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.

Development of reusable electrochemiluminescence sensing microchip for detection of vomitoxin.

In this work, a reusable DNA sensing microchip was developed for detection of vomitoxin (deoxynivalenol, DON) in sorghum using Cd-based core-shell CdSe@CdS quantum dots (QDs) as promising electrochemiluminescence (ECL) emitter. The size-adjustable aqueous phase CdSe@CdS QDs were prepared through homogeneous method, exhibiting strong cathodic ECL emission with a central wavelength of 520 nm in S2O82- coreactant. And gold nanoparticles-modified iron cobalt cyanide hydrate (Fe-Co-Au) was introduced as an accelerator to amplify the ECL signal. ECL signal was quenched after the formation of a double-stranded (dsDNA) S1-S2 by generating an electron transfer system between the emitter and ferrocene (Fc), which are modified on the aptamer (ssDNA S1) and its complement sequence (ssDNA S2), respectively. When the target DON is presence, the aptamer ssDNA S1 will bind to the DON and trigger the unbinding of double strands DNA and the release of the ssDNA S2, thus the signal can be generated. This approach offers a feasible method for the detection of DON within the range of 1 ng/mL to 200 ng/mL.

Inhibition of the thioredoxin system for radiosensitization therapy of cancer.

Radiotherapy (RT) stands as a cornerstone in the clinical armamentarium against various cancers due to its proven efficacy. However, the intrinsic radiation resistance exhibited by cancer cells, coupled with the adverse effects of RT on normal tissues, often compromises its therapeutic potential and leads to unwanted side effects. This comprehensive review aims to consolidate our understanding of how radiosensitizers inhibit the thioredoxin (Trx) system in cellular contexts. Notable radiosensitizers, including gold nanoparticles (GNPs), gold triethylphosphine cyanide ([Au(SCN) (PEt3)]), auranofin, ceria nanoparticles (CONPs), curcumin and its derivatives, piperlongamide, indolequinone derivatives, micheliolide, motexafin gadolinium, and ethane selenide selenidazole derivatives (SeDs), are meticulously elucidated in terms of their applications in radiotherapy. In this review, the sensitization mechanisms and the current research progress of these radiosensitizers are discussed in detail, with the overall aim of providing valuable insights for the judicious application of Trx system inhibitors in the field of cancer radiosensitization therapy.

O-phthalaldehyde based quantification of polysaccharide modification in conjugate vaccines.

Polysaccharide-based vaccines cannot stimulate long-lasting immune response in infants due to their inability to elicit a T-cell-dependent immune response. This has been addressed using conjugation technology, where conjugates were produced by coupling a carrier protein to polysaccharides using different conjugation chemistries, such as cyanylation, reductive amination, ethylene diamine reaction, and others. Many glycoconjugate vaccines that are manufactured using different conjugation technologies are already in the market for neonates, infants and young children (e.g., Haemophilus influenzae type-b, Streptococcus pneumoniae and Neisseria meningitidis vaccines), and all of them elicit a T-cell dependent immune response. To manufacture glycoconjugate vaccines, the capsular polysaccharide is first activated by converting its hydroxyl groups to aldehyde-, cyanyl-, or cyanate ester groups, depending on the conjugation chemistry selected. The oxidized and reduced aldehyde functional groups of the polysaccharides are subsequently reacted with the amino groups of carrier protein by reductive amination to form a stable amide bond. In CDAP-based conjugation, the polysaccharide -OH groups are activated to form cyanyl-, or cyanate ester groups to react with the amino groups of carrier protein and forms an isourea bond. Understanding the extent of polysaccharide activation/modification is essential since it directly influences the molar mass of the conjugate, its stability, and the immunogenicity of the product. Reported methods are available to estimate the aldehyde groups of polysaccharides generated by reductive amination. However, no method is available to quantify the cyanyl or cyanate ester (-OCN) groups generated by cyanylation with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP). We report a novel strategy using an O-phthalaldehyde (OPA) derivatization process followed by size-exclusion chromatography (SEC) high-performance liquid chromatography (HPLC) separation and UV detection. The cyanate ester groups on the activated polysaccharide directly reveal the extent of polysaccharide activation/modification and the residual activated groups in the purified conjugates. This method would be useful for conjugate vaccine manufacturing using CDAP chemistry.

