Detoxifying Cyanides Using Cyanase Enzyme Complexes Composed of Carbonic Anhydrase via Irreversible Covalent Bonds.

Cyanase is a possible solution to reduce the environmental impact of cyanide. However, the enzyme's dependence on HCO3- limits its industrial applications. To overcome this problem, carbonic anhydrase is utilized in this study. Three types of Catcher/Tag systems were introduced into the cyanase (psCYN) from Pseudomonas stutzeri and the carbonic anhydrase (hmCA) from Hydrogenovibrio marinus to construct enzyme complexes via irreversible covalent bonds. Initially, a cyanase complex with the aid of scaffolding proteins was designed. The results of cyanase complexes using scaffolding proteins were similar to or inferior to those of the two free enzymes. To address this, the two enzymes were manipulated to form a direct bioconjugation without the need for scaffolding proteins. The two enzymes forming a direct conjugation showed activity more than 2.5 times higher than that of cyanase alone. In conclusion, this outcome will contribute to solving problems related to residual cyanides in food and the environment.

Structural mechanism of Escherichia coli cyanase.

Cyanase plays a vital role in the detoxification of cyanate and supplies a continuous nitrogen source for soil microbes by converting cyanate to ammonia and carbon dioxide in a bicarbonate-dependent reaction. The structures of cyanase complexed with dianion inhibitors, in conjunction with biochemical studies, suggest putative binding sites for substrates. However, the substrate-recognition and reaction mechanisms of cyanase remain unclear. Here, crystal structures of cyanase from Escherichia coli were determined in the native form and in complexes with cyanate, bicarbonate and intermediates at 1.5-1.9 Šresolution using synchrotron X-rays and an X-ray free-electron laser. Cyanate and bicarbonate interact with the highly conserved Arg96, Ser122 and Ala123 in the active site. In the presence of a mixture of cyanate and bicarbonate, three different electron densities for intermediates were observed in the cyanase structures. Moreover, the observed electron density could explain the dynamics of the substrate or product. In addition to conformational changes in the substrate-binding pocket, dynamic movement of Leu151 was observed, which functions as a gate for the passage of substrates or products. These findings provide a structural mechanism for the substrate-binding and reaction process of cyanase.

LACC1 bridges NOS2 and polyamine metabolism in inflammatory macrophages.

The mammalian immune system uses various pattern recognition receptors to recognize invaders and host damage and transmits this information to downstream immunometabolic signalling outcomes. Laccase domain-containing 1 (LACC1) protein is an enzyme highly expressed in inflammatory macrophages and serves a central regulatory role in multiple inflammatory diseases such as inflammatory bowel diseases, arthritis and clearance of microbial infection1-4. However, the biochemical roles required for LACC1 functions remain largely undefined. Here we elucidated a shared biochemical function of LACC1 in mice and humans, converting L-citrulline to L-ornithine (L-Orn) and isocyanic acid and serving as a bridge between proinflammatory nitric oxide synthase (NOS2) and polyamine immunometabolism. We validated the genetic and mechanistic connections among NOS2, LACC1 and ornithine decarboxylase 1 (ODC1) in mouse models and bone marrow-derived macrophages infected by Salmonella enterica Typhimurium. Strikingly, LACC1 phenotypes required upstream NOS2 and downstream ODC1, and Lacc1-/- chemical complementation with its product L-Orn significantly restored wild-type activities. Our findings illuminate a previously unidentified pathway in inflammatory macrophages, explain why its deficiency may contribute to human inflammatory diseases and suggest that L-Orn could serve as a nutraceutical to ameliorate LACC1-associated immunological dysfunctions such as arthritis or inflammatory bowel disease.

Synergistic metabolism of carbon and nitrogen: Cyanate drives nitrogen cycle to conserve nitrogen in composting system.

In this study, HCO3- was used as a co-substrate for cyanate metabolism to investigate its effect on nitrogen cycle in composting. The results showed that the carbamate content in experimental group (T) with HCO3- added was higher than that in control group (CP) during cooling period. Actinobacteria and Proteobacteria were the dominant phyla for cyanate metabolism, and the process was mediated by cyanase gene (cynS). The cynS abundance was 16.6% higher in T than CP. In cooling period, the nitrification gene hao in T was 8.125% higher than CP. Denitrification genes narG, narH, nirK, norB, and nosZ were 25.64%, 35.33%, 45.93%, 36.62%, and 36.12% less than CP, respectively. The nitrogen fixation gene nifH in T was consistently higher than CP in the late composting period. Conclusively, cyanate metabolism drove the nitrogen cycle by promoting nitrification, nitrogen fixation, and inhibiting denitrification, which improved nitrogen retention and compost quality.

