In silico structural features of the CgNR5A: CgDAX complex and its role in regulating gene expression of CYP target genes in Crassostrea gigas.

Contamination of aquatic environments has been steadily increasing due to human activities. The Pacific oyster Crassostrea gigas has been used as a key species in studies assessing the impacts of contaminants on human health and the aquatic biome. In this context, cytochrome P450 (CYPs) play a crucial role in xenobiotic metabolism. In vertebrates many of these CYPs are regulated by nuclear receptors (NRs) and little is known about the NRs role in C. gigas. Particularly, the CgNR5A represents a homologue of SF1 and LRH-1 found in vertebrates. Members of this group can regulate genes of CYPs involved in lipid/steroid metabolism, with their activity regulated by other NR, called as DAX-1, generating a NR complex on DNA response elements (REs). As C. gigas does not exhibit steroid biosynthesis pathways, CgNR5A may play other physiological roles. To clarify this issue, we conducted an in silico investigation of the interaction between CgNR5A and DNA to identify potential C. gigas CYP target genes. Using molecular docking and dynamics simulations of the CgNR5A on DNA molecules, we identified a monomeric interaction with extended REs. This RE was found in the promoter region of 30 CYP genes and also the NR CgDAX. When the upstream regulatory region was analyzed, CYP2C39, CYP3A11, CYP4C21, CYP7A1, CYP17A1, and CYP27C1 were mapped as the main genes regulated by CgNR5A. These identified CYPs belong to families known for their involvement in xenobiotic and lipid/steroid metabolism. Furthermore, we reconstructed a trimeric complex, previously proposed for vertebrates, with CgNR5A:CgDAX and subjected it to molecular dynamics simulations analysis. Heterotrimeric complex remained stable during the simulations, suggesting that CgDAX may modulate CgNR5A transcriptional activity. This study provides insights into the potential physiological processes involving these NRs in the regulation of CYPs associated with xenobiotic and steroid/lipid metabolism.

Nuclear receptor superfamily structural diversity in pacific oyster: In silico identification of estradiol binding candidates.

The increasing presence of anthropogenic contaminants in aquatic environments poses challenges for species inhabiting contaminated sites. Due to their structural binding characteristics to ligands that inhibit or activate gene transcription, these xenobiotic compounds frequently target the nuclear receptor superfamily. The present work aims to understand the potential interaction between the hormone 17-ß-estradiol, an environmental contaminant, and the nuclear receptors of Crassostrea gigas, the Pacific oyster. This filter-feeding, sessile oyster species is subject to environmental changes and exposure to contaminants. In the Pacific oyster, the estrogen-binding nuclear receptor is not able to bind this hormone as it does in vertebrates. However, another receptor may exhibit responsiveness to estrogen-like molecules and derivatives. We employed high-performance in silico methodologies, including three-dimensional modeling, molecular docking and atomistic molecular dynamics to identify likely binding candidates with the target moecule. Our approach revealed that among the C. gigas nuclear receptor superfamily, candidates with the most favorable interaction with the molecule of interest belonged to the NR1D, NR1H, NR1P, NR2E, NHR42, and NR0B groups. Interestingly, NR1H and NR0B were associated with planktonic/larval life cycle stages, while NR1P, NR2E, and NR0B were associated with sessile/adult life stages. The application of this computational methodological strategy demonstrated high performance in the virtual screening of candidates for binding with the target xenobiotic molecule and can be employed in other studies in the field of ecotoxicology in non-model organisms.

Emergence of Two Distinct SARS-CoV-2 Gamma Variants and the Rapid Spread of P.1-like-II SARS-CoV-2 during the Second Wave of COVID-19 in Santa Catarina, Southern Brazil.

The western mesoregion of the state of Santa Catarina (SC), Southern Brazil, was heavily affected as a whole by the COVID-19 pandemic in early 2021. This study aimed to evaluate the dynamics of the SARS-CoV-2 virus spreading patterns in the SC state from March 2020 to April 2021 using genomic surveillance. During this period, there were 23 distinct variants, including Beta and Gamma, among which the Gamma and related lineages were predominant in the second pandemic wave within SC. A regionalization of P.1-like-II in the Western SC region was observed, concomitant to the increase in cases, mortality, and the case fatality rate (CFR) index. This is the first evidence of the regionalization of the SARS-CoV-2 transmission in SC and it highlights the importance of tracking the variants, dispersion, and impact of SARS-CoV-2 on the public health systems.