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.

Design, Synthesis and Biological Activities of (Thio)Urea Benzothiazole Derivatives.

(Thio)ureas ((T)Us) and benzothiazoles (BTs) each have demonstrated to have a great variety of biological activities. When these groups come together, the 2-(thio)ureabenzothizoles [(T)UBTs] are formed, improving the physicochemical as well as the biological properties, making these compounds very interesting in medicinal chemistry. Frentizole, bentaluron and methabenzthiazuron are examples of UBTs used for treatment of rheumatoid arthritis and as wood preservatives and herbicides in winter corn crops, respectively. With this antecedent, we recently reported a bibliographic review about the synthesis of this class of compounds, from the reaction of substituted 2-aminobenzothiazoles (ABTs) with iso(thio)cyanates, (thio)phosgenes, (thio)carbamoyl chlorides, 1,1'-(thio)carbonyldiimidazoles, and carbon disulfide. Herein, we prepared a bibliographic review about those features of design, chemical synthesis, and biological activities relating to (T)UBTs as potential therapeutic agents. This review is about synthetic methodologies generated from 1968 to the present day, highlighting the focus to transform (T)UBTs to compounds containing a range substituents, as illustrated with 37 schemes and 11 figures and concluded with 148 references. In this topic, the scientists dedicated to medicinal chemistry and pharmaceutical industry will find useful information for the design and synthesis of this interesting group of compounds with the aim of repurposing these compounds.

Potential interference of in vitro carbamylation on C-reactive protein laboratory measurement.

Carbamylation is a nonenzymatic post-translational modification observed during the reaction between cyanate and amino acids and/or proteins that may occur during some pathologies such as chronic kidney disease. Evidence suggests that carbamylation may interfere with the quantification of some analytes measured using immunoturbidimetric assays. C-reactive protein (CRP) is an inflammatory response protein that is commonly quantified through immunoturbidimetry in clinical laboratories. Because the presence of modified proteins in serum can lead to impaired quantification, this study aimed to verify the impact of in vitro carbamylation on the measurement of CRP in a CRP standard solution and serum pool. The samples were incubated with 150 nM, 150 µM, or 150 mM potassium cyanate (KOCN) or 20, 100, or 500 mg/dL urea at 37 °C for 24 h. CRP concentrations were measured using an immunoturbidimetric assay. The results showed a 61%-72% decrease in the CRP detection rate after incubation with KOCN. Incubation with urea resulted in a 0.7%-8% lower CRP detection rate. The results of this study indicate that high concentrations of cyanate can lead to falsely decreased CRP levels, as measured by immunoturbidimetry.

Synthesis and antitumor activity of some cholesterol-based selenocyanate compounds.

The introduction of selenium-containing functional groups into steroids to study the biological activities of related derivatives is rarely reported in the literature. In the present study, using cholesterol as raw material, four cholesterol-3-selenocyanoates and eight B-norcholesterol selenocyanate derivatives were synthesized, respectively. The structures of the compounds were characterized by NMR and MS. The results of the in vitro antiproliferative activity test showed that the cholesterol-3-selenocyanoate derivatives did not exhibit obvious inhibitory on the tested tumor cell lines. However, the B-norcholesterol selenocyanate derivatives obtained by structural modification of cholesterol showed good inhibitory activity against the proliferation of tumor cell. Among them, compounds 9b-c, 9f and 12 showed similar inhibitory activity against tested tumor cells as positive control 2-methoxyestradiol, and better than Abiraterone. At the same time, these B-norcholesterol selenocyanate derivatives displayed a strong selective inhibitory against Sk-Ov-3 cell line. Except for compound 9g, the IC50 value of all B-norcholesterol selenocyanate compounds against Sk-Ov-3 cells was less than 10 µM, and compound 9d was 3.4 µM. In addition, Annexin V-FITC/PI double staining was used to analyze the cell death mechanism. The results showed that compound 9c could induce Sk-Ov-3 cells to enter programmed apoptosis in a dose-dependent manner. Furthermore, the in vivo antitumor experiments of compound 9f against zebrafish xenograft tumor showed that 9f displayed obvious inhibitory effect on the growth of human cervical cancer (HeLa) xenograft tumor in zebrafish. Our results provide new thinking for the study of such compounds as new antitumor drugs.

