Molecular, kinetic, thermodynamic, and structural analyses of Mycobacterium tuberculosis hisD-encoded metal-dependent dimeric histidinol dehydrogenase (EC 1.1.1.23)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Activation of Ca2+-independent membrane-damaging activity in Lys49-phospholipase A(2) promoted by amphiphilic molecules

Association of class-II phospholipase A(2) (PLA(2)) with aggregated phospholipid substrate results in elevated levels of the Ca2+-dependent hydrolytic activity. The Asp49 residue participates in coordination of the Ca2+ ion cofactor, however, in Lys49-PLA(2) homologues (Lys49-PLA(2)S), substitution of the Asp49 by Lys results in loss of Ca2+ binding and lack of detectable phospholipid hydrolysis. Nevertheless, Lys49-PLA2S cause Ca2+-independent damage of liposome membranes. Bothropstoxin-I is a homodimeric Lys49-PLA(2) from the venom of Bothrops jararacussu, and in fluorescent marker release and dynamic light scattering experiments with DPPC liposomes we demonstrate activation of the Ca2+-independent membrane damaging activity by similar to4 molecules of sodium dodecyl sulphate (SDS) per protein monomer. Activation is accomparlied by significant changes in the intrinsic tryptophan fluorescence emission (ITFE) and near UV circular dichroism (UVCD) spectra of the protein. Subsequent binding of 7-10 SDS molecules results in further alterations in the ITFE and far UVCD spectra. Reduction in the rate of N-bromosuccinimide modification of Trp77 at the dimer interface suggests that initial binding of SDS to this region accompanies the activation of the membrane damaging activity. 1-anilinonaphthalene-8-sulphonic acid binding studies indicate that subsequent SDS binding to the active site is concomitant with the second structural transition. These results provide insights in the structural basis of amphiphile/protein coupling in class-II PLA(2)s. (C) 2004 Published by Elsevier B.V.

Localization of peroxidase activity in blood mononuclear phagocytes in pacu fish (Piaractus mesopotamicus)

The localization of peroxidase activity in different cell regions is used as a criterion for classifying the stage of maturity of mammalian mononuclear phagocytes, with a positive peroxidase reaction indicating the presence of monoblasts, promonocytes, monocytes, and macrophages. Peroxidase activity was observed ultrastructurally in the circulating blood of pacu fish (Piaractus mesopotamicus), identifying monoblasts, promonocytes, monocytes, and macrophages. These observations suggest that differentiation of mononuclear phagocytes occurs in the blood circulation of fish, whereas in mammals, monoblasts and promonocytes are detected in bone marrow, with only monocytes detected in circulating blood and differentiation into macrophages occurring in other body compartments.

Chorismate synthase: An attractive target for drug development against orphan diseases

The increase in incidence of infectious diseases worldwide, particularly in developing countries, is worrying. Each year, 14 million people are killed by infectious diseases, mainly HIV/AIDS, respiratory infections, malaria and tuberculosis. Despite the great burden in the poor countries, drug discovery to treat tropical diseases has come to a standstill. There is no interest by the pharmaceutical industry in drug development against the major diseases of the poor countries, since the financial return cannot be guaranteed. This has created an urgent need for new therapeutics to neglected diseases. A possible approach has been the exploitation of the inhibition of unique targets, vital to the pathogen such as the shikimate pathway enzymes, which are present in bacteria, fungi and apicomplexan parasites but are absent in mammals. The chorismate synthase (CS) catalyses the seventh step in this pathway, the conversion of 5-enolpyruvylshikimate-3-phosphate to chorismate. The strict requirement for a reduced flavin mononucleotide and the anti 1,4 elimination are both unusual aspects which make CS reaction unique among flavin-dependent enzymes, representing an important target for the chemotherapeutic agents development. In this review we present the main biochemical features of CS from bacterial and fungal sources and their difference from the apicomplexan CS. The CS mechanisms proposed are discussed and compared with structural data. The CS structures of some organisms are compared and their distinct features analyzed. Some known CS inhibitors are presented and the main characteristics are discussed. The structural and kinetics data reviewed here can be useful for the design of inhibitors. © 2007 Bentham Science Publishers Ltd.

