Structural identification and comprehension of human ALDH1L1-Gossypol complex.

The folate metabolism enzyme ALDH1L1 catalyzed 10-formyltetrahydrofolate to tetrahydrofolate and CO2. Non-small cell lung cancer cells (NSCLC) strongly express ALDH1L1. Gossypol binds to an allosteric site and disrupts the folate metabolism by preventing NADP+ binding. The Cryo-EM structures of tetrameric C-terminal aldehyde dehydrogenase human ALDH1L1 complex with gossypol were examined. Gossypol-bound ALDH1L1 interfered with NADP+ by shifting the allosteric site of the structural conformation, producing a closed-form NADP+ binding site. In addition, the inhibition activity of ALDH1L1 was targeted with gossypol in NSCLC. The gossypol treatment had anti-cancer effects on NSCLC by blocking NADPH and ATP production. These findings emphasize the structure characterizing ALDH1L1 with gossypol.

Curcumin-based-fluorescent probes targeting ALDH1A3 as a promising tool for glioblastoma precision surgery and early diagnosis.

Glioblastoma (GBM) is the most aggressive primary brain tumour for which both effective treatments and efficient tools for an early-stage diagnosis are lacking. Herein, we present curcumin-based fluorescent probes that are able to bind to aldehyde dehydrogenase 1A3 (ALDH1A3), an enzyme overexpressed in glioma stem cells (GSCs) and associated with stemness and invasiveness of GBM. Two compounds are selective versus ALDH1A3, without showing any appreciable interaction with other ALDH1A isoenzymes. Indeed, their fluorescent signal is detectable only in our positive controls in vitro and absent in cells that lack ALDH1A3. Remarkably, in vivo, our Probe selectively accumulate in glioblastoma cells, allowing the identification of the growing tumour mass. The significant specificity of our compounds is the necessary premise for their further development into glioblastoma cells detecting probes to be possibly used during neurosurgical operations.

Thioester synthesis by a designed nickel enzyme models prebiotic energy conversion.

The formation of carbon-carbon bonds from prebiotic precursors such as carbon dioxide represents the foundation of all primordial life processes. In extant organisms, this reaction is carried out by the carbon monoxide dehydrogenase (CODH)/acetyl coenzyme A synthase (ACS) enzyme, which performs the cornerstone reaction in the ancient Wood-Ljungdahl metabolic pathway to synthesize the key biological metabolite, acetyl-CoA. Despite its significance, a fundamental understanding of this transformation is lacking, hampering efforts to harness analogous chemistry. To address these knowledge gaps, we have designed an artificial metalloenzyme within the azurin protein scaffold as a structural, functional, and mechanistic model of ACS. We demonstrate the intermediacy of the NiI species and requirement for ordered substrate binding in the bioorganometallic carbon-carbon bond-forming reaction from the one-carbon ACS substrates. The electronic and geometric structures of the nickel-acetyl intermediate have been characterized using time-resolved optical, electron paramagnetic resonance, and X-ray absorption spectroscopy in conjunction with quantum chemical calculations. Moreover, we demonstrate that the nickel-acetyl species is chemically competent for selective acyl transfer upon thiol addition to biosynthesize an activated thioester. Drawing an analogy to the native enzyme, a mechanism for thioester generation by this ACS model has been proposed. The fundamental insight into the enzymatic process provided by this rudimentary ACS model has implications for the evolution of primitive ACS-like proteins. Ultimately, these findings offer strategies for development of highly active catalysts for sustainable generation of liquid fuels from one-carbon substrates, with potential for broad applications across diverse fields ranging from energy storage to environmental remediation.

Visualizing the gas channel of a monofunctional carbon monoxide dehydrogenase.