Comparison of quantification of selenocyanate and thiocyanate in cultured mammalian cells between HPLC-fluorescence detector and HPLC-inductively coupled plasma mass spectrometer.

The simultaneous detection of cyanide (CN), thiocyanate (SCN), and selenocyanate (SeCN) by a HPLC-fluorescence detector (FLD) with the post-column König reaction was recently reported. SCN and SeCN are also detectable by HPLC-inductively coupled plasma mass spectrometry (HPLC-ICP-MS) because sulfur and selenium can be detected, respectively, without any pre- or post-treatment. ICP-MS has high sensitivity for selenium and sulfur detection and is robust to sample matrices. In this study, we compared HPLC-FLD with the post-column König reaction and HPLC-ICP-MS in terms of SCN and SeCN detection sensitivity and linearity. The limit of detection (LOD) for SCN indicated that HPLC-FLD with the post-column König reaction was 354 times more sensitive than HPLC-ICP-MS. Likewise, the LOD for SeCN indicated that HPLC-FLD was 51 times more sensitive than HPLC-ICP-MS. These results demonstrated that HPLC-FLD was a more suitable technique for SeCN and SCN detection than HPLC-ICP-MS. We previously reported that SeCN was generated in selenite-exposed mammalian cells to detoxify excess selenite. HPLC-FLD with the post-column König reaction enabled good separation and detection for quantifying SCN and SeCN in mammalian cell lines exposed to selenite. The intracellular SCN and SeCN concentrations determined by this technique suggested differences in the metabolic capacity for selenite to form SeCN among the cell lines. In addition, since the amount of intracellular SCN and SeCN were significantly decreased by pretreatment of myeloperoxidase (MPO) inhibitors, SCN and SeCN were resulted from the interaction of sulfur and selenium with endogenous CN, respectively, generated with MPO.

Detecting the nature and solving the crystal structure of a contaminant protein from an opportunistic pathogen.

The unintentional crystallization of contaminant proteins in the place of target recombinant proteins is sporadically reported, despite the availability of stringent expression/purification protocols and of software for the detection of contaminants. Typically, the contaminant protein originates from the expression organism (for example Escherichia coli), but in rare circumstances contaminants from different sources have been reported. Here, a case of contamination from a Serratia bacterial strain that occurred while attempting to crystallize an unrelated protein from Burkholderia pseudomallei (overexpressed in E. coli) is presented. The contamination led to the unintended crystallization and structure analysis of a cyanase hydratase from a bacterial strain of the Serratia genus, an opportunistic enterobacterium that grows under conditions similar to those of E. coli and that is found in a variety of habitats, including the laboratory environment. In this context, the procedures that were adopted to identify the contaminant based on crystallographic data only are presented and the crystal structure of Serrata spp. cyanase hydratase is briefly discussed.

Effect of gut microflora on nutritional availability of selenium.

Since selenium (Se) is an essential mineral, animals must be able to metabolize the various selenocompounds in meat, fish and vegetables. It is unclear how animals, including humans, utilize selenocompound efficiently, but we hypothesized that gut microflora might contribute to these processes. In this study, we revealed that Se-methylselenocysteine and selenocyanate were metabolized to selenomethionine (SeMet) by intestinal microflora, suggesting selenocompounds might be metabolized to SeMet, which can be used by the host organism. The major urinary selenosugar, 1ß-methylseleno-N-acetyl-d-galactosamine, was utilized less in microflora-suppressed than healthy rats, suggesting that this sugar can be transformed to a nutritionally available form by gut microflora in animals with a healthy microbiota. We concluded that, in rats at least, gut microflora has a role in the metabolism of Se in the host animal, and this finding might be worth investigating in humans.

Identification of the biliary selenium metabolite and the biological significance of selenium enterohepatic circulation.

Although selenium (Se) is mainly excreted in urine, it has been reported that an unknown Se metabolite is excreted in bile. When we administered selenomethionine (SeMet), selenocyanate or selenite to rats, a common biliary selenometabolite was detected 10 min after administration. The amount of the selenometabolite originating from SeMet was less than that originating from the two inorganic Se compounds, selenocyanate and selenite, suggesting that the transformation from the methylated organic selenocompound, i.e., SeMet, was less efficient than that from the inorganic Se compounds. The common biliary selenometabolite was concretely identified as selenodiglutathione (GSSeSG) by two types of mass spectrometry, i.e., LC-inductively coupled mass spectrometry (ICP-MS) and LC-ESI-Q/TOF. The bile-drained rats had lower urinary Se levels than the sham-operated rats. In addition, the Se amounts in urine plus bile of the bile-drained rats were comparable to the Se amount in the urine of the sham-operated rats. These results suggest that the biliary selenometabolite, GSSeSG, was reabsorbed in the gut and finally excreted in urine. Enterohepatic circulation occurs to maintain Se status in the body.