Oleate Hydratase in Lactobacillus delbrueckii subsp. bulgaricus LBP UFSC 2230 Catalyzes the Reversible Conversion between Linoleic Acid and Ricinoleic Acid.

Conjugated linoleic acid (CLA) has been the subject of numerous studies in recent decades because of its associated health benefits. CLA is an intermediate product of the biohydrogenation pathway of linoleic acid (LA) in bacteria. Several bacterial species capable of efficiently converting LA into CLA have been widely reported in the literature, among them Lactobacillus delbrueckii subsp. bulgaricus LBP UFSC 2230. Over the last few years, a multicomponent enzymatic system consisting of three enzymes involved in the biohydrogenation process of LA has been proposed. Sequencing the genome of L. delbrueckii subsp. bulgaricus LBP UFSC 2230 revealed only one gene capable of encoding an oleate hydratase (OleH), unlike the presence of multiple genes typically found in similar strains. This study investigated the biological effect of the OleH enzyme of L. delbrueckii subsp. bulgaricus LBP UFSC 2230 on the hydration of LA and dehydration of ricinoleic acid (RA) and its possible role in the production of CLA. The OleH was cloned, expressed, purified, and characterized. Fatty acid measurements were made by an internal standard method using a gas chromatography-coupled flame ionization detector (GC-FID) system. It was found that the enzyme is a hydratase/dehydratase, leading to a reversible transformation between LA and RA. In addition, the results showed that L. delbrueckii subsp. bulgaricus LBP UFSC 2230 OleH protein plays a role in stress tolerance in Escherichia coli. In conclusion, the OleH of L. delbrueckii subsp. bulgaricus LBP UFSC 2230 catalyzes the initial stage of saturation metabolism of LA, although it has not converted the substrates directly into CLA. IMPORTANCE This study provides insight into the enzymatic mechanism of CLA synthesis in L. delbrueckii subsp. bulgaricus and broadens our understanding of the bioconversion of LA and RA by OleH. The impact of OleH on the production of the c9, t11 CLA isomer and stress tolerance by E. coli has been assisted. The results provide an understanding of the factors which influence OleH activity. L. delbrueckii subsp. bulgaricus LBP UFSC 2230 OleH presented two putative fatty acid-binding sites. Recombinant OleH catalyzed both LA hydration and RA dehydration. OleH was shown to play a role in bacterial growth performance in the presence of LA.

Characterization of a fatty acid-binding protein from the Pacific oyster (Crassostrea gigas): pharmaceutical and toxicological implications.

Pharmaceuticals and their metabolites constitute a class of xenobiotics commonly found in aquatic environments which may cause toxic effects in aquatic organisms. Several different lipophilic molecules, including some pharmaceuticals, can bind to fatty acid-binding proteins (FABPs), a group of evolutionarily related cytoplasmic proteins that belong to the intracellular lipid-binding protein (iLBP) family. An oyster FABP genome-wide investigation was not available until a recent study on gene organization, protein structure, and phylogeny of Crassostrea gigas iLBPs. Higher transcript levels of the C. gigas FABP2 gene were found after exposure to sewage and pharmaceuticals. Because of its relevance as a potential biomarker of aquatic contamination, in this study, recombinant FABP2 from C. gigas (CgFABP2) was successfully cloned, expressed, and purified, and in vitro and in silico assays were performed using lipids and pharmaceuticals. This is the first characterization of a protein from the iLBP family in C. gigas. Homology modeling and molecular docking were used to evaluate the binding affinities of natural ligands (palmitic, oleic, and arachidonic acids) and pharmaceuticals (ibuprofen, sodium diclofenac, and acetaminophen). Among the tested fatty acids, CgFABP2 showed preference for palmitic acid. The selected pharmaceuticals presented a biphasic-binding mode, suggesting a different binding affinity with a preference for diclofenac. Therefore, the approach using circular dichroism and in silico data might be useful for ligand-binding screening in an invertebrate model organism.

Probucol Protects Neuronal Cells Against Peroxide-Induced Damage and Directly Activates Glutathione Peroxidase-1.