Carbon monoxide dehydrogenase (CODH) plays an important role in the processing of the one­carbon gases carbon monoxide and carbon dioxide. In CODH enzymes, these gases are channeled to and from the Ni-Fe-S active sites using hydrophobic cavities. In this work, we investigate these gas channels in a monofunctional CODH from Desulfovibrio vulgaris, which is unusual among CODHs for its oxygen-tolerance. By pressurizing D. vulgaris CODH protein crystals with xenon and solving the structure to 2.10 Å resolution, we identify 12 xenon sites per CODH monomer, thereby elucidating hydrophobic gas channels. We find that D. vulgaris CODH has one gas channel that has not been experimentally validated previously in a CODH, and a second channel that is shared with Moorella thermoacetica carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS). This experimental visualization of D. vulgaris CODH gas channels lays groundwork for further exploration of factors contributing to oxygen-tolerance in this CODH, as well as study of channels in other CODHs. We dedicate this publication to the memory of Dick Holm, whose early studies of the Ni-Fe-S clusters of CODH inspired us all.

A Morphing [4Fe-3S-nO]-Cluster within a Carbon Monoxide Dehydrogenase Scaffold.

Ni,Fe-containing carbon monoxide dehydrogenases (CODHs) catalyze the reversible reduction of CO2 to CO. Several anaerobic microorganisms encode multiple CODHs in their genome, of which some, despite being annotated as CODHs, lack a cysteine of the canonical binding motif for the active site Ni,Fe-cluster. Here, we report on the structure and reactivity of such a deviant enzyme, termed CooS-VCh . Its structure reveals the typical CODH scaffold, but contains an iron-sulfur-oxo hybrid-cluster. Although closely related to true CODHs, CooS-VCh catalyzes neither CO oxidation, nor CO2 reduction. The active site of CooS-VCh undergoes a redox-dependent restructuring between a reduced [4Fe-3S]-cluster and an oxidized [4Fe-2S-S*-2O-2(H2 O)]-cluster. Hydroxylamine, a slow-turnover substrate of CooS-VCh , oxidizes the hybrid-cluster in two structurally distinct steps. Overall, minor changes in CODHs are sufficient to accommodate a Fe/S/O-cluster in place of the Ni,Fe-heterocubane-cluster of CODHs.

GSNOR regulates ganoderic acid content in Ganoderma lucidum under heat stress through S-nitrosylation of catalase.

As a master regulator of the balance between NO signaling and protein S-nitrosylation, S-nitrosoglutathione (GSNO) reductase (GSNOR) is involved in various developmental processes and stress responses. However, the proteins and specific sites that can be S-nitrosylated, especially in microorganisms, and the physiological functions of S-nitrosylated proteins remain unclear. Herein, we show that the ganoderic acid (GA) content in GSNOR-silenced (GSNORi) strains is significantly lower (by 25%) than in wild type (WT) under heat stress (HS). Additionally, silencing GSNOR results in an 80% increase in catalase (CAT) activity, which consequently decreases GA accumulation via inhibition of ROS signaling. The mechanism of GSNOR-mediated control of CAT activity may be via protein S-nitrosylation. In support of this possibility, we show that CAT is S-nitrosylated (as shown via recombinant protein in vitro and via GSNORi strains in vivo). Additionally, Cys (cysteine) 401, Cys642 and Cys653 in CAT are S-nitrosylation sites (assayed via mass spectrometry analysis), and Cys401 may play a pivotal role in CAT activity. These findings indicate a mechanism by which GSNOR responds to stress and regulates secondary metabolite content through protein S-nitrosylation. Our results also define a new S-nitrosylation site and the function of an S-nitrosylated protein regulated by GSNOR in microorganisms.

Gas channel rerouting in a primordial enzyme: Structural insights of the carbon-monoxide dehydrogenase/acetyl-CoA synthase complex from the acetogen Clostridium autoethanogenum.

