Jorge Caldeira

Crystal structure of the first dissimilatory nitrate reductase at 1.9 Å solved by MAD methods

BACKGROUND The periplasmic nitrate reductase (NAP) from the sulphate reducing bacterium Desulfovibrio desulfuricans ATCC 27774 is induced by growth on nitrate and catalyses the reduction of nitrate to nitrite for respiration. NAP is a molybdenum-containing enzyme with one bis-molybdopterin guanine dinucleotide (MGD) cofactor and one [4Fe-4S] cluster in a single polypeptide chain of 723 amino acid residues. To date, there is no crystal structure of a nitrate reductase. RESULTS The first crystal structure of a dissimilatory (respiratory) nitrate reductase was determined at 1.9 A resolution by multiwavelength anomalous diffraction (MAD) methods. The structure is folded into four domains with an alpha/beta-type topology and all four domains are involved in cofactor binding. The [4Fe-4S] centre is located near the periphery of the molecule, whereas the MGD cofactor extends across the interior of the molecule interacting with residues from all four domains. The molybdenum atom is located at the bottom of a 15 A deep crevice, and is positioned 12 A from the [4Fe-4S] cluster. The structure of NAP reveals the details of the catalytic molybdenum site, which is coordinated to two MGD cofactors, Cys140, and a water/hydroxo ligand. A facile electron-transfer pathway through bonds connects the molybdenum and the [4Fe-4S] cluster. CONCLUSIONS The polypeptide fold of NAP and the arrangement of the cofactors is related to that of Escherichia coli formate dehydrogenase (FDH) and distantly resembles dimethylsulphoxide reductase. The close structural homology of NAP and FDH shows how small changes in the vicinity of the molybdenum catalytic site are sufficient for the substrate specificity.

Identification of novel GAPDH-derived antimicrobial peptides secreted by Saccharomyces cerevisiae and involved in wine microbial interactions

Saccharomyces cerevisiae plays a primordial role in alcoholic fermentation and has a vast worldwide application in the production of fuel-ethanol, food and beverages. The dominance of S. cerevisiae over other microbial species during alcoholic fermentations has been traditionally ascribed to its higher ethanol tolerance. However, recent studies suggested that other phenomena, such as microbial interactions mediated by killer-like toxins, might play an important role. Here we show that S. cerevisiae secretes antimicrobial peptides (AMPs) during alcoholic fermentation that are active against a wide variety of wine-related yeasts (e.g. Dekkera bruxellensis) and bacteria (e.g. Oenococcus oeni). Mass spectrometry analyses revealed that these AMPs correspond to fragments of the S. cerevisiae glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein. The involvement of GAPDH-derived peptides in wine microbial interactions was further sustained by results obtained in mixed cultures performed with S. cerevisiae single mutants deleted in each of the GAPDH codifying genes (TDH1-3) and also with a S. cerevisiae mutant deleted in the YCA1 gene, which codifies the apoptosis-involved enzyme metacaspase. These findings are discussed in the context of wine microbial interactions, biopreservation potential and the role of GAPDH in the defence system of S. cerevisiae.

Clean synthesis of molecular recognition polymeric materials with chiral sensing capability using supercritical fluid technology. Application as HPLC stationary phases

Molecularly imprinted polymers (MIPs) of poly(ethylene glycol dimethacrylate) and poly(N-isopropylacrylamide-co-ethylene glycol dimethacrylate) were synthesized for the first time in supercritical carbon dioxide (scCO(2)), using Boc-L-tryptophan as template. Supercritical fluid technology provides a clean and one-step synthetic route for the preparation of affinity polymeric materials with sensing capability for specific molecules. The polymeric materials were tested as stationary HPLC phases for the enantiomeric separation of L- and D-tryptophan. HPLC results prove that the synthesized MIPs are able to recognize the template molecule towards its enantiomer which opens up potential applications in chromatographic chiral separation.

Oxidative stress and histological changes following exposure to diamond nanoparticles in the freshwater Asian clam Corbicula fluminea (Müller, 1774).

Recently, the scientific community became aware of the potential ability of nanoparticles to cause toxicity in living organisms. Therefore, many of the implications for aquatic ecosystems and its effects on living organisms are still to be evaluated and fully understood. In this study, the toxicity of nanodiamonds (NDs) was assessed in the freshwater bivalve (Corbicula fluminea) following exposure to different nominal concentrations of NDs (0.01, 0.1, 1, and 10 mg l(-1)) throughout 14 days. The NDs were characterized (gravimetry, pH, zeta potential, electron microscopy, and atomic force microscopy) confirming manufacturer information and showing NDs with a size of 4-6 nm. Oxidative stress enzymes activities (glutathione-S-transferase, catalase) and lipid peroxidation were determined. The results show a trend to increase in GST activities after seven days of exposure in bivalves exposed to NDs concentrations (>0.1 mg l(-1)), while for catalase a significant increase was found in bivalves exposed from 0.01 to 1.0 mg l(-1) following an exposure of 14 days. The histological analysis revealed alterations in digestive gland cells, such as vacuolization and thickening. The lipid peroxidation showed a trend to increase for the different tested NDs concentrations which is compatible with the observed cellular damage.