Biotinylated selenocyanates: Potent and selective cytostatic agents.

Most of the currently available cytotoxic agents for tackling cancer are devoid of selectivity, thus causing severe side-effects. This situation stimulated us to develop new antiproliferative agents with enhanced affinity towards tumour cells. We focused our attention on novel chalcogen-containing compounds (thiosemicarbazones, disulfides, selenoureas, thio- and selenocyanates), and particularly on selenium derivatives, as it has been documented that this kind of compounds might act as prodrugs releasing selenium-based reactive species on tumour cells. Particularly interesting in terms of potency and selectivity was a pharmacophore comprised by a selenocyanato-alkyl fragment connected to a p-phenylenediamine residue, where the nature of the second amino moiety (free, Boc-protected, enamine-protected) provided a wide variety of antiproliferative activities, ranging from the low micromolar to the nanomolar values. The optimized structure was in turn conjugated through a peptide linkage with biotin (vitamin B7), a cellular growth promoter, whose receptor is overexpressed in numerous cancer cells; the purpose was to develop a selective vector towards malignant cells. Such biotinylated derivative behaved as a very strong antiproliferative agent, achieving GI50 values in the low nM range for most of the tested cancer cells; moreover, it was featured with an outstanding selectivity, with GI50 > 100 µM against human fibroblasts. Mechanistic studies on the mode of inhibition of the biotinylated selenocyanate revealed (Annexin-V assay) a remarkable increase in the number of apoptotic cells compared to the control experiment; moreover, depolarization of the mitochondrial membrane was detected by flow cytometry analysis, and with fluorescent microscopy, what supports the apoptotic cell death. Prior to the apoptotic events, cytostatic effects were observed against SW1573 cells using label-free cell-living imaging; therefore, tumour cell division was prevented. Multidrug resistant cell lines exhibited a reduced sensitivity towards the biotinylated selenocyanate, probably due to its P-gp-mediated efflux. Remarkably, antiproliferative levels could be restored by co-administration with tariquidar, a P-gp inhibitor; this approach can, therefore, overcome multidrug resistance mediated by the P-gp efflux system.

New Method to Biomonitor Workers Exposed to 1,6-Hexamethylene Diisocyanate.

Isocyanates such as 1,6-hexamethylene diisocyanate (HDI), 4,4'-methylenediphenyl diisocyanate, and toluene diisocyanate are highly reactive compounds that have a variety of commercial applications, including manufacturing polyurethane foam, elastomers, paints, adhesives, coatings, insecticides, and many other products. Their primary route of occupational exposure is through inhalation. Due to their high chemical reactivity, they are toxic and have adverse effects at the cellular and subcellular levels, leading to irritative and immunological reactions associated with lung disease. High concentrations of isocyanates are strong respiratory irritants. Bronchial sensitization and asthma are among the major adverse clinical reactions associated with low-level chronic exposure to isocyanates. Albumin adducts have been linked to the mechanism of occupational asthma caused by isocyanates. Isocyanates react in vivo with albumin, which is recognized by the immune system. Albumin adducts of isocyanates trigger immune responses and are probably the antigenic basis for isocyanate asthma. Sensitization to isocyanates is the main pathway for adverse health effects. Therefore, markers for the biologically effective dose such as albumin adducts of HDI are needed. A new isocyanate adduct of HDI with lysine─Nε-[(6-amino-hexyl-amino)carbonyl]-lysine (HDI-Lys)─was synthesized and characterized by 1H-NMR, 13C-NMR, and mass spectrometry (MS). Appropriate internal standards─HDI-Lys-4,4'-5,5'-d4 (HDI-d4-Lys) and Nε-[(7-amino-heptyl-amino)carbonyl]-lysine (Hep-Lys)─were synthesized to establish a LC-MS/MS method for the analysis of HDI adducts in in vitro modified albumin and in workers. The presence of HDI-Lys was found after pronase digestion of albumin and confirmed by two independent chromatographic approaches: with a C8 reversed-phase column and with a hydrophilic interaction liquid chromatography column. Quantification was performed with positive electrospray ionization (ESI)-MS. The adduct peak found in vivo was confirmed with the less sensitive negative ESI-MS. In summary, these are new compounds and methods to determine isocyanate-specific adducts with albumin in workers exposed to HDI.

