RESUMO
DapE is a Zn2+-metallohydrolase recognized as a drug target for bacterial control. It is a homodimer that requires the exchange of interface strands by an induced fit essential for catalysis. Identifying novel anti-DapE agents requires greater structural details. Most of the characterized DapEs are from the Gram-negative group. Here, two high-resolution DapE crystal structures from Enterococcus faecium are presented for the first time with novel aspects. A loosened enzyme intermediate between the open and closed conformations is observed. Substrates may bind to loose state, subsequently it closes, where hydrolysis occurs, and finally, the change to the open state leads to the release of the products. Mutation of His352 suggests a role, along with His194, in the oxyanion stabilization in the mono-metalated Zn2+ isoform, while in the di-metalated isoform, the metal center 2 complements it function. An aromatic-π box potentially involved in the interaction of DapE with other proteins, and a peptide flip could determine the specificity in the Gram-positive ArgE/DapE group. Finally, details of two extra-catalytic cavities whose geometry changes depending on the conformational state of the enzyme are presented. These cavities could be a target for developing non-competitive agents that trap the enzyme in an inactive state.
Assuntos
Proteínas de Bactérias , Enterococcus faecium , Enterococcus faecium/enzimologia , Especificidade por Substrato , Ligantes , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Modelos Moleculares , Conformação Proteica , Zinco/química , Zinco/metabolismo , Domínio Catalítico , Amidoidrolases/química , Amidoidrolases/metabolismo , Amidoidrolases/genética , Cristalografia por Raios X , Sequência de Aminoácidos , Ligação ProteicaRESUMO
In higher eukaryotes and plants, the last two sequential steps in the de novo biosynthesis of uridine 5'-monophosphate (UMP) are catalyzed by a bifunctional natural chimeric protein called UMP synthase (UMPS). In higher plants, UMPS consists of two naturally fused enzymes: orotate phosphoribosyltransferase (OPRTase) at N-terminal and orotidine-5'-monophosphate decarboxylase (ODCase) at C-terminal. In this work, we obtained the full functional recombinant protein UMPS from Coffea arabica (CaUMPS) and studied its structure-function relationships. A biochemical and structural characterization of a plant UMPS with its two functional domains is described together with the presentation of the first crystal structure of a plant ODCase at 1.4 Å resolution. The kinetic parameters measured of CaOPRTase and CaODCase domains were comparable to those reported. The crystallographic structure revealed that CaODCase is a dimer that conserves the typical fold observed in other ODCases from prokaryote and eukaryote with a 1-deoxy-ribofuranose-5'-phosphate molecule bound in the active site of one subunit induced a closed conformation. Our results add to the knowledge of one of the key enzymes of the de novo biosynthesis of pyrimidines in plant metabolism and open the door to future applications.
Assuntos
Carboxiliases , Coffea , Orotato Fosforribosiltransferase/química , Orotato Fosforribosiltransferase/metabolismo , Orotidina-5'-Fosfato Descarboxilase/genética , Orotidina-5'-Fosfato Descarboxilase/química , Orotidina-5'-Fosfato Descarboxilase/metabolismo , Complexos Multienzimáticos/química , Proteínas Recombinantes/genética , Uridina MonofosfatoRESUMO
Psychological distress induces oxidative stress and alters mitochondrial metabolism in the nervous and immune systems. Psychological distress promotes alterations in brain metabolism and neurochemistry in wild-type (WT) rats in a similar manner as in Parkinsonian rats lacking endogenous PTEN-induced kinase 1 (PINK1), a serine/threonine kinase mutated in a recessive forms of Parkinson's disease. PINK1 has been extensively studied in the brain, but its physiological role in peripheral tissues and the extent to which it intersects with the neuroimmune axis is not clear. We surmised that PINK1 modulates the bioenergetics of peripheral blood mononuclear cells (PBMCs) under basal conditions or in situations that promote oxidative stress as psychological distress. By using an XF metabolic bioanalyzer, PINK1-KO-PBMCs showed significantly increased oxidative phosphorylation and basal glycolysis compared to WT cells and correlated with motor dysfunction. In addition, psychological distress enhanced the glycolytic capacity in PINK1-KO-PBMCs but not in WT-PBMCs. The level of antioxidant markers and brain-derived neurotrophic factor were altered in PINK1-KO-PBMCs and by psychological distress. In summary, our data suggest that PINK1 is critical for modulating the bioenergetics and antioxidant responses in PBMCs whereas lack of PINK1 upregulates compensatory glycolysis in response to oxidative stress induced by psychological distress.
