds was obtained using a 50% dose of foliar nitrogen. Increasing nitrogen doses resulted in a decrease in the crude fat yield in seeds. However, a significant increase in this nutrient content was observed after application of 50% foliar nitrogen. Soil nitrogen fertilization increased the enzymatic activity of dehydrogenases, particularly at the N80 dose. The activity of this enzyme was positively correlated with the obtained mustard seed yield, as well as with its quality measured by total phosphorus and crude fat content. Full article

The global gold extraction industry has been reported to use cyanide-based recovery processes, which pose environmental effects on water resources. The study examined Coptodon zillii liver rhodanese from a gold mining-impacted reservoir with a specific focus on the enzyme’s critical function in cyanide detoxification. Rhodanese was purified using successive chromatographic techniques with 5.4 U/mg specific activity and 3.1-fold purification. The molecular weight of the native enzyme was 36 kDa, and the subunits were 17 kDa, indicative of a dimeric structure. Optimal enzymatic activity was recorded at pH 8.0 and 50 °C. The effect of metal ions was significantly varied: the activity was inhibited by BaCl₂, CaCl₂, NaCl, and MgCl₂, and KCl enhanced performance. The kinetic determinations showed Michaelis-Menten kinetics with a Km of 20.0 mM for sodium thiosulfate and 25.0 mM for potassium cyanide. The enzyme’s minimal activity was identified toward 2-mercaptoethanol, ammonium persulfate, and ammonium sulfate, but with evidence of preference for thiosulfate utilization under the substrate specificity tests. The major interactions between the enzyme and the substrate were revealed by the molecular docking experiments. These showed Glu159, Gln161, and Arg173 formed important hydrogen bonds with thiosulfate, while Arg156 and Val172 were also involved. Other substrates are bound to Gln121 and Trp139 residues with much lower binding energy than thiosulfate. The findings increase our understanding of biochemical adaptation process knowledge in anthropogenically stressed environments, showing strategies of ecological resilience. The characterized enzymatic features showed potent cyanide detoxification potential, and the possible applications are in bioremediation strategies for mining-impacted aquatic ecosystems. Full article

The co-occurrence of rare terpenoid thiocyanates (R-SCN), structurally similar to their more common isothiocyanate isomers (R-NCS), poses an enigma: how does the accepted path, terpenyl cation R⁺ → R-NC → R-NCS, accommodate R-SCN? The mystery can now be rationalized by the consideration of three biosynthetic motifs: terpenoid carbocation (R⁺) capture by cyanoformate, NC-COOH (itself in equilibrium with NC⁻ and CO₂); co-localized rhodanese (a dual-function enzyme) that can both convert fugitive inorganic NC⁻ to thiocyanate ion, NCS⁻, and alkyl isonitriles to alkyl isothiocyanate (R-NC → R-NCS) and adventitious capture of the NCS⁻ by R⁺. The former two scenarios explain the preponderance of isothiocyanates, R-NCS, as products of a linear reaction path—the α-addition of S⁰ to R-NC—and the third scenario explains minor, less stable thiocyanates, R-SCN, as products of the adventitious capture of liberated NCS⁻ by the penultimate R⁺ precursor. DFT calculations support this proposal and eliminate other possibilities, e.g., the isomerization of R-NCS to R-SCN. Full article

In vascular plants, the final photosynthetic electron transfer from ferredoxin (Fd) to NADP⁺ is catalyzed by the flavoenzyme ferredoxin:NADP⁺ oxidoreductase (FNR). FNR is recruited to thylakoid membranes via an integral membrane protein TROL (thylakoid rhodanese-like protein) and the membrane associated protein Tic62. We have previously demonstrated that the absence of TROL triggers a very efficient superoxide (O₂^•−) removal mechanism. The dynamic TROL–FNR interaction has been shown to be an apparently overlooked mechanism that maintains linear electron flow before alternative pathway(s) is(are) activated. In this work, we aimed to further test our hypothesis that the FNR–TROL pair could be the source element that triggers various downstream networks of chloroplast ROS scavenging. Tandem affinity purification followed by the MS analysis confirmed the TROL–FNR interaction and revealed possible interaction of TROL with the thylakoid form of the enzyme ascorbate peroxidase (tAPX), which catalyzes the H₂O₂-dependent oxidation of ascorbate and is, therefore, the crucial component of the redox homeostasis system in plants. Further, EPR analyses using superoxide spin trap DMPO showed that, in comparison with the wild type, plants overexpressing TROL (TROL OX) propagate more O₂^•− when exposed to high light stress. This indicates an increased sensitivity to oxidative stress in conditions when there is an excess of membrane-bound FNR and less free FNR is found in the stroma. Finally, immunohistochemical analyses of glutathione in different Arabidopsis leaf cell compartments showed highly elevated glutathione levels in TROL OX, indicating an increased demand for this ROS scavenger in these plants, likely needed to prevent the damage of important cellular components caused by reactive oxygen species. Full article