Experimental evidence has shown that probucol, a hypocholesterolemic agent, is also able to increase glutathione peroxidase (GPx) activity. However, there is a lack of knowledge about the mechanism(s) involved in this event. In this study, in vitro experiments with purified GPx1 from bovine erythrocytes and cultured SH-SY5Y neuroblastoma cells, as well as in silico studies with GPx1, were performed in order to elucidate mechanisms mediating the stimulatory effect of probucol on GPx activity and to investigate the relevance of this event in terms of susceptibility against peroxide-induced cytotoxicity. In vitro experiments with purified GPx1 showed a direct stimulatory effect of probucol on the activity of GPx1, which was related to an increase in Vmax with no changes in KM. Probucol also increased GPx activity in cultured SH-SY5Y neuroblastoma cells, while the levels of GPx1 expression were not changed, corroborating the results found with the purified enzyme. In addition, probucol rendered SH-SY5Y cells more resistant to hydroperoxide-induced cytotoxicity, and this event was abolished in GPx1 knocked-down cells. In silico studies with GPx1 pointed to a potential binding site for probucol at the close vicinity of the GSH pocket. Collectively, the results presented herein indicate that GPx1 plays a central role in the probucol-induced protective effects against peroxide toxicity. This highlights a novel target (GPx1) and a new mechanism of action (direct activation) for an "old drug." The relevance of such results for in vivo conditions deserves further investigation.

Ceramides bind VDAC2 to trigger mitochondrial apoptosis.

Ceramides draw wide attention as tumor suppressor lipids that act directly on mitochondria to trigger apoptotic cell death. However, molecular details of the underlying mechanism are largely unknown. Using a photoactivatable ceramide probe, we here identify the voltage-dependent anion channels VDAC1 and VDAC2 as mitochondrial ceramide binding proteins. Coarse-grain molecular dynamics simulations reveal that both channels harbor a ceramide binding site on one side of the barrel wall. This site includes a membrane-buried glutamate that mediates direct contact with the ceramide head group. Substitution or chemical modification of this residue abolishes photolabeling of both channels with the ceramide probe. Unlike VDAC1 removal, loss of VDAC2 or replacing its membrane-facing glutamate with glutamine renders human colon cancer cells largely resistant to ceramide-induced apoptosis. Collectively, our data support a role of VDAC2 as direct effector of ceramide-mediated cell death, providing a molecular framework for how ceramides exert their anti-neoplastic activity.

Experimentally-induced maternal hypothyroidism alters enzyme activities and the sensorimotor cortex of the offspring rats.

In this study, we used an experimental model of congenital hypothyroidism to show that deficient thyroid hormones (TH) disrupt different neurochemical, morphological and functional aspects in the cerebral cortex of 15-day-old offspring. Our results showing decreased glutamine synthetase (GS) activity and Ca2+ overload in the cerebral cortex of hypothyroid pups suggest misregulated glutamate metabolism associated with developmentally induced TH deficiency. The 14C-MeAIB accumulation indicates upregulated System A activity and glutamine uptake by neurons. Energy metabolism in hypothyroid cortical slices was preserved, as demonstrated by unaltered glucose metabolism. We also found upregulated acetylcholinesterase activity, depleting acetylcholine from the synaptic cleft, pointing to disrupted cholinergic system. Increased reactive oxygen species (ROS) generation, lipid peroxidation, glutathione (GSH) depletion, which were associated with glutathione peroxidase, superoxide dismutase and gamma-glutamyltransferase downregulation suggest redox imbalance. Disrupted astrocyte cytoskeleton was evidenced by downregulated and hyperphosphorylated glial fibrillary acidic protein (GFAP). Morphological and structural characterization of the sensorimotor cerebral cortex (SCC) showed unaltered thickness of the SCC. However, decreased size of neurons on the layers II & III and IV in the right SCC and increased NeuN positive neurons in specific SCC layers, suggest that they are differently affected by the low TH levels during neurodevelopment. Hypothyroid pups presented increased number of foot-faults in the gridwalk test indicating affected motor functions. Taken together, our results show that congenital hypothyroidism disrupts glutamatergic and cholinergic neurotransmission, Ca2+ equilibrium, redox balance, cytoskeleton integrity, morphological and functional aspects in the cerebral cortex of young rats.