Synthesis of Spiro[5.5]trienones- and Spiro[4.5]trienones-Fused Selenocyanates via Electrophilic Selenocyanogen Cyclization and Dearomative Spirocyclization.

A practical strategy for the synthesis of spiro[5.5]trienones-fused selenocyanates and spiro[4.5]trienones-fused selenocyanates through electrophilic selenocyanogen cyclization and dearomative spirocyclization is reported. This approach was conducted under mild conditions with broad substrate scope and good functional group tolerance. The utility of this procedure is exhibited in the late-stage functionalization of nature product and drug molecules.

Direct Access to Thiocyano-Thioesters from Cyclic Thioacetals via Photoredox Catalysis: An Introduction of Two Functional Groups in One Pot.

The cyanation of organic compounds is an important synthetic transformation and mainly relies on a toxic CN source. Undeniably, thiocyanate salt has emerged as a very mild and environmentally benign CN source, yet its synthetic utility for cyanation is highly limited to very few types of organic compounds. Herein, we report the direct cyanation of cyclic thioacetals for accessing compounds with two different functional groups (thiocyano-thioesters) in one pot using sodium thiocyanate via photoredox catalysis. The protocol has been further extended for the direct cyanation of disulfides and diselenide to access aryl thiocyanates and aryl selenocyanate. A plausible mechanism has been proposed based on a series of control experiments, cyclic voltammetry and Stern-Volmer studies.

Synthesis and Biological Importance of 2-(thio)ureabenzothiazoles.

The (thio)urea and benzothiazole (BT) derivatives have been shown to have a broad spectrum of biological activities. These groups, when bonded, result in the 2-(thio)ureabenzothizoles (TBT and UBT), which could favor the physicochemical and biological properties. UBTs and TBTs are compounds of great importance in medicinal chemistry. For instance, Frentizole is a UBT derivative used for the treatment of rheumatoid arthritis and systemic lupus erythematosus. The UBTs Bentaluron and Bethabenthiazuron are commercial fungicides used as wood preservatives and herbicides in winter corn crops. On these bases, we prepared this bibliography review, which covers chemical aspects of UBTs and TBTs as potential therapeutic agents as well as their studies on the mechanisms of a variety of pharmacological activities. This work covers synthetic methodologies from 1935 to nowadays, highlighting the most recent approaches to afford UBTs and TBTs with a variety of substituents as illustrated in 42 schemes and 13 figures and concluded with 187 references. In addition, this interesting review is designed on chemical reactions of 2-aminobenzothiazoles (2ABTs) with (thio)phosgenes, iso(thio)cyanates, 1,1'-(thio)carbonyldiimidazoles [(T)CDI]s, (thio)carbamoyl chlorides, and carbon disulfide. This topic will provide information of utility for medicinal chemists dedicated to the design and synthesis of this class of compounds to be tested with respect to their biological activities and be proposed as new pharmacophores.

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.

Nickel-Catalyzed Urea Electrolysis: From Nitrite and Cyanate as Major Products to Nitrogen Evolution.