Recently, a CRISPR-Cas9 genome-editing system was developed with introduced sequential ‘driver’ mutations in the WNT, MAPK, TGF-β, TP53 and PI3K pathways into organoids derived from normal human intestinal epithelial cells. Prior studies have demonstrated that isogenic organoids harboring mutations in the tumor suppressor genes APC, SMAD4 and TP53, as well as the oncogene KRAS, assumed more proliferative and invasive properties in vitro and in vivo. A separate body of studies implicates the role of various hydrogen sulfide (H₂S)-producing enzymes in the pathogenesis of colon cancer. The current study was designed to determine if the sequential mutations in the above pathway affect the expression of various H₂S producing enzymes. Western blotting was used to detect the expression of the H₂S-producing enzymes cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST), as well as several key enzymes involved in H₂S degradation such as thiosulfate sulfurtransferase/rhodanese (TST), ethylmalonic encephalopathy 1 protein/persulfide dioxygenase (ETHE1) and sulfide-quinone oxidoreductase (SQR). H₂S levels were detected by live-cell imaging using a fluorescent H₂S probe. Bioenergetic parameters were assessed by Extracellular Flux Analysis; markers of epithelial-mesenchymal transition (EMT) were assessed by Western blotting. The results show that the consecutive mutations produced gradual upregulations in CBS expression—in particular in its truncated (45 kDa) form—as well as in CSE and 3-MST expression. In more advanced organoids, when the upregulation of H₂S-producing enzymes coincided with the downregulation of the H₂S-degrading enzyme SQR, increased H₂S generation was also detected. This effect coincided with the upregulation of cellular bioenergetics (mitochondrial respiration and/or glycolysis) and an upregulation of the Wnt/β-catenin pathway, a key effector of EMT. Thus sequential mutations in colon epithelial cells according to the Vogelstein sequence are associated with a gradual upregulation of multiple H₂S generating pathways, which, in turn, translates into functional changes in cellular bioenergetics and dedifferentiation, producing more aggressive and more invasive colon cancer phenotypes. Full article

Thiosulfate: cyanide sulfurtransferase (TST), also named rhodanese, is an enzyme widely distributed in both prokaryotes and eukaryotes, where it plays a relevant role in mitochondrial function. TST enzyme is involved in several biochemical processes such as: cyanide detoxification, the transport of sulfur and selenium in biologically available forms, the restoration of iron–sulfur clusters, redox system maintenance and the mitochondrial import of 5S rRNA. Recently, the relevance of TST in metabolic diseases, such as diabetes, has been highlighted, opening the way for research on important aspects of sulfur metabolism in diabetes. This review underlines the structural and functional characteristics of TST, describing the physiological role and biomedical and biotechnological applications of this essential enzyme. Full article

Fresh cassava root is not recommended for animal feeding due to high quantities of hydrocyanic acid (HCN), which produces symptoms of poisoning. The purpose of this study was to find out how a rhodanese enzyme addition affects rumen fermentation, HCN content, feed utilization, and blood metabolites in beef calves fed fresh cassava root. Four Thai native beef cattle with an initial body weight (BW) of 95 ± 10.0 kg (1–1.5 years old) were randomly allocated to receive fresh cassava root containing HCN at 0, 300, 450, and 600 ppm according to a 4 × 4 Latin square design. Rice straw was the basal diet. The rhodanese enzyme was combined with concentrated feeds at a concentration of 1 mg/10⁴ ppm HCN. The fresh cassava root was cleaned to remove dirt and chopped into 3 to 5 mm sized pieces before being fed to the animals at their various levels. The total feed intake of beef cattle increased when fed with fresh cassava root (p < 0.05). The digestibility of crude protein (CP) was different among various fresh cassava root levels (p < 0.05). Ruminal ammonia-N levels were measured 4 hours after feeding, and the average concentration declined considerably in animals fed fresh cassava root at 300–600 ppm HCN (p < 0.05). Cyanide concentration in the rumen was linearly increased by 270.6% (p < 0.05) when it was supplemented with a high level of fresh cassava root. Blood urea-N concentration was altered and decreased when supplemented with fresh cassava root (p < 0.01). The blood thiocyanate concentration was altered by the levels of fresh cassava root and rhodanese enzyme, which ranged from 4.1 to 27.9 mg/dL (p < 0.01). Cattle given fresh cassava root showed no influence on total volatile fatty acid, acetic acid, or butyric acid concentrations in the rumen (p > 0.05). However, the concentration of propionic acid increased slightly (p < 0.05) 4 hours after feeding. Supplementing fresh cassava root up to 600 ppm HCN/day improved N absorption, retention, and the proportion of N retention to N intake (p < 0.05). Therefore, increasing the inclusion of fresh cassava root with a rhodanese enzyme addition improves total feed intake, CP digestibility, nitrogen utilization, blood thiocyanate, and propionate concentrations, which may remove HCN without harming animal health. Full article