Proteomic response of gill microsomes of Crassostrea brasiliana exposed to diesel fuel water-accommodated fraction.

Diesel fuel water-accommodated fraction (diesel-WAF) is a complex mixture of organic compounds that may cause harmful effects to marine invertebrates. Expression of microsomal proteins can be changed by oil exposure, causing functional alterations in endoplasmic reticulum (ER). The aim of this study was to investigate changes in protein expression signatures in microsomes of oysterl Crassostrea brasiliana (=C.gasar) gill after exposure to 10% diesel-WAF for 24 and 72 h. Protein expression signatures of gills of oysters exposed to diesel-WAF were compared to those of unexposed oysters using two-dimensional electrophoresis (2-DE) to identify differentially expressed proteins. A total of 458 protein spots with molecular weights between 30-75 kDa were detected by 2-DE in six replicates of exposed oyster proteomes compared to unexposed ones. Fourteen differentially expressed proteins (six up-regulated and eight down-regulated) were identified. They are: proteins related to xenobiotic biotransformation (cytochrome P450 6 A, NADPH-cytochrome P450 reductase); cytoskeleton (α-tubulin, ß-tubulin, gelsolin); processing and degradation of proteins pathways (thioredoxin domain-containing protein E3 ubiquitin-protein ligase MIB2); involved in the biosynthesis of glycolipids and glycoproteins (beta-1,3-galactosyltransferase 1); associated with stress responses (glutamate receptor 4 and 14-3-3 protein zeta, corticotropin-releasing factor-binding protein); plasmalogen biosynthesis (fatty acyl-CoA reductase 1), and sodium-and chloride-dependent glycine transporter 2 and glyoxylate reductase/hydroxypyruvate reductase. Different patterns of protein responses were observed between 24 and 72 h-exposed groups. Expression pattern of microsomal proteins provided a first insight on the potential diesel-WAF effects at protein level in microsomal fraction of oyster gills and indicated new potential biomarkers of exposure and effect. The present work can be a basis for future ecotoxicological studies in oysters aiming to elucidate the molecular mechanisms behind diesel-WAF toxicity and for environmental monitoring programs.

Reverse T3 interacts with αvß3 integrin receptor and restores enzyme activities in the hippocampus of hypothyroid developing rats: Insight on signaling mechanisms.

In the present study we provide evidence that 3,3',5'-triiodothyronine (reverse T3, rT3) restores neurochemical parameters induced by congenital hypothyroidism in rat hippocampus. Congenital hypothyroidism was induced by adding 0.05% propylthiouracil in the drinking water from gestation day 8 and continually up to lactation day 15. In the in vivo rT3 exposure, hypothyroid 12-day old pups were daily injected with rT3 (50 ng/kg body weight) or saline until day 14. In the ex vivo rT3 treatment, hippocampal slices from 15-day-old hypothyroid pups were incubated for 30 min with or without rT3 (1 nM). We found that ex vivo and/or in vivo exposure to rT3 failed in restoring the decreased 14C-glutamate uptake; however, restored the phosphorylation of glial fibrillary acidic protein (GFAP), 45Ca2+ influx, aspartate transaminase (AST), glutamine synthetase (GS) and gamma-glutamate transferase (GGT) activities, as well as glutathione (GSH) levels in hypothyroid hippocampus. In addition, rT3 improved 14C-2-deoxy-D-glucose uptake and lactate dehydrogenase (LDH) activity. Receptor agonists/antagonists (RGD peptide and AP-5), kinase inhibitors of p38MAPK, ERK1/2, CaMKII, PKA (SB239063, PD98059, KN93 and H89, respectively), L-type voltage-dependent calcium channel blocker (nifedipine) and intracellular calcium chelator (BAPTA-AM) were used to determine the mechanisms of the nongenomic rT3 action on GGT activity. Using molecular docking analysis, we found rT3 interaction with αvß3 integrin receptors, nongenomically activating signaling pathways (PKA, CaMKII, p38MAPK) that restored GGT activity. We provide evidence that rT3 is an active TH metabolite and our results represent an important contribution to elucidate the nonclassical mechanism of action of this metabolite in hypothyroidism.