Clostridium autoethanogenum, the bacterial model for biological conversion of waste gases into biofuels, grows under extreme carbon-monoxide (CO) concentrations. The strictly anaerobic bacterium derives its entire cellular energy and carbon from this poisonous gas, therefore requiring efficient molecular machineries for CO-conversion. Here, we structurally and biochemically characterized the key enzyme of the CO-converting metabolism: the CO-dehydrogenase/Acetyl-CoA synthase (CODH/ACS). We obtained crystal structures of natively isolated complexes from fructose-grown and CO-grown C. autoethanogenum cultures. Both contain the same isoforms and if the overall structure adopts the classic α2ß2 architecture, comparable to the model enzyme from Moorella thermoacetica, the ACS binds a different position on the CODH core. The structural characterization of a proteolyzed complex and the conservation of the binding interface in close homologs rejected the possibility of a crystallization artefact. Therefore, the internal CO-channeling system, critical to transfer CO generated at the C-cluster to the ACS active site, drastically differs in the complex from C. autoethanogenum. The 1.9-Å structure of the CODH alone provides an accurate picture of the new CO-routes, leading to the ACS core and reaching the surface. Increased gas accessibility would allow the simultaneous CO-oxidation and acetyl-CoA production. Biochemical experiments showed higher flexibility of the ACS subunit from C. autoethanogenum compared to M. thermoacetica, albeit monitoring similar CO-oxidation and formation rates. These results show a reshuffling of internal CO-tunnels during evolution of these Firmicutes, putatively leading to a bidirectional complex that ensure a high flux of CO-conversion toward energy conservation, acting as the main cellular powerplant.

Targeting NAD-dependent dehydrogenases in drug discovery against infectious diseases and cancer.

Dehydrogenases are oxidoreductase enzymes that play a variety of fundamental functions in the living organisms and have primary roles in pathogen survival and infection processes as well as in cancer development. We review here a sub-set of NAD-dependent dehydrogenases involved in human diseases and the recent advancements in drug development targeting pathogen-associated NAD-dependent dehydrogenases. We focus also on the molecular aspects of the inhibition process listing the structures of the most relevant molecules targeting this enzyme family. Our aim is to review the most impacting findings regarding the discovery of novel inhibitory compounds targeting the selected NAD-dependent dehydrogenases involved in cancer and infectious diseases.

Imidazo[1,2-a]pyridine Derivatives as Aldehyde Dehydrogenase Inhibitors: Novel Chemotypes to Target Glioblastoma Stem Cells.

Glioblastoma multiforme (GBM) is the deadliest form of brain tumor. It is known for its ability to escape the therapeutic options available to date thanks to the presence of a subset of cells endowed with stem-like properties and ability to resist to cytotoxic treatments. As the cytosolic enzyme aldehyde dehydrogenase 1A3 turns out to be overexpressed in these kinds of cells, playing a key role for their vitality, treatments targeting this enzyme may represent a successful strategy to fight GBM. In this work, we describe a novel class of imidazo[1,2-a]pyridine derivatives as aldehyde dehydrogenase 1A3 inhibitors, reporting the evidence of their significance as novel drug candidates for the treatment of GBM. Besides showing an interesting functional profile, in terms of activity against the target enzyme and selectivity toward highly homologous isoenzymes, representative examples of the series also showed a nanomolar to picomolar efficacy against patient-derived GBM stem-like cells, thus proving the concept that targeting aldehyde dehydrogenase might represent a novel and promising way to combat GBM by striking its ability to divide immortally.

QM/MM computations reveal details of the acetyl-CoA synthase catalytic center.

The "open" (Aopen) and "closed" (Aclosed) A-clusters of the acteyl-CoA synthase (ACS) enzyme from Moorella thermoacetica have been studied using a combined quantum mechanical (QM)/molecular mechanical (MM) approach. Geometry optimizations of the oxidized, one- and two-electron reduced Aopen state have been carried out for the fully solvated ACS enzyme, and the CO ligand has been modeled in the reduced models. Using a combination of both αopen and αclosed protein scaffolds and the positions of metal atoms in these structures, we have been able to piece together critical parts of the catalytic cycle of ACS. We have replaced the unidentified exogenous ligand in the crystal structure with CO using both a square planar and tetrahedral proximal Ni atom. A one-electron reduced A-cluster that is characterized by a proximal Ni atom in a tetrahedral coordination pattern observed in both the Aopen (lower occupancy proximal Ni) and Aclosed (proximal Zn atom) geometries with three cysteine thiolates and a modeled CO ligand demonstrates excellent agreement with the crystal structure atomic positions, particularly with the displacement of the side chain ring of Phe512 which appears to serve as a structural gate for ligand binding. The QM/MM optimized geometry of the A-cluster of ACS with an uncoordinated, oxidized proximal nickel atom in a square planar geometry demonstrates poor agreement with the atomic coordinates taken from the crystal structure. Based on these calculations, we conclude that the square planar proximal nickel coordination that has been captured in the Aopen structure does not correspond to the ligand-free, oxidized [Fe4S4]2+ - Nip2+ - Nid2+ state. Overall, these computations shed further light on the mechanistic details of protein conformational changes and electronic transitions involved in the ACS catalytic cycle.