Mitochondria are the key organelles of Fe–S cluster synthesis. They contain the enzyme cysteine desulfurase, a scaffold protein, iron and electron donors, and specific chaperons all required for the formation of Fe–S clusters. The newly formed cluster can be utilized by mitochondrial Fe–S protein synthesis or undergo further transformation. Mitochondrial Fe–S cluster biogenesis components are required in the cytosolic iron–sulfur cluster assembly machinery for cytosolic and nuclear cluster supplies. Clusters that are the key components of Fe–S proteins are vulnerable and prone to degradation whenever exposed to oxidative stress. However, once degraded, the Fe–S cluster can be resynthesized or repaired. It has been proposed that sulfurtransferases, rhodanese, and 3-mercaptopyruvate sulfurtransferase, responsible for sulfur transfer from donor to nucleophilic acceptor, are involved in the Fe–S cluster formation, maturation, or reconstitution. In the present paper, we attempt to sum up our knowledge on the involvement of sulfurtransferases not only in sulfur administration but also in the Fe–S cluster formation in mammals and yeasts, and on reconstitution-damaged cluster or restoration of enzyme’s attenuated activity. Full article

The use of cyanide-containing feed (HCN) is restricted because it causes prussic acid poisoning in animals. The objective of this study was to see how adding rhodanese enzyme to an HCN-containing diet affected gas dynamics, in vitro ruminal fermentation, HCN concentration reduction, and nutrient digestibility. A 3 × 4 factorial arrangement in a completely randomized design was used for the experiment. Factor A was the three levels of potassium cyanide (KCN) at 300, 450, and 600 ppm. Factor B was the four doses of rhodanese enzyme at 0, 0.65, 1, and 1.35 mg/10⁴ ppm KCN, respectively. At 96 h of incubation, gas production from an insoluble fraction (b), potential extent (omit gas) (a + b), and cumulative gas were similar between KCN additions of 300 to 450 ppm (p > 0.05), whereas increasing KCN to 600 ppm significantly decreased those kinetics of gas (p < 0.05). Supplementation of rhodanese enzymes at 1.0 to 1.35 mg/10⁴ ppm KCN enhanced cumulative gas when compared to the control group (p < 0.05). Increasing the dose of rhodanese up to 1.0 mg/10⁴ ppm KCN significantly increased the rate of ruminal HCN degradation efficiency (DE) by 70% (p < 0.05). However, no further between the two factors was detected on ruminal fermentation and in vitro digestibility (p > 0.05). The concentration of ammonia-nitrogen (NH₃-N) increased with increasing doses of KCN (p < 0.05), but remained unchanged with varying levels of rhodanese enzymes (p > 0.05). The in vitro dry matter digestibility (IVDMD) was suppressed when increasing doses of KCH were administered at 600 ppm, whereas supplementation of rhodanese enzymes at 1.0–1.35 mg/10⁴ ppm KCN enhanced IVDMD (p < 0.05). Increasing doses of KCN affected reduced total volatile fatty acids (TVFA) concentration, which was lowest when 600 ppm was added (p < 0.05). Nevertheless, the concentration of TVFAs increased when rhodanese enzymes were included by 1.0–1.35 mg/10⁴ ppm KCN (p < 0.05). Based on this study, it could be concluded that supplementation of rhodaneses enzyme at 1.0–1.35 mg/10⁴ ppm KCN could enhance cumulative gas, digestibility, and TVAF, as well as lowering ruminal HCN concentration. Full article