Candidate cytochrome P450 genes for ethoxyresorufin O-deethylase activity in oyster Crassostrea gigas.

Vertebrate cytochrome P450 1 (CYP1) enzymes metabolize endogenous and xenobiotic compounds and usually demonstrate a substrate-inducible response. Ethoxyresorufin O-deethylase activity (EROD) is a common method to quantify CYP1 enzymes activity in these organisms. Despite the absence of this gene family in protostomes, CYP1-like genes were identified in several species, even though no evolutionary relationship has been established with the vertebrate CYP1 family. In the present study, EROD activity was evaluated in microsomal fraction of gills, digestive gland and mantle of Crassostrea gigas. Enzyme activity was quantified in gills, although no activity was detected in digestive gland and mantle. EROD kinetic characterization in gills using typical Michaelis-Menten equation demonstrated an apparent Km of 1.15µM and Vmax of 229.2 fmol.min-1mg.protein -1. EROD activity was analyzed in the presence of CYP1 inhibitors, ellipticine (ELP), furafylline (FRF), clotrimazole (CTZ), α-naphthoflavone (ANF), and the non-ionic surfactant Triton X-100. CTZ inhibited EROD activity in all tested concentrations while Triton X-100 (0.5mM) caused 16% inhibition. Transcript levels of four CYP1-like genes were determined in gills, digestive gland and mantle. In general, CYP1-like genes showed higher transcript levels in gills compared to other tissues. The transcript levels of CYP1-like 1 and 2, analyzed together, positively correlated with EROD activity observed in gills, suggesting the possible involvement of these two gene products in EROD activity in this tissue. Homology models of translated CYP1-like 1 and 2 were generated based on human CYP1A1 structure and were similar to the general canonical cytochrome P450 fold. Molecular docking analysis showed that the two putative oyster CYP1-like structures have the potential to metabolize 7-ethoxyresorufin (7-ER), although the contribution of other CYP1-like genes needs to be investigated. Proteins encoded by CYP1-like 1 and 2 genes are plausible candidates for EROD activity observed in gills of C. gigas.

Developmental exposure to glyphosate-based herbicide and depressive-like behavior in adult offspring: Implication of glutamate excitotoxicity and oxidative stress.

We have previously demonstrated that maternal exposure to glyphosate-based herbicide (GBH) leads to glutamate excitotoxicity in 15-day-old rat hippocampus. The present study was conducted in order to investigate the effects of subchronic exposure to GBH on some neurochemical and behavioral parameters in immature and adult offspring. Rats were exposed to 1% GBH in drinking water (corresponding to 0.36% of glyphosate) from gestational day 5 until postnatal day (PND)-15 or PND60. Results showed that GBH exposure during both prenatal and postnatal periods causes oxidative stress, affects cholinergic and glutamatergic neurotransmission in offspring hippocampus from immature and adult rats. The subchronic exposure to the pesticide decreased L-[14C]-glutamate uptake and increased 45Ca2+ influx in 60-day-old rat hippocampus, suggesting a persistent glutamate excitotoxicity from developmental period (PND15) to adulthood (PND60). Moreover, GBH exposure alters the serum levels of the astrocytic protein S100B. The effects of GBH exposure were associated with oxidative stress and depressive-like behavior in offspring on PND60, as demonstrated by the prolonged immobility time and decreased time of climbing observed in forced swimming test. The mechanisms underlying the GBH-induced neurotoxicity involve the NMDA receptor activation, impairment of cholinergic transmission, astrocyte dysfunction, ERK1/2 overactivation, decreased p65 NF-κB phosphorylation, which are associated with oxidative stress and glutamate excitotoxicity. These neurochemical events may contribute, at least in part, to the depressive-like behavior observed in adult offspring.

Intracellular lipid binding protein family diversity from Oyster Crassostrea gigas: genomic and structural features of invertebrate lipid transporters.