The electrochemical urea oxidation reaction (UOR) to N2 represents an efficient route to simultaneous nitrogen removal from N-enriched waste and production of renewable fuels at the cathode. However, the overoxidation of urea to NOx - usually dominates over its oxidation to N2 at Ni(OH)2 -based anodes. Furthermore, detailed reaction mechanisms of UOR remain unclear, hindering the rational catalyst design. We found that UOR to NOx - on Ni(OH)2 is accompanied by the formation of near stoichiometric amount of cyanate (NCO- ), which enabled the elucidation of UOR mechanisms. Based on our experimental and computational findings, we show that the formation of NOx - and N2 follows two distinct vacancy-dependent pathways. We also demonstrate that the reaction selectivity can be steered towards N2 formation by altering the composition of the catalyst, e.g., doping the catalyst with copper (Ni0.8 Cu0.2 (OH)2 ) increases the faradaic efficiency of N2 from 30 % to 55 %.

Thermal desorption bridged the gap between dielectric barrier discharge ionization and dried plasma spot samples for sensitive and rapid detection of fentanyl analogs in mass spectrometry.

The urgent threat of new psychoactive substances worldwide promotes rapid detection and identification demand for public security. Ambient ionization mass spectrometry (AIMS) has become mainstream among various detection techniques. Still, scant successful applications have been fulfilled toward dried blood spot (DBS) or plasma spot (DPS) as an easy-to-implement sampling format in AIMS. This work bridged the gap between dielectric barrier discharge ionization mass spectrometry (DBDI-MS) and DPS/DBS samples by thermal desorption (TD) assistance. It made the impossible mission of direct DBDI-MS measurement on DPS/DBS samples containing fentanyl analogs (FTNs) possible. Guided by finite element simulations and a customized three-dimensional printing interface, we constructed a semi-covered flat-TD surface for sufficient desorption and ionization of FTNs from DPS/DBS samples without any sample pretreatment or sample separation. We successfully quantified eight FTNs in DPS samples using deuterated fentanyl as internal standard by triple quadrupole tandem mass spectrometry (MS/MS) and proved its practical applicability in the fentanyl-exposed rat plasma samples. This DBDI-TD-MS method also fits well with DBS samples, and it only took 20 s to analyze each sample. Further, based on summarized fragmentation characteristics of twenty FTNs, we established a backbone alerting ion-guided screening rule for suspect screening of FTNs in DPS samples via quadrupole time-of-flight MS/MS and built a chemometric approach for convenient mutual verification of screening "unknown" artificial samples. We hope this ideal DBDI-TD-MS method finds its valuable role in national security, doping control, and public health for routine large-scale analysis or on-site detection.

Non-Native Anionic Ligand Binding and Reactivity in Engineered Variants of the Fe(II)- and α-Ketoglutarate-Dependent Oxygenase, SadA.

Mononuclear non-heme Fe(II)- and α-ketoglutarate-dependent oxygenases (FeDOs) catalyze a site-selective C-H hydroxylation. Variants of these enzymes in which a conserved Asp/Glu residue in the Fe(II)-binding facial triad is replaced by Ala/Gly can, in some cases, bind various anionic ligands and catalyze non-native chlorination and bromination reactions. In this study, we explore the binding of different anions to an FeDO facial triad variant, SadX, and the effects of that binding on HO• vs X• rebound. We establish not only that chloride and bromide enable non-native halogenation reactions but also that all anions investigated, including azide, cyanate, formate, and fluoride, significantly accelerate and influence the site selectivity of SadX hydroxylation catalysis. Azide and cyanate also lead to the formation of products resulting from N3•, NCO•, and OCN• rebound. While fluoride rebound is not observed, the rate acceleration provided by this ligand leads us to calculate barriers for HO• and F• rebound from a putative Fe(III)(OH)(F) intermediate. These calculations suggest that the lack of fluorination is due to the relative barriers of the HO• and F• rebound transition states rather than an inaccessible barrier for F• rebound. Together, these results improve our understanding of the FeDO facial triad variant tolerance of different anionic ligands, their ability to promote rebound involving these ligands, and inherent rebound preferences relative to HO• that will aid efforts to develop non-native catalysis using these enzymes.

Selenocyanate derived Se-incorporation into the nitrogenase Fe protein cluster.

The nitrogenase Fe protein mediates ATP-dependent electron transfer to the nitrogenase MoFe protein during nitrogen fixation, in addition to catalyzing MoFe protein-independent substrate (CO2) reduction and facilitating MoFe protein metallocluster biosynthesis. The precise role(s) of the Fe protein Fe4S4 cluster in some of these processes remains ill-defined. Herein, we report crystallographic data demonstrating ATP-dependent chalcogenide exchange at the Fe4S4 cluster of the nitrogenase Fe protein when potassium selenocyanate is used as the selenium source, an unexpected result as the Fe protein cluster is not traditionally perceived as a site of substrate binding within nitrogenase. The observed chalcogenide exchange illustrates that this Fe4S4 cluster is capable of core substitution reactions under certain conditions, adding to the Fe protein's repertoire of unique properties.

Many of the molecules that form the building blocks of life contain nitrogen. This element makes up most of the gas in the atmosphere, but in this form, it does not easily react, and most organisms cannot incorporate atmospheric nitrogen into biological molecules. To get around this problem, some species of bacteria produce an enzyme complex called nitrogenase that can transform nitrogen from the air into ammonia. This process is called nitrogen fixation, and it converts nitrogen into a form that can be used to sustain life. The nitrogenase complex is made up of two proteins: the MoFe protein, which contains the active site that binds nitrogen, turning it into ammonia; and the Fe protein, which drives the reaction. Besides the nitrogen fixation reaction, the Fe protein is involved in other biological processes, but it was not thought to bind directly to nitrogen, or to any of the other small molecules that the nitrogenase complex acts on. The Fe protein contains a cluster of iron and sulfur ions that is required to drive the nitrogen fixation reaction, but the role of this cluster in the other reactions performed by the Fe protein remains unclear. To better understand the role of this iron sulfur cluster, Buscagan, Kaiser and Rees used X-ray crystallography, a technique that can determine the structure of molecules. This approach revealed for the first time that when nitrogenase reacts with a small molecule called selenocyanate, the selenium in this molecule can replace the sulfur ions of the iron sulfur cluster in the Fe protein. Buscagan, Kaiser and Rees also demonstrated that the Fe protein could still incorporate selenium ions in the absence of the MoFe protein, which has traditionally been thought to provide the site essential for transforming small molecules. These results indicate that the iron sulfur cluster in the Fe protein may bind directly to small molecules that react with nitrogenase. In the future, these findings could lead to the development of new molecules that artificially produce ammonia from nitrogen, an important process for fertilizer manufacturing. In addition, the iron sulfur cluster found in the Fe protein is also present in many other proteins, so Buscagan, Kaiser and Rees' experiments may shed light on the factors that control other biological reactions.

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.

Infrared Spectroscopy Elucidates the Inhibitor Binding Sites in a Metal-Dependent Formate Dehydrogenase.

Biological carbon dioxide (CO2 ) reduction is an important step by which organisms form valuable energy-richer molecules required for further metabolic processes. The Mo-dependent formate dehydrogenase (FDH) from Rhodobacter capsulatus catalyzes reversible formate oxidation to CO2 at a bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor. To elucidate potential substrate binding sites relevant for the mechanism, we studied herein the interaction with the inhibitory molecules azide and cyanate, which are isoelectronic to CO2 and charged as formate. We employed infrared (IR) spectroscopy in combination with density functional theory (DFT) and inhibition kinetics. One distinct inhibitory molecule was found to bind to either a non-competitive or a competitive binding site in the secondary coordination sphere of the active site. Site-directed mutagenesis of key amino acid residues in the vicinity of the bis-MGD cofactor revealed changes in both non-competitive and competitive binding, whereby the inhibitor is in case of the latter interaction presumably bound between the cofactor and the adjacent Arg587.