Microbial (Enzymatic) Degradation of Cyanide to Produce Pterins as Cofactors.

Cyanide is one of the most poisonous substances in the environment, which may have originated from natural and anthropogenic sources. There are many enzymes produced by microorganisms which can degrade and utilize cyanide. The major byproducts of cyanide degradation are alanine, glutamic acid, alpha-amino-butyric acid, beta-cyanoalanine, pterin etc. These products have many pharmaceutical and medicinal applications. For the degradation of cyanide, microbes produce necessary cofactors which catalyze the degradation pathways. Pterin is one of the cofactors for cyanide degradation. There are many pathways involved for the degradation of cyanide, cyanate, and thiocyanate. Some of the microorganisms possess resistance to cyanide, since they have developed adaptive alternative pathways for the production of ATP by utilization of cyanide as carbon and nitrogen sources. In this review, we summarized different enzymes, their mechanisms, and corresponding pathways for the degradation of cyanide and production of pterins during cyanide degradation. We aim to enlighten different types of pterin, its classification, and biological significance through this literature review.

Could cyanogenic glycoside rich diet cause increased risk for carbamylation-induced protein damage in individuals with chronic inflammatory diseases?

Cyanogenic glycosides are found in a diverse group of plants and are metabolized into thiocyanate by the intestines and liver. Conversion of plant derived thiocyanates into cyanide and isocyanic acid occurs by the activity of neutrophil-derived enzyme myeloperoxidase. Therefore, increased intake of cyanogenic glycoside rich plant based diet may lead to increased isocyanic acid induced protein carbamylation in chronic inflammatory states (increased myeloperoxidase activity). As there is a close relationship between non-enzymatic post-translational modification and protein function, carbamylation induced structural changes also affect the functions of proteins. Carbamylation induced structural alterations of proteins have recently drawn a great attention in the current literature, especially regarding the alterations of proteins with long half-life such as type I collagen, elastin, α-crystallin. We hypothesize that a plant-based natural diet, rich in cyanogenic glycosides, may have unintended consequences on native protein structure/function in individuals with chronic inflammatory diseases such as chronic kidney and rheumatological diseases because of the higher rate of transformation of plant derived thiocyanates into isocyanic acid by the increased activity of neutrophil-derived enzyme myeloperoxidase. Regulation of myeloperoxidase activity or moderation of cyanogenic glycoside rich diet might be important in the prevention/modulation of dangerous protein carbamylation process, especially in this patient group.

Tailored cell sheet engineering using microstereolithography and electrochemical cell transfer.

Postoperative adhesion and occlusion remain a serious issue associated with various surgeries, including endoscopic surgery, in which proliferated fibrous tissues stick to adjacent tissues and often cause severe complications. Cell sheet engineering has emerged as an effective approach not only for cell transplantation but also for the treatment of postoperative adhesion and occlusion. However, as the tissues in the body, such as middle ear and small intestine, and typical operative sites are non-flat and spatially complicated, tailored cell sheets with three-dimensional (3D) configurations may lead to widespread use of this approach. In the present study, we used microstereolithography, biocompatible gold plating, and electrochemical cell detachment to achieve this purpose. Various objects with dimensions ranging from millimeter- to micrometer-scale were fabricated with photocurable resin using lab-made equipment for microstereolithography. To coat the fabricated objects with a thin gold layer, conventional cyanide-based gold plating was unusable because it severely damaged almost all cells. Electroless non-cyanide gold plating we prepared was cytocompatible and suitable for electrochemical cell detachment. Cell sheets on the gold-plated substrate could be directly transplanted into a mouse intraperitoneally using electrochemical cell detachment. We further demonstrated that cell sheets grown on gold-coated 3D objects were rapidly detached along with the desorption of electroactive-oligopeptide monolayer and transferred to a surrounding hydrogel. This approach may provide a promising strategy to prepare and directly transplant tailor-made cell sheets with suitable configurations.

Cyanate Assimilation by the Alkaliphilic Cyanide-Degrading Bacterium Pseudomonas pseudoalcaligenes CECT5344: Mutational Analysis of the cyn Gene Cluster.