A Self-Powered Biosensor with a Flake Electrochromic Display for Electrochemical and Colorimetric Formaldehyde Detection.

The formaldehyde biosensors with the features of cost effectiveness, high specificity, easy operation, and simplicity are urgently desired in routing and field detection of formaldehyde. Here, we report a new design of an enzymatic self-powered biosensor (ESPB) toward formaldehyde detection. The ESPB involves a formaldehyde dehydrogenase/poly-methylene green/buckypaper bioanode as the sensing electrode and a Prussian blue/Au nanoparticles/carbon fiber paper cathode as the electrochromic display. Formaldehyde acts as the fuel to drive the ESPB, relying on that the concentration of formaldehyde can be determined with the ESPB by both directly measuring the variance in short circuit current and observing the color change of the cathode. By measuring the variance in short circuit current, a linear detection range from 0.01 to 0.35 mM and a calculated detection limit of 0.006 mM are obtained, comparable to or better than those reported before. The color change of the cathode can be distinguished easily and exactly via the naked eye after immersing the ESPB in formaldehyde solution for 90 s with the concentration up to 0.35 mM, covering the permissive level of formaldehyde in some standards associated with environmental quality control. Specially, the formaldehyde concentration can be precisely quantified by analyzing the color change of the cathode digitally using the equation of B/(R + G + B). In the following test of real spiked samples of tap water and lake water, the recovery ratios of formaldehyde with the concentrations from 0.010 to 0.045 mM are tested to be between 95 and 100% by both measuring the variance in short circuit current and analyzing the color change of the cathode digitally. In addition, the ESPB exhibits negligible interference from acetaldehyde and ethanol and can be stored at 4 °C for 21 days with a loss of less than 8% in its initial value of short circuit current. Therefore, the ESPB with the capability of working like disposable test paper can be expected as a sensitive, simple, rapid, cost-effective colorimetric method with high selectivity in routing and field formaldehyde detection.

Energetics for the Mechanism of Nickel-Containing Carbon Monoxide Dehydrogenase.

Nickel-containing carbon monoxide (CO) dehydrogenase is an enzyme that catalyzes the important reversible carbon dioxide reduction. Several high-resolution structures have been determined at various stages of the reduction, which can be used as good starting points for the present computational study. The cluster model is used in combination with a systematic application of the density functional theory as recently described. The results are in very good agreement with experimental evidence. There are a few important results. To explain why the X-ray structure for the reduced Cred1 state has an empty site on nickel, it is here suggested that the cluster has been over-reduced by X-rays and is therefore not the desired reduced state, which instead contains a bound CO on nickel. After an additional reduction, a hydride bound to nickel is suggested to play a role. In order to obtain energetics in agreement with experiments, it is concluded that one sulfide bridge in the Ni-Fe cluster should be protonated. The best test of the accuracy obtained is to compare the computed rate for reduction using -0.6 V with that for oxidation using -0.3 V, where good agreement was obtained. Obtaining a mechanism that is easily reversible is another demanding aspect of the modeling. Nickel oscillates between nickel(II) and nickel(I), while nickel(0) never comes in.

Redox-dependent rearrangements of the NiFeS cluster of carbon monoxide dehydrogenase.