Assuntos
Metabolismo Energético , Leucócitos Mononucleares/metabolismo , Proteínas Quinases/deficiência , Angústia Psicológica , Animais , Antioxidantes/metabolismo , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Respiração Celular , Feminino , Regulação da Expressão Gênica , Glicólise , Masculino , Mitocôndrias/metabolismo , RatosRESUMO
Chronic psychological stress is an important public health issue which generates behavioral changes, anxiety, immunosuppression and oxidative damage. Piracetam is a cognitive enhancer, at cellular level it protects from oxidative stress. The aim of this study was to evaluate the effect of psychological stress and of piracetam on circulating mononuclear cells by analyzing the biochemical spectrome using Synchrotron Radiation Fourier Transform Infrared Microspectroscopy (SR-µFTIR). Rats were exposed for five days to a stressor (cat odor) under oral administration of piracetam (600â¯mg/kg). SR-µFTIR analysis showed a decrease in bands associated to the lipids region (2852â¯cm-1, 2923â¯cm-1 and 2962â¯cm-1) and an increase absorption of the amide I band (1654â¯cm-1) under stress conditions. The principal component analysis showed increase oxidation of lipids (decrease of 3010â¯cm-1, 2923â¯cm-1 and 2852â¯cm-1 bands) as well as proteins denaturation (increase of 1610â¯cm-1 and 1690â¯cm-1 bands) under stress. Piracetam provided protection to polyunsaturated lipids (pâ¯≤â¯0.001) and lipids/proteins ratio (pâ¯≤â¯0.001). Behaviorally, this drug diminished fear and anxiety in stressed animals by the plus maze test (pâ¯≤â¯0.002). However, this drug induced oxidative stress in mononuclear cells from unstressed animals and altered their behavior.
Assuntos
Leucócitos Mononucleares/efeitos dos fármacos , Nootrópicos , Piracetam , Estresse Psicológico/sangue , Administração Oral , Animais , Biomarcadores/sangue , Feminino , Peroxidação de Lipídeos/efeitos dos fármacos , Lipídeos/sangue , Nootrópicos/administração & dosagem , Nootrópicos/farmacologia , Piracetam/administração & dosagem , Piracetam/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
The plasma membrane Ca2+-ATPase (PMCA) removes Ca2+ from the cytosol into the extracellular space. Its catalytic activity can be stimulated by calmodulin (CaM) or by limited proteolysis. We evaluated the effect of chlorpromazine (CPZ) and dimethyl sulfoxide (DMSO) over the hydrolytic activity of PMCA. Activity was monitored in three different forms: native, CaM-activated and proteolyzed by trypsin. CPZ appears to inhibit PMCA without directly interfering with the C-terminal site, since it is affected by CaM and proteolysis. Although the treatment of PMCA with trypsin and CaM produces an activation, it also produces an enzymatic form that is more sensitive to inhibition by CPZ. The same case was observed in the DMSO inhibition experiments. In the absence of CPZ, DMSO produces a progressive loss of activity, but in the presence of CPZ the profile of activity against DMSO changes and produces a recovery of activity, indicating a possible partition of CPZ by the solvent. Increasing Ca2+ concentrations indicated that CPZ interacts with PMCA rather than with CaM. This observation is supported by docking analysis that suggests that the CPZ-PMCA interaction is non-competitive. We propose that CPZ interacts with the state of lower affinity for Ca2 +.