Intracellular lipid binding proteins (iLBPs) play a role in the transport and cellular uptake of fatty acids and gene expression regulation. The aim of this work was to characterize the iLBP gene family of the Pacific oyster Crassostrea gigas, one of the most cultivated marine bivalves in the world, using bioinformatics and molecular biology approaches. A total of 26 different iLBPs transcripts were identified in the Pacific oyster genome, including alternative splicing and gene duplication events. The oyster iLBP gene family seems to be more expanded than in other invertebrates. Furthermore, 3D structural modeling and molecular docking analysis mapped the main amino acids involved in ligand interactions, and comparisons to available protein structures from vertebrate families revealed new binding cavities. Ten different CgiLBPs were analyzed by quantitative PCR in various tissues of C. gigas, which suggested differential prevalent gene expression of CgiLBPs among tissue groups. The data indicate a wider repertoire of iLBPs in labial palps, a food-sorting tissue. The different gene transcription profiles and reported docking systems suggest that the iLBPs are a non-generalist ligand binding protein family with specific functions.

Thiol oxidation of hemolymph proteins in oysters Crassostrea brasiliana as markers of oxidative damage induced by urban sewage exposure.

Urban sewage is a concerning issue worldwide, threatening both wildlife and human health. The present study investigated protein oxidation in mangrove oysters (Crassostrea brasiliana) exposed to seawater from Balneário Camboriú, an important tourist destination in Brazil that is affected by urban sewage. Oysters were exposed for 24 h to seawater collected close to the Camboriú River (CAM1) or 1 km away (CAM2). Seawater from an aquaculture laboratory was used as a reference. Local sewage input was marked by higher levels of coliforms, nitrogen, and phosphorus in seawater, as well as polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), linear alkylbenzenes (LABs), and fecal steroid in sediments at CAM1. Exposure of oysters to CAM1 caused marked bioaccumulation of LABs and decreased PAH and PCB concentrations after exposure to both CAM1 and CAM2. Protein thiol oxidation in gills, digestive gland, and hemolymph was evaluated. Lower levels of reduced protein thiols were detected in hemolymph from CAM1, and actin, segon, and dominin were identified as targets of protein thiol oxidation. Dominin susceptibility to oxidation was confirmed in vitro by exposure to peroxides and hypochlorous acid, and 2 cysteine residues were identified as potential sites of oxidation. Overall, these data indicate that urban sewage contamination in local waters has a toxic potential and that protein thiol oxidation in hemolymph could be a useful biomarker of oxidative stress in bivalves exposed to contaminants. Environ Toxicol Chem 2017;36:1833-1845. © 2016 SETAC.

New potential eukaryotic substrates of the mycobacterial protein tyrosine phosphatase PtpA: hints of a bacterial modulation of macrophage bioenergetics state.

The bacterial protein tyrosine phosphatase PtpA is a key virulence factor released by Mycobacterium tuberculosis in the cytosol of infected macrophages. So far only two unrelated macrophage components (VPS33B, GSK3α) have been identified as PtpA substrates. As tyrosine phosphatases are capable of using multiple substrates, we developed an improved methodology to pull down novel PtpA substrates from an enriched P-Y macrophage extract using the mutant PtpA D126A. This methodology reduced non-specific protein interactions allowing the identification of four novel putative PtpA substrates by MALDI-TOF-MS and nano LC-MS: three mitochondrial proteins - the trifunctional enzyme (TFP), the ATP synthase, and the sulfide quinone oxidoreductase - and the cytosolic 6-phosphofructokinase. All these proteins play a relevant role in cell energy metabolism. Using surface plasmon resonance, PtpA was found to bind immunopurified human TFP through its catalytic site since TFP-PtpA association was inhibited by a specific phosphatase inhibitor. Moreover, PtpA wt was capable of dephosphorylating immunopurified human TFP in vitro supporting that TFP may be a bona fide PtpA susbtrate. Overall, these results suggest a novel scenario where PtpA-mediated dephosphorylation may affect pathways involved in cell energy metabolism, particularly the beta oxidation of fatty acids through modulation of TFP activity and/or cell distribution.

S-nitrosylation of Mycobacterium tuberculosis tyrosine phosphatase A (PtpA) induces its structural instability.