The inhibition of 5-enolpyruvylshikimate-3-phosphate synthase as a model for development of novel antimicrobials

EPSP synthase (EPSPS) is an essential enzyme in the shikimate pathway, transferring the enolpyruvyl group of phosphoenolpyruvate to shikimate-3-phosphate to form 5-enolpyruvyl-3-shikimate phosphate and inorganic phosphate. This enzyme is composed of two domains, which are formed by three copies of βαβαββ-folding units; in between there are two crossover chain segments hinging the nearly topologically symmetrical domains together and allowing conformational changes necessary for substrate conversion. The reaction is ordered with shikimate-3-phosphate binding first, followed by phosphoenolpyruvate, and then by the subsequent release of phosphate and EPSP. N-[phosphomethyl]glycine (glyphosate) is the commercial inhibitor of this enzyme. Apparently, the binding of shikimate-3-phosphate is necessary for glyphosate binding, since it induces the closure of the two domains to form the active site in the interdomain cleft. However, it is somehow controversial whether binding of shikimate-3-phosphate alone is enough to induce the complete conversion to the closed state. The phosphoenolpyruvate binding site seems to be located mainly on the C-terminal domain, while the binding site of shikimate-3-phosphate is located primarily in the N-terminal domain residues. However, recent results demonstrate that the active site of the enzyme undergoes structural changes upon inhibitor binding on a scale that cannot be predicted by conventional computational methods. Studies of molecular docking based on the interaction of known EPSPS structures with (R)- phosphonate TI analogue reveal that more experimental data on the structure and dynamics of various EPSPS-ligand complexes are needed to more effectively apply structure-based drug design of this enzyme in the future. © 2007 Bentham Science Publishers Ltd.

The Mycobacterium tuberculosis Rv2540c DNA sequence encodes a bifunctional chorismate synthase

Background. The emergence of multi- and extensively-drug resistant Mycobacterium tuberculosis strains has created an urgent need for new agents to treat tuberculosis (TB). The enzymes of shikimate pathway are attractive targets to the development of antitubercular agents because it is essential for M. tuberculosis and is absent from humans. Chorismate synthase (CS) is the seventh enzyme of this route and catalyzes the NADH- and FMN-dependent synthesis of chorismate, a precursor of aromatic amino acids, naphthoquinones, menaquinones, and mycobactins. Although the M. tuberculosis Rv2540c (aroF) sequence has been annotated to encode a chorismate synthase, there has been no report on its correct assignment and functional characterization of its protein product. Results. In the present work, we describe DNA amplification of aroF-encoded CS from M. tuberculosis (MtCS), molecular cloning, protein expression, and purification to homogeneity. N-terminal amino acid sequencing, mass spectrometry and gel filtration chromatography were employed to determine identity, subunit molecular weight and oligomeric state in solution of homogeneous recombinant MtCS. The bifunctionality of MtCS was determined by measurements of both chorismate synthase and NADH:FMN oxidoreductase activities. The flavin reductase activity was characterized, showing the existence of a complex between FMN ox and MtCS. FMNox and NADH equilibrium binding was measured. Primary deuterium, solvent and multiple kinetic isotope effects are described and suggest distinct steps for hydride and proton transfers, with the former being more rate-limiting. Conclusion. This is the first report showing that a bacterial CS is bifunctional. Primary deuterium kinetic isotope effects show that C4-proS hydrogen is being transferred during the reduction of FMNox by NADH and that hydride transfer contributes significantly to the rate-limiting step of FMN reduction reaction. Solvent kinetic isotope effects and proton inventory results indicate that proton transfer from solvent partially limits the rate of FMN reduction and that a single proton transfer gives rise to the observed solvent isotope effect. Multiple isotope effects suggest a stepwise mechanism for the reduction of FMNox. The results on enzyme kinetics described here provide evidence for the mode of action of MtCS and should thus pave the way for the rational design of antitubercular agents. © 2008 Ely et al; licensee BioMed Central Ltd.

The kinetic mechanism of human uridine phosphorylase 1: Towards the development of enzyme inhibitors for cancer chemotherapy

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Mechanism and control of Genipa americana seed germination

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Structural insights into the catalytic mechanism of sphingomyelinases D and evolutionary relationship to glycerophosphodiester phosphodiesterases

Spider venom sphingomyelinases D catalyze the hydrolysis of sphingomyelin via an Mg2+ ion-dependent acid-base catalytic mechanism which involves two histidines. In the crystal structure of the sulfate free enzyme determined at 1.85 angstrom resolution, the metal ion is tetrahedrally coordinated instead of the trigonal-bipyramidal coordination observed in the sulfate bound form. The observed hyperpolarized state of His47 requires a revision of the previously suggested catalytic mechanism. Molecular modeling indicates that the fundamental structural features important for catalysis are fully conserved in both classes of SMases D and that the Class II SMases D contain an additional intra-chain disulphide bridge (Cys53-Cys201). Structural analysis suggests that the highly homologous enzyme from Loxosceles bonetti is unable to hydrolyze sphingomyelin due to the 95G1y -> Asn and 134Pro -> Glu mutations that modify