Further characterization of two different, reversible aldehyde oxidoreductases from Clostridium formicoaceticum, one containing tungsten and the other molybdenum

The tungsten- and the molybdenum-containing aldehyde oxidoreductases from Clostridium formicoaceticum show, for aldehydes, Km values<30 μM and Ki values of millimolar concentrations. The tungsten-containing aldehyde oxidoreductase is inactivated to 50% by 3 mM KCN within 1 min, by 1 mM ferricyanide within 5 min, and by 0.05 mM chloralhydrate within 30 s. The molybdenum-containing AOR shows 50% inactivation within 1 min only with 70 mM KCN. The tungsten-containing enzyme is very sensitive to oxygen, especially in the reduced state, whereas the molybdenum-containing enzyme exhibits only moderate oxygen sensitivity without being markedly influenced by the redox state of the enzyme. The tungsten in the aldehyde oxidoreductase is bound to a pterin cofactor (Wco) of the mononucleotide form that is known for molybdopterin cofactor (Moco). The nature of the molybdenum cofactor in the molybdenum-containing aldehyde oxidoreductase is still unclear. The UV/VIS spectrum of the tungsten-containing aldehyde oxidoreductase shows a broad absorption in the range of 400 nm with a millimolar absorption coefficient of 18.1 (reduced form) and 24.8 (dehydrogenated form) at 396 nm. The epr spectrum exhibits two different W(V) signals with the following g values for signal A: 2.035, 1.959, 1.899 and signal B: 2.028, 2.017, 2.002. Dithionite-reduced enzyme shows signals of 4Fe−4S or 2Fe−2S clusters. Initial rate studies with different substrates for the carboxylate reduction led to a Bi Uni Uni Bi mechanism.

Cell-to-cell contact and antimicrobial peptides play a combined role in the death of Lachanchea thermotolerans during mixed-culture alcoholic fermentation with Saccharomyces cerevisiae

The roles of cell-to-cell contact and antimicrobial peptides in the early death of Lachanchea thermotolerans CBS2803 during anaerobic, mixed-culture fermentations with Saccharomyces cerevisiae S101 were investigated using a commercially available, double-compartment fermentation system separated by cellulose membranes with different pore sizes, i.e. 1000 kDa for mixed- and single-culture fermentations, and 1000 and 3.5-5 kDa for compartmentalized-culture fermentations. SDS-PAGE and gel filtration chromatography were used to determine an antimicrobial peptidic fraction in the fermentations. Our results showed comparable amounts of the antimicrobial peptidic fraction in the inner compartments of the mixed-culture and 1000 kDa compartmentalized-culture fermentations containing L. thermotolerans after 4 days of fermentation, but a lower death rate of L. thermotolerans in the 1000 kDa compartmentalized-culture fermentation than in the mixed-culture fermentation. Furthermore, L. thermotolerans died off even more slowly in the 3.5-5 kDa than in the 1000 kDa compartmentalized-culture fermentation, which coincided with the presence of less of the antimicrobial peptidic fraction in the inner compartment of that fermentation than of the 1000 kDa compartmentalized-culture fermentation. Taken together, these results indicate that the death of L. thermotolerans in mixed cultures with S. cerevisiae is caused by a combination of cell-to-cell contact and antimicrobial peptides.

Characterization of a nematic mixture by reversed‐phase HPLC and UV spectroscopy: an application to phase behaviour studies in liquid crystal–CO2 systems

In the present work a simple but selective reversed‐phase high performance liquid chromatographic method (HPLC) was developed for the analysis of the nematic liquid crystal mixture E7 to determine precisely the composition of the liquid crystal mixture used in PDLC film preparation. Ultraviolet absorption spectrophotometry experiments were carried out to determine the HPLC detection wavelength and to characterize the absorptivity constants of E7 constituents. The technique developed is applied in the case of equilibrium solubility studies for E7 in supercritical carbon dioxide (scCO2). The results indicate unambiguously that scCO2 can fractionate the mixture towards the E7 components. The four single component peaks of the E7 mixture were distinctively separated by this method, which enabled the determination of the solubility of E7 constituents in the scCO2.