Hypertension and age are key risk factors for cardiovascular morbidity and mortality. Hydrogen sulfide (H₂S), a gaseous transmitter, contributes significantly to regulating arterial blood pressure and aging processes. This study evaluated the effects of hypertension and aging on the hepatic metabolism of sulfur-containing compounds, the activity of the enzymes involved in sulfur homeostasis, and the liver’s ability to generate H₂S. Livers isolated from 16- and 60-week-old normotensive Wistar Kyoto rats (WKY) and Spontaneously Hypertensive Rats (SHR) were used to evaluate gene expression using RT-PCR, and the activity of enzymes participating in H₂S metabolism, including thiosulfate sulfurtransferase (rhodanese; TST), cystathionine gamma-lyase (CTH), and 3-mercaptopyruvate sulfurtransferase (MPST). The levels of cysteine, cystine, reduced and oxidized glutathione were measured using RP-HPLC. SHR livers from both age groups showed a higher capacity to generate H₂S than livers from WKY. The gene expression and activity of enzymes involved in sulfur metabolism differed between WKY and SHR, and between the age groups. For example, 16-week-old SHR had significantly higher activity of TST than 16-week-old WKY. Furthermore, differences between younger and older WKY rats in the expression and/or activity of TST and MPST were present. In conclusion, our study shows that arterial hypertension and aging affect hepatic sulfur metabolism and H₂S production in rats. These findings pave the way for interventional studies evaluating a potential causal relation between liver sulfur metabolism, hypertension and aging. Full article

The heat-shock protein, Hsp60, is one of the most abundant proteins in Helicobacter pylori. Given its sequence homology to the Escherichia coli Hsp60 or GroEL, Hsp60 from H. pylori would be expected to function as a molecular chaperone in this organism. H. pylori is a type of bacteria that grows on the gastric epithelium, where the pH can fluctuate between neutral and 4.5, and the intracellular pH can be as low as 5.0. We previously showed that Hsp60 functions as a chaperone under acidic conditions. However, no reports have been made on the ability of Hsp60 to function as a molecular chaperone under other stressful conditions, such as heat stress or elevated temperatures. We report here that Hsp60 could suppress the heat-induced aggregation of the enzymes rhodanese, malate dehydrogenase, citrate synthase, and lactate dehydrogenase. Moreover, Hsp60 was found to have a potassium and magnesium-dependent ATPase activity that was stimulated at elevated temperatures. Although, Hsp60 was found to bind GTP, the hydrolysis of this nucleotide could not be observed. Our results show that Hsp60 from H. pylori can function as a molecular chaperone under conditions of heat stress. Full article

Urm1 (ubiquitin related modifier 1) is a molecular fossil in the class of ubiquitin-like proteins (UBLs). It encompasses characteristics of classical UBLs, such as ubiquitin or SUMO (small ubiquitin-related modifier), but also of bacterial sulfur-carrier proteins (SCP). Since its main function is to modify tRNA, Urm1 acts in a non-canonical manner. Uba4, the activating enzyme of Urm1, contains two domains: a classical E1-like domain (AD), which activates Urm1, and a rhodanese homology domain (RHD). This sulfurtransferase domain catalyzes the formation of a C-terminal thiocarboxylate on Urm1. Thiocarboxylated Urm1 is the sulfur donor for 5-methoxycarbonylmethyl-2-thiouridine (mcm⁵s²U), a chemical nucleotide modification at the wobble position in tRNA. This thio-modification is conserved in all domains of life and optimizes translation. The absence of Urm1 increases stress sensitivity in yeast triggered by defects in protein homeostasis, a hallmark of neurological defects in higher organisms. In contrast, elevated levels of tRNA modifying enzymes promote the appearance of certain types of cancer and the formation of metastasis. Here, we summarize recent findings on the unique features that place Urm1 at the intersection of UBL and SCP and make Urm1 an excellent model for studying the evolution of protein conjugation and sulfur-carrier systems. Full article

We have been studying the general aspects of the functions of H₂S and polysulfides, and the enzymes involved in their biosynthesis, for more than 20 years. Our aim has been to elucidate novel physiological and pathological functions of H₂S and polysulfides, and unravel the regulation of the enzymes involved in their biosynthesis, including cystathionine β-synthase (EC 4.2.1.22), cystathionine γ-lyase (EC 4.4.1.1), thiosulfate sulfurtransferase (rhodanese, EC 2.8.1.1), and 3-mercaptopyruvate sulfurtransferase (EC 2.8.1.2). Physiological and pathological functions, alternative biosynthetic processes, and additional functions of H₂S and polysulfides have been reported. Further, the structure and reaction mechanisms of related enzymes have also been reported. We expect this issue to advance scientific knowledge regarding the detailed functions of H₂S and polysulfides as well as the general properties and regulation of the enzymes involved in their metabolism. We would like to cover four topics: the physiological and pathological functions of H₂S and polysulfides, the mechanisms of the biosynthesis of H₂S and polysulfides, the properties of the biosynthetic enzymes, and the regulation of enzymatic activity. The knockout mouse technique is a useful tool to determine new physiological functions, especially those of H₂S and polysulfides. In the future, we shall take a closer look at symptoms in the human congenital deficiency of each enzyme. Further studies on the regulation of enzymatic activity by in vivo substances may be the key to finding new functions of H₂S and polysulfides. Full article