Assuntos
Clorpromazina/farmacologia , Dimetil Sulfóxido/farmacologia , Membrana Eritrocítica/enzimologia , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Biocatálise/efeitos dos fármacos , Calmodulina/farmacologia , Antagonistas de Dopamina/farmacologia , Interações Medicamentosas , Humanos , Simulação de Acoplamento Molecular , Tripsina/farmacologiaRESUMO
The digestive enzymes-polyphenolic compounds (PCs) interactions behind the inhibition of these enzymes have not been completely studied. The existing studies have mainly analyzed polyphenolic extracts and reported inhibition percentages of catalytic activities determined by UV-Vis spectroscopy techniques. Recently, pure PCs and new methods such as isothermal titration calorimetry and circular dichroism have been applied to describe these interactions. The present review focuses on PCs structural characteristics behind the inhibition of digestive enzymes, and progress of the used methods. Some characteristics such as molecular weight, number and position of substitution, and glycosylation of flavonoids seem to be related to the inhibitory effect of PCs; also, this effect seems to be different for carbohydrate-hydrolyzing enzymes and proteases. The digestive enzyme-PCs molecular interactions have shown that non-covalent binding, mostly by van der Waals forces, hydrogen binding, hydrophobic binding, and other electrostatic forces regulate them. These interactions were mainly associated to non-competitive type inhibitions of the enzymatic activities. The present review emphasizes on the digestive enzymes such as α-glycosidase (AG), α-amylase (PA), lipase (PL), pepsin (PE), trypsin (TP), and chymotrypsin (CT). Existing studies conducted in vitro allow one to elucidate the characteristics of the structure-function relationships, where differences between the structures of PCs might be the reason for different in vivo effects.
Assuntos
Hidrolases/química , Polifenóis/química , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Ligação Proteica , TermodinâmicaRESUMO
The inhibitory activity and binding characteristics of caffeic acid, p-coumaric acid, quercetin and capsaicin, four phenolic compounds found in hot pepper, against porcine pancreatic lipase activity were studied and compared to hot pepper extract. Quercetin was the strongest inhibitor (IC50=(6.1±2.4) µM), followed by p-coumaric acid ((170.2±20.6) µM) and caffeic acid ((401.5±32.1) µM), while capsaicin and a hot pepper extract had very low inhibitory activity. All polyphenolic compounds showed a mixed-type inhibition. Fluorescence spectroscopy studies showed that polyphenolic compounds had the ability to quench the intrinsic fluorescence of pancreatic lipase by a static mechanism. The sequence of Stern-Volmer constant was quercetin, followed by caffeic and p-coumaric acids. Molecular docking studies showed that caffeic acid, quercetin and p-coumaric acid bound near the active site, while capsaicin bound far away from the active site. Hydrogen bonds and π-stacking hydrophobic interactions are the main pancreatic lipase-polyphenolic compound interactions observed.
RESUMO
Mutations the in human DJ-1 (hDJ-1) gene are associated with early-onset autosomal recessive forms of Parkinson's disease (PD). hDJ-1/parkinsonism associated deglycase (PARK7) is a cytoprotective multi-functional protein that contains a conserved cysteine-protease domain. Given that cysteine-proteases can act on both amide and ester substrates, we surmised that hDJ-1 possessed cysteine-mediated esterase activity. To test this hypothesis, hDJ-1 was overexpressed, purified and tested for activity towards 4-nitrophenyl acetate (pNPA) as µmol of pNPA hydrolyzed/min/mg·protein (U/mg protein). hDJ-1 showed maximum reaction velocity esterase activity (Vmax = 235.10 ± 12.00 U/mg protein), with a sigmoidal fit (S0.5 = 0.55 ± 0.040 mM) and apparent positive cooperativity (Hill coefficient of 2.05 ± 0.28). A PD-associated mutant of DJ-1 (M26I) lacked activity. Unlike its protease activity which is inactivated by reactive oxygen species (ROS), esterase activity of hDJ-1 is enhanced upon exposure to low concentrations of hydrogen peroxide (<10 µM) and plateaus at elevated concentrations (>100 µM) suggesting that its activity is resistant to oxidative stress. Esterase activity of DJ-1 requires oxidation of catalytic cysteines, as chemically protecting cysteines blocked its activity whereas an oxido-mimetic mutant of DJ-1 (C106D) exhibited robust esterase activity. Molecular docking studies suggest that C106 and L126 within its catalytic site interact with esterase substrates. Overall, our data show that hDJ-1 contains intrinsic redox-sensitive esterase activity that is abolished in a PD-associated mutant form of the hDJ-1 protein.