The alkaliphilic bacterium Pseudomonas pseudoalcaligenes CECT5344 can grow with cyanate, cyanide, or cyanide-containing industrial residues as the sole nitrogen source, but the assimilation of cyanide and cyanate takes place through independent pathways. Therefore, cyanide degradation involves a chemical reaction between cyanide and oxaloacetate to form a nitrile that is hydrolyzed to ammonium by the nitrilase NitC, whereas cyanate assimilation requires a cyanase that catalyzes cyanate decomposition to ammonium and carbon dioxide. The P. pseudoalcaligenes CECT5344 cynFABDS gene cluster codes for the putative transcriptional regulator CynF, the ABC-type cyanate transporter CynABD, and the cyanase CynS. In this study, transcriptional analysis revealed that the structural cynABDS genes constitute a single transcriptional unit, which was induced by cyanate and repressed by ammonium. Mutational characterization of the cyn genes indicated that CynF was essential for cynABDS gene expression and that nitrate/nitrite transporters may be involved in cyanate uptake, in addition to the CynABD transport system. Biodegradation of hazardous jewelry wastewater containing high amounts of cyanide and metals was achieved in a batch reactor operating at an alkaline pH after chemical treatment with hydrogen peroxide to oxidize cyanide to cyanate.

Interaction of cyanate uptake by rice seedlings with nitrate assimilation: gene expression analysis.

Cyanate (CNO-) has been produced in the environment through either natural or anthropogenic sources. However, due to industrialization, it has been led more over-loads. In this study, interaction of CNO- uptake by rice seedlings with nitrate assimilation was investigated using gene expression analysis after an acute phytotoxicity assay. Our results showed that CNO- exposure caused inhibition on relative growth rates of plants. CNO- analysis demonstrated that rice seedlings had higher potential for CNO- uptake and the removal rates showed a zero-order kinetic. PCR analysis exposed that OsCYN transcript was not significantly induced by CNO- treatments in rice tissues and CNO- exposure also repressed gene expression of the collaborative enzyme carbonic anhydrase (CA), suggesting that assimilation of CNO- initiated by the enzyme cyanase (CYN) in rice seedlings was an enzyme-limitation reaction. Gene expression of other enzymes involved in nitrate metabolism was tissue-specific under CNO- exposure, suggesting that rice seedlings were able to trigger its intrinsic regulative and responsive mechanisms to cope up with uneven N conditions. Significant upregulation of three OsGDH isogenes, except for OsGDH1 in roots, was detected in both rice materials with enhancing CNO- concentrations, suggesting that GDH may play a primary role to maintain the balance of C and N in plants under CNO- exposure. In conclusion, because the innate pool of CYN activity was non-sufficient to degrade exogenous CNO- by rice seedlings, CNO-derived ammonium only can serve as a supporting N source to support growth of rice seedling under non-effective doses of CNO- exposure.

Clinical and pathophysiologic relevance of autoantibodies in rheumatoid arthritis.

Rheumatoid arthritis (RA) is an autoimmune/inflammatory disease affecting 0.5 to 1% of adults worldwide and frequently leads to joint destruction and disability. Early diagnosis and early and effective therapy may prevent joint damage and lead to better long-term results. Therefore, reliable biomarkers and outcome measures are needed. Refinement of the understanding of molecular pathways involved in disease pathogenesis have been achieved by combining knowledge on RA-associated genes, environmental factors and the presence of serological elements. The presence of autoantibodies is a distinctive feature of RA. Rheumatoid Factor and Anti-Citrullinated Protein Antibodies are the two most remarkable autoantibodies in RA and provide different clinical and pathophysiological information. They precede the onset of disease symptoms and predict a more severe disease course, indicating a pathogenetic role in RA. Therefore, they promote a more accurate prognosis and contribute for a better disease management. Several RA-associated autoantibody systems have been identified: Anti-Carbamylated Antibodies, Anti-BRAF, Anti-Acetylated, Anti-PAD4 antibodies and others. Hopefully, the characterization of a comprehensive array of novel autoantibody systems in RA will provide unique pathogenic insights of relevance for the development of diagnostic and prognostic approaches compatible with an effective personalized medicine.

Cyanate and urea are substrates for nitrification by Thaumarchaeota in the marine environment.