EPR and Mössbauer Spectroscopic Studies on Enoate Reductase

Enoate reductase (EC) is a protein isolated from Clostridium tyrobutyricum that contains iron, labile sulfide, FAD, and FMN. The enzyme reduces the α,β carbon-carbon double bond of nonactivated 2-enoates and in a reversible way that of 2-enals at the expense of NADH or reduced methyl viologen. UV-visible and EPR potentiometric titrations detect a semiquinone species in redox intermediate states characterized by an isotropic EPR signal at g = 2.0 without contribution at 580 nm. EPR redox titration shows two widely spread mid-point redox potentials (−190 and −350 mV at pH 7.0), and a nearly stoichiometric amount of this species is detected. The data suggest the semiquinone radical has an anionic nature. In the reduced form, the [Fe-S] moiety is characterized by a single rhombic EPR spectrum, observed in a wide range of temperatures (4.2-60 K) with g values at 2.013, 1.943, and 1.860 (−180 mV at pH 7.0). The gmax value is low when compared with what has been reported for other iron-sulfur clusters. Mössbauer studies reveal the presence of a [4Fe-4S]+2/+1 center. One of the subcomponents of the spectrum shows an unusually large value of quadrupole splitting (ferrous character) in both the oxidized and reduced states. Substrate binding to the reduced enzyme induces subtle changes in the spectroscopic Mössbauer parameters. The Mössbauer data together with known kinetic information suggest the involvement of this iron-sulfur center in the enzyme mechanism.

Antimicrobial properties and death-inducing mechanisms of saccharomycin, a biocide secreted by Saccharomyces cerevisiae

We recently found that Saccharomyces cerevisiae (strain CCMI 885) secretes antimicrobial peptides (AMPs) derived from the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) that are active against various wine-related yeast and bacteria. Here, we show that several other S. cerevisiae strains also secrete natural biocide fractions during alcoholic fermentation, although at different levels, which correlates with the antagonistic effect exerted against non-Saccharomyces yeasts. We, therefore, term this biocide saccharomycin. The native AMPs were purified by gel-filtration chromatography and its antimicrobial activity was compared to that exhibited by chemically synthesized analogues (AMP1 and AMP2/3). Results show that the antimicrobial activity of the native AMPs is significantly higher than that of the synthetic analogues (AMP1 and AMP2/3), but a conjugated action of the two synthetic peptides is observed. Moreover, while the natural AMPs are active at pH 3.5, the synthetic peptides are not, since they are anionic and cannot dissolve at this acidic pH. These findings suggest that the molecular structure of the native biocide probably involves the formation of aggregates of several peptides that render them soluble under acidic conditions. The death mechanisms induced by the AMPs were also evaluated by means of epifluorescence microscopy-based methods. Sensitive yeast cells treated with the synthetic AMPs show cell membrane disruption, apoptotic molecular markers, and internalization of the AMPs. In conclusion, our work shows that saccharomycin is a natural biocide secreted by S. cerevisiae whose activity depends on the conjugated action of GAPDH-derived peptides. This study also reveals that S. cerevisiae secretes GAPDH-derived peptides as a strategy to combat other microbial species during alcoholic fermentations.

Postprandial insulin resistance in Zucker diabetic fatty rats is associated with parasympathetic-nitric oxide axis deficiencies.

The Zucker diabetic fatty (ZDF) rat is an obesity and type 2 diabetes model. Progression to diabetes is well characterised in ZDF rats, but only in the fasted state. We evaluated the mechanisms underlying postprandial insulin resistance in young ZDF rats. We tested the hypothesis that the overall postprandial action of insulin is affected in ZDF rats as a result of impairment of the hepatic parasympathetic‐nitric oxide (PSN‐NO) axis and/or glutathione (GSH), resulting in decreased indirect (PSN‐NO axis) and direct actions of insulin. Nine‐week‐old male ZDF rats and lean Zucker rats (LZR, controls) were used. The action of insulin was assessed in the fed state before and after parasympathetic antagonism atropine. Basal hepatic NO and GSH were measured, as well as NO synthase (NOS) and γ‐glutamyl‐cysteine synthethase (GCS) activity and expression. ZDF rats presented postprandial hyperglycaemia (ZDF, 201.4 ± 12.9 mg/dl; LZR, 107.7 ± 4.3 mg/dl), but not insulinopaenia (ZDF, 5.9 ± 0.8 ng/ml; LZR, 1.5 ± 0.3 ng/ml). Total postprandial insulin resistance was observed (ZDF, 78.6 ± 7.5 mg glucose/kg; LZR, 289.2 ± 24.7 mg glucose/kg), with a decrease in both the direct action of insulin (ZDF, 54.8 ± 7.0 mg glucose/kg; LZR, 173.3 ± 20.5 mg glucose/kg) and the PSN‐NO axis (ZDF, 24.5 ± 3.9 mg glucose/kg; LZR, 115.9 ± 19.4 mg glucose/kg). Hepatic NO (ZDF, 117.2 ± 11.4 μmol/g tissue; LZR, 164.6 ± 4.9 μmol/g tissue) and GSH (ZDF, 4.9 ± 0.3 μmol/g; LZR, 5.9 ± 0.2 μmol/g) were also compromised as a result of decreased NOS and GCS activity, respectively. These results suggest a compromise of the mechanism responsible for potentiating insulin action after a meal in ZDF rats. We show that defective PSN‐NO axis and GSH synthesis, together with an impaired direct action of insulin, appears to contribute to postprandial insulin resistance in this model.