S-nitrosylation is associated with signal transduction and microbicidal activity of nitric oxide (NO). We have recently described the S-nitrosylation of Mycobacterium tuberculosis protein tyrosine phosphatase A, PtpA, an enzyme that plays an important role in mycobacteria survival inside macrophages. This post-translational modification decreases the activity of the enzyme upon modification of a single Cys residue, C53. The aim of the present work was the investigation of the effect of S-nitrosylation in PtpA kinetic parameters, thermal stability and structure. It was observed that the K(M) of nitrosylated PtpA was similar to its unmodified form, but the V(max) was significantly reduced. In contrast, treatment of PtpA C53A with GSNO, did not alter either K(M) or V(max). These results confirmed that PtpA S-nitrosylation occurs specifically in the non-catalytic C53 and that this modification does not affect substrate affinity. Using circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy techniques it was shown that PtpA S-nitrosylation decreased protein thermal stability and promoted a local effect in the surroundings of the C53 residue, which interfered in both protein stability and function.

Spectroscopic and catalytic characterization of a functional Fe(III)Fe(II) biomimetic for the active site of uteroferrin and protein cleavage.

A mixed-valence complex, [Fe(III)Fe(II)L1(µ-OAc)(2)]BF(4)·H(2)O, where the ligand H(2)L1 = 2-{[[3-[((bis(pyridin-2-ylmethyl)amino)methyl)-2-hydroxy-5-methylbenzyl](pyridin-2-ylmethyl)amino]methyl]phenol}, has been studied with a range of techniques, and, where possible, its properties have been compared to those of the corresponding enzyme system purple acid phosphatase. The Fe(III)Fe(II) and Fe(III)(2) oxidized species were studied spectroelectrochemically. The temperature-dependent population of the S = 3/2 spin states of the heterovalent system, observed using magnetic circular dichroism, confirmed that the dinuclear center is weakly antiferromagnetically coupled (H = -2JS(1)·S(2), where J = -5.6 cm(-1)) in a frozen solution. The ligand-to-metal charge-transfer transitions are correlated with density functional theory calculations. The Fe(III)Fe(II) complex is electron paramagnetic resonance (EPR)-silent, except at very low temperatures (<2 K), because of the broadening caused by the exchange coupling and zero-field-splitting parameters being of comparable magnitude and rapid spin-lattice relaxation. However, a phosphate-bound Fe(III)(2) complex showed an EPR spectrum due to population of the S(tot) = 3 state (J= -3.5 cm(-1)). The phosphatase activity of the Fe(III)Fe(II) complex in hydrolysis of bis(2,4-dinitrophenyl)phosphate (k(cat.) = 1.88 × 10(-3) s(-1); K(m) = 4.63 × 10(-3) mol L(-1)) is similar to that of other bimetallic heterovalent complexes with the same ligand. Analysis of the kinetic data supports a mechanism where the initiating nucleophile in the phosphatase reaction is a hydroxide, terminally bound to Fe(III). It is interesting to note that aqueous solutions of [Fe(III)Fe(II)L1(µ-OAc)(2)](+) are also capable of protein cleavage, at mild temperature and pH conditions, thus further expanding the scope of this complex's catalytic promiscuity.

Heat stability of Proteobacterial PII protein facilitate purification using a single chromatography step.

The P(II) proteins comprise a family of widely distributed signal transduction proteins that integrate the signals of cellular nitrogen, carbon and energy status, and then regulate, by protein-protein interaction, the activity of a variety of target proteins including enzymes, transcriptional regulators and membrane transporters. We have previously shown that the P(II) proteins from Azospirillum brasilense, GlnB and GlnZ, do not alter their migration behavior under native gel electrophoresis following incubated for a few minutes at 95°C. This data suggested that P(II) proteins were either resistant to high temperatures and/or that they could return to their native state after having been unfolded by heat. Here we used (1)H NMR to show that the A. brasilense GlnB is stable up to 70°C. The melting temperature (Tm) of GlnB was determined to be 84°C using the fluorescent dye Sypro-Orange. P(II) proteins from other Proteobacteria also showed a high Tm. We exploited the thermo stability of P(II) by introducing a thermal treatment step in the P(II) purification protocol, this step significantly improved the homogeneity of A. brasilense GlnB and GlnZ, Herbaspirillum seropedicae GlnB and GlnK, and of Escherichia coli GlnK. Only a single chromatography step was necessary to obtain homogeneities higher than 95%. NMR(1) and in vitro uridylylation analysis showed that A. brasilense GlnB purified using the thermal treatment maintained its folding and activity. The purification protocol described here can facilitate the study of P(II) protein family members.