Assuntos
Esterases/química , Esterases/metabolismo , Doença de Parkinson/enzimologia , Esterases/genética , Humanos , Peróxido de Hidrogênio/farmacologia , Simulação de Acoplamento Molecular , Mutação , Nitrofenóis/metabolismo , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Doença de Parkinson/genética , Proteína Desglicase DJ-1/química , Proteína Desglicase DJ-1/genética , Proteína Desglicase DJ-1/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Plants express chitinase and chitinase-like proteins (CLPs) belonging to the glycosyl hydrolases of the GH18 and GH19 families, which exhibit varied functions. CLPs in the GH18 family have been structurally and functionally characterized; however, there are no structures available for any member of the GH19 family. In this study, two CLPs of the GH19 family from the rubber tree Hevea brasiliensis (HbCLP1 and HbCLP2) were cloned, expressed and characterized. HbCLP1 was identical to the allergen Hev b 11.0101 previously described by others, while HbCLP2 was a novel isoform exhibiting an unusual half chitin-binding domain before the catalytic domain. Sequence alignments showed that in the two proteins the catalytic residues Glu117 and Glu147 in HbCLP1 and HbCLP2, respectively, were mutated to Ala, accounting for the lack of activity. Nonetheless, both CLPs bound chitin and chitotriose (GlcNAc)3 with high affinities, as evaluated with chitin-affinity chromatography and tryptophan fluorescence experiments. The chitin-binding domains also bound chitotriose with even higher affinities. The crystal structures of the HbCLP1-isolated domains were determined at high resolution. The analysis of the crystallographic models and docking experiments using (GlcNAc)6 oligosaccharides provides evidence of the residues involved in sugar binding. Endochitinase activity was restored in both proteins by mutating residues A117E (HbCLP1) and A147E (HbCLP2); the distance between the catalytic proton donor and the catalytic nucleophile in the in silico mutated residues was 9.5 Å, as occurs in inverting enzymes. HbCLP1 and HbCLP2 were highly thermostable and exhibited antifungal activity against Alternaria alternata, suggesting their participation in plant defense mechanisms.
Assuntos
Quitina/química , Quitinases/química , Hevea/enzimologia , Proteínas de Plantas/química , Alternaria/efeitos dos fármacos , Sequência de Aminoácidos , Antifúngicos/química , Antifúngicos/farmacologia , Sequência de Bases , Domínio Catalítico , Quitinases/biossíntese , Quitinases/genética , Quitinases/farmacologia , Clonagem Molecular , Sequência Conservada , Cristalografia por Raios X , Estabilidade Enzimática , Hidrólise , Testes de Sensibilidade Microbiana , Anotação de Sequência Molecular , Dados de Sequência Molecular , Folhas de Planta/enzimologia , Proteínas de Plantas/biossíntese , Proteínas de Plantas/genética , Proteínas de Plantas/farmacologia , Ligação Proteica , Estrutura Secundária de ProteínaRESUMO
Potassium ions are non-essential activators of several aldehyde dehydrogenases (ALDHs), whereas a few others require the cation for activity. Two kinds of cation-binding sites, which we named intra-subunit and inter-subunit, have been observed in crystal structures of ALDHs, and based on reported crystallographic data, we here propose the existence of a third kind located in the central cavity of some tetrameric ALDHs. Given the high structural similarity between these enzymes, cation-binding sites may be present in many other members of this superfamily. To explore the prevalence of these sites, we compared 37 known crystal structures from 13 different ALDH families and evaluated the possible existence of a cation on the basis of the number, distance and geometry of its potential interactions, as well as of B-factor values of modeled cations obtained in new refinements of some reported crystal structures. Also, by performing multiple alignments of 855 non-redundant amino acid sequences, we assessed the degree of conservation in their respective families of the amino acid residues putatively relevant for cation binding. Among the ALDH enzymes studied, and according to our analyses, potential intra-subunit cation-binding sites seem to be present in most members of ALDH2, ALDH1L, ALDH4, ALDH5, ALDH7, ALDH10, and ALDH25 families, as well as in the bacterial and fungal members of the ALDH9 family and in a few ALDH1, ALDH6, ALDH11 and ALDH26 enzymes; potential inter-subunit sites in members of ALDH1L, ALDH3, ALDH4 from bacillales, ALDH5, ALDH7, ALDH9, ALDH10, ALDH11 and ALDH25 families; and potential central-cavity sites only in some bacterial and animal ALDH9s and in most members of the ALDH1L family. Because potassium is the most abundant intracellular cation, we propose that these are potassium-binding sites, but the specific structural and/or functional roles of the cation bound to these different sites remain to be investigated.