The C-cluster of the enzyme carbon monoxide dehydrogenase (CODH) is a structurally distinctive Ni-Fe-S cluster employed to catalyze the reduction of CO2 to CO as part of the Wood-Ljungdahl carbon fixation pathway. Using X-ray crystallography, we have observed unprecedented conformational dynamics in the C-cluster of the CODH from Desulfovibrio vulgaris, providing the first view of an oxidized state of the cluster. Combined with supporting spectroscopic data, our structures reveal that this novel, oxidized cluster arrangement plays a role in avoiding irreversible oxidative degradation at the C-cluster. Furthermore, mutagenesis of a conserved cysteine residue that binds the C-cluster in the oxidized state but not in the reduced state suggests that the oxidized conformation could be important for proper cluster assembly, in particular Ni incorporation. Together, these results lay a foundation for future investigations of C-cluster activation and assembly, and contribute to an emerging paradigm of metallocluster plasticity.

PPARgamma-mediated ALDH1A3 suppression exerts anti-proliferative effects in lung cancer by inducing lipid peroxidation.

CONTEXT: The metabolic function of peroxisome proliferator-activated receptor gamma (PPARγ) in lung cancer remains unclear. OBJECTIVES: To determine the relationship of PPARγ on ALDH1A3-induced lipid peroxidation to inhibit lung cancer cell growth. MATERIALS AND METHODS: In silico analysis using microarray dataset was performed to screen the positive correlation between PPARγ and all ALDH isoforms. NUBIscan software and ChIP assay were used to identify the binding sites (BSs) of PPARγ on ALDH1A3 promoter. The expression of ALDH1A3 under thiazolidinedione (TZD) treatment was evaluated by QPCR and Western Blot in HBEC and H1993 cell lines. Upon treatment of TZD, colony formation assay was used to check cell growth inhibition and 4-hydroxy-2-nonenal (4HNE) production as lipid peroxidation marker was determined by Western Blot in PPARγ positive cell H1993 and PPARγ negative cell H1299. RESULTS: Compared to other ALDH isoforms, ALDH1A3 showed the highest positive correlation to PPARγ expression. ALDH1A3 upregulated PPARγ expression while PPARγ activation suppressed ALDH1A3. Among 2 potential screened PPARγ response elements, BS 1 and 2 in the promoter of ALDH1A3 gene, PPARγ bound directly to BS2. Ligand activation of PPARγ suppressed mRNA and protein expression of ALDH1A3. Growth inhibition was observed in H1993 (PPARγ positive cell) treated with PPARγ activator and ALDH inhibitor compared to H1299 (PPARγ negative cell). PPARγ activation increased 4HNE which is known to be suppressed by ALDH1A3. CONCLUSIONS: ALDH1A3 suppression could be one of PPARγ tumor suppressive function. This study provides a better understanding of the role of PPARγ in lung cancer.

Chloroacetaldehyde dehydrogenase from Ancylobacter aquaticus UV5: Cloning, expression, characterization and molecular modeling.

1,2-Dichloroethane (1,2-DCE) is oxidatively converted to a carcinogenic intermediate compound, chloroacetaldehyde by chloroacetaldehyde dehydrogenase (CAldA) during its biodegradation by many bacterial strains, including Xanthobacter autotrophicus and Ancylobacter aquaticus. In this study, a 55kDa NAD-dependent CAldA expressed by chromosomally encoded aldA gene, is reported in an indigenous Ancylobacter aquaticus UV5. A. aquaticus UV5 aldA gene was found to be 99% homologous to the plasmid (pXAU1) encoded aldA gene reported in X. autotrophicus GJ10. Pulse-field gel electrophoresis (PFGE) and PCR experiments revealed the absence of pXAU1 in A. aquaticus UV5 and that aldA was chromosomal encoded. A 6× His-tag fused CAldA cloned in pET15b, overexpressed and purified on Co-agarose affinity column using AKTA purification system showed Mr of 57,526. CAldA was active optimally at pH9 and 30°C. The Km and vmax for the substrate, acetaldehyde were found to be 115µM and 650mU/mg, respectively. CAldA substrate specificity was found to be low for chloroacetaldehyde, formaldehyde, propionaldehyde, butyraldehyde, benzaldehyde and glutaraldehyde as compared to acetaldehyde. Computational modeling revealed a predicted structure of CAldA consisting of five ß-sheets that comprise seven antiparallel ß-strands and 11 mix strands. The Molecular Dynamics and Docking studies showed that acetaldehyde bind to CaldA more tightly as compared to chloroacetaldehyde.