Ammonia-oxidizing archaea of the phylum Thaumarchaeota are among the most abundant marine microorganisms1. These organisms thrive in the oceans despite ammonium being present at low nanomolar concentrations2,3. Some Thaumarchaeota isolates have been shown to utilize urea and cyanate as energy and N sources through intracellular conversion to ammonium4-6. Yet, it is unclear whether patterns observed in culture extend to marine Thaumarchaeota, and whether Thaumarchaeota in the ocean directly utilize urea and cyanate or rely on co-occurring microorganisms to break these substrates down to ammonium. Urea utilization has been reported for marine ammonia-oxidizing communities7-10, but no evidence of cyanate utilization exists for marine ammonia oxidizers. Here, we demonstrate that in the Gulf of Mexico, Thaumarchaeota use urea and cyanate both directly and indirectly as energy and N sources. We observed substantial and linear rates of nitrite production from urea and cyanate additions, which often persisted even when ammonium was added to micromolar concentrations. Furthermore, single-cell analysis revealed that the Thaumarchaeota incorporated ammonium-, urea- and cyanate-derived N at significantly higher rates than most other microorganisms. Yet, no cyanases were detected in thaumarchaeal genomic data from the Gulf of Mexico. Therefore, we tested cyanate utilization in Nitrosopumilus maritimus, which also lacks a canonical cyanase, and showed that cyanate was oxidized to nitrite. Our findings demonstrate that marine Thaumarchaeota can use urea and cyanate as both an energy and N source. On the basis of these results, we hypothesize that urea and cyanate are substrates for ammonia-oxidizing Thaumarchaeota throughout the ocean.

Cyanase-independent utilization of cyanate as a nitrogen source in ascomycete yeasts.

The occurrence of putative cyanases (EC 4.2.1.104) in the genomes of yeasts belonging to the ascomycete sub-phyla Saccharomycotina (budding yeasts) and Taphrinomycotina (fission yeasts) was investigated. Predicted gene products displaying significant sequence similarity to previously characterized cyanases were identified in the genomes of the budding yeast Lipomyces starkeyi and the fission yeasts Protomyces lactucaedebilis, Saitoella complicata and Taphrina deformans. Li. starkeyi and Sai. complicata were further tested for their ability to utilize cyanate as a nitrogen source. However, neither species displayed significant growth when cyanate was provided as the sole nitrogen source. Cyanate utilization assays of 15 yeast species whose genomes lack detectable cyanase homologs unexpectedly resulted in consistently strong growth in six species as well as variable growth in an additional three species. The present study represents the first known report of cyanase-independent utilization of cyanate as a nitrogen source in ascomycete yeasts. Implications of cyanate utilization for the ecological niches occupied by ascomycete yeasts are discussed.

Utilization of urea and cyanate in waters overlying and within the eastern tropical north Pacific oxygen deficient zone.

In marine oxygen deficient zones (ODZs), which contribute up to half of marine N loss, microbes use nitrogen (N) for assimilatory and dissimilatory processes. Here, we examine N utilization above and within the ODZ of the Eastern Tropical North Pacific Ocean, focusing on distribution, uptake and genes for the utilization of two simple organic N compounds, urea and cyanate. Ammonium, urea and cyanate concentrations generally peaked in the oxycline while uptake rates were highest in the surface. Within the ODZ, concentrations were lower, but urea N and C and cyanate C were taken up. All identified autotrophs had an N assimilation pathway that did not require external ammonium: ODZ Prochlorococcus possessed genes to assimilate nitrate, nitrite and urea; nitrite oxidizers (Nitrospina) possessed genes to assimilate nitrite, urea and cyanate; anammox bacteria (Scalindua) possessed genes to utilize cyanate; and ammonia-oxidizing Thaumarchaeota possessed genes to utilize urea. Urease genes were present in 20% of microbes, including SAR11, suggesting the urea utilization capacity was widespread. In the ODZ core, cyanate genes were largely (∼95%) associated with Scalindua, suggesting that, within this ODZ, cyanate N is primarily used for N loss via anammox (cyanammox), and that anammox does not require ammonium for N loss.

[Protein carbamylation: what it is and why it concerns nephrologists].

Spontaneous urea dissociation in water solution is a prominent source of protein carbamylation in our body. Protein carbamylation is a well-known phenomenon since early seventies. Some years ago, much interest in the diagnostic power of carbamylated protein arouse. Recently the target of the researches focused on its potential cardiovascular pathogenicity. Some authors claimed that this could be a reason for higher cardiovascular mortality in uremic patients. Nutritional therapy, amino acids supplementation and intensive dialysis regimen are some of the therapeutic tools tested to lower the carbamylation burst in this population.