S-Nitrosoglutathione Reductase Dysfunction Contributes to Obesity-Associated Hepatic Insulin Resistance via Regulating Autophagy.

Obesity is associated with elevated intracellular nitric oxide (NO) production, which promotes nitrosative stress in metabolic tissues such as liver and skeletal muscle, contributing to insulin resistance. The onset of obesity-associated insulin resistance is due, in part, to the compromise of hepatic autophagy, a process that leads to lysosomal degradation of cellular components. However, it is not known how NO bioactivity might impact autophagy in obesity. Here, we establish that S-nitrosoglutathione reductase (GSNOR), a major protein denitrosylase, provides a key regulatory link between inflammation and autophagy, which is disrupted in obesity and diabetes. We demonstrate that obesity promotes S-nitrosylation of lysosomal proteins in the liver, thereby impairing lysosomal enzyme activities. Moreover, in mice and humans, obesity and diabetes are accompanied by decreases in GSNOR activity, engendering nitrosative stress. In mice with a GSNOR deletion, diet-induced obesity increases lysosomal nitrosative stress and impairs autophagy in the liver, leading to hepatic insulin resistance. Conversely, liver-specific overexpression of GSNOR in obese mice markedly enhances lysosomal function and autophagy and, remarkably, improves insulin action and glucose homeostasis. Furthermore, overexpression of S-nitrosylation-resistant variants of lysosomal enzymes enhances autophagy, and pharmacologically and genetically enhancing autophagy improves hepatic insulin sensitivity in GSNOR-deficient hepatocytes. Taken together, our data indicate that obesity-induced protein S-nitrosylation is a key mechanism compromising the hepatic autophagy, contributing to hepatic insulin resistance.

Immobilization of formaldehyde dehydrogenase in tailored siliceous mesostructured cellular foams and evaluation of its activity for conversion of formate to formaldehyde.

Formaldehyde dehydrogenase (FaldDH) is used as a catalyst to reduce formate to formaldehyde in a cascade reaction to convert CO2 to methanol. This enzyme, however, has low activity and is sensitive to substrate/product concentration and pH. To improve the performance of FaldDH, it can be immobilized through physical adsorption in siliceous mesostructured cellular foams (MCF), which physical properties are suitable for the immobilization of large molecules as FaldDH (molecular size of 8.6 × 8.6 × 19 nm). In this work two MCF materials were synthesized: MCF1 with a pore size of 26.8 nm and window size of 10.5 nm; and MCF2 with a pore size of 32.9 nm and window size of 13.0 nm. The surfaces of the materials were functionalized with octyl, mercaptopropyl or chloromethyl groups. FaldDH was successfully immobilized inside all the materials, yielding enzyme loadings of about 300 mg g-1 in MCF1 and more than 750 mg g-1 in MCF2. However, the enzyme was inactive upon immobilization on MCF1, whereas on MCF2 the enzyme retained its catalytic activity presumably owing to the larger pores of this material and the need for the enzyme to undergo configurational changes during the reaction. Using MCF2 functionalized with mercaptopropyl groups the activity of FaldDH was enhanced beyond that of the free enzyme. Additionally, low leakage of the enzyme from the MCF2 was observed during the reactions. Thus, tailored MCF is a highly attractive material for employment of the FaldDH enzyme.

Redox regulation of plant S-nitrosoglutathione reductase activity through post-translational modifications of cysteine residues.

Nitric oxide (NO) is considered as a signalling molecule involved in a variety of important physiological and pathological processes in plant and animal systems. The major pathway of NO reactions in vivo represents S-nitrosation of thiols to form S-nitrosothiols. S-nitrosoglutathione reductase (GSNOR) is the key enzyme in the degradation pathway of S-nitrosoglutathione (GSNO), a low-molecular weight adduct of NO and glutathione. GSNOR indirectly regulates the level of protein S-nitrosothiol in the cells. This study was focused on the dynamic regulation of the activity of plant GSNORs through reversible S-nitrosation and/or oxidative modifications of target cysteine residues. Pre-incubation with NO/NO- donors or hydrogen peroxide resulted in a decreased reductase and dehydrogenase activity of all studied plant GSNORs. Incubation with thiol reducing agent completely reversed inhibitory effects of nitrosative modifications and partially also oxidative inhibition. In biotin-labelled samples, S-nitrosation of plant GSNORs was confirmed after immunodetection and using mass spectrometry S-nitrosation of conserved Cys271 was identified in tomato GSNOR. Negative regulation of constitutive GSNOR activity in vivo by nitrosative or oxidative modifications might present an important mechanism to control GSNO levels, a critical mediator of the downstream signalling effects of NO, as well as for formaldehyde detoxification in dehydrogenase reaction mode.

The CO dehydrogenase accessory protein CooT is a novel nickel-binding protein.

In Rhodospirillum rubrum, maturation of Carbon Monoxide Dehydrogenase (CODH) requires three accessory proteins, CooC, CooT and CooJ, dedicated to nickel insertion into the active site, which is constituted by a distorted [NiFe3S4] cubane coordinated with a mononuclear Fe site. CooC is an ATPase proposed to provide the energy required for the maturation process, while CooJ is described as a metallochaperone with 16 histidines and 2 cysteines at the C-terminus, likely involved in metal binding and/or storage. Prior to the present study, no information was available on CooT at the molecular level. Here, the X-ray structure of RrCooT was obtained, which revealed that this protein is a homodimer featuring a fold that resembles an Sm-like domain, suggesting a role in RNA metabolism that was however not supported by experimental observations. Biochemical and biophysical evidence based on circular dichroism spectroscopy, light scattering, isothermal titration calorimetry and site-directed mutagenesis showed that RrCooT specifically binds a single Ni(ii) per dimer, with a dissociation constant of 9 nM, through the pair of Cys2, highly conserved residues, located at the dimer interface. Despite its role in the activation of RrCODH in vivo, CooT was thought to be a unique protein, found only in R. rubrum, with an unclear function. In this study, we extended the biological impact of CooT, establishing that this protein is a member of a novel Ni(ii)-binding protein family with 111 homologues, linked to anaerobic metabolism in bacteria and archaea, and in most cases to the presence of CODH.

Pyrenyl carbon nanostructures for ultrasensitive measurements of formaldehyde in urine.

Measurement of ultra-low (e.g., parts-per-billion) levels of small-molecule markers in body fluids (e.g., serum, urine, saliva) involves a considerable challenge in view of designing assay strategies with sensitivity and selectivity. Herein we report for the first time an amperometric nano-bioelectrode design that uniquely combines 1-pyrenebutyric acid units pi-pi stacked with carboxylated multiwalled carbon nanotubes on the surface of gold screen printed electrodes for covalent attachment of NAD+ dependent formaldehyde dehydrogenase (FDH). The designed enzyme bioelectrode offered 6 ppb formaldehyde detection in 10-times diluted urine with a wide dynamic range of 10 ppb to 10 ppm. Fourier transform infrared, Raman, and electrochemical impedance spectroscopic characterizations confirmed the successful design of the FDH bioelectrode. Flow injection analysis provided lower detection limit and greater affinity for formaldehyde (apparent KM 9.6 ± 1.2 ppm) when compared with stirred solution method (apparent KM 19.9 ± 4.6 ppm). Selectivity assays revealed that the bioelectrode was selective toward formaldehyde with a moderate cross-reactivity for acetaldehyde (∼25%) and negligible cross-reactivity toward propanaldehyde, acetone, methanol, and ethanol. Formaldehyde is an indoor pollutant, and studies have indicated neurotoxic characteristics and systemic toxic effects of this compound upon chronic and high doses of exposure. Moreover, reported chromatography and mass spectrometry methods identified elevated urine formaldehyde levels in patients with bladder cancer, dementia, and early stages of cognitive impairments compared to healthy people. Results demonstrate that pyrenyl carbon nanostructures-based FDH bioelectrode design represents novelty and simplicity for enzyme-selective electrochemical quantitation of small 30 Da formaldehyde. Broader applicability of the presented approach for other small-molecule markers is feasible that requires only the design of appropriate marker-specific enzyme systems or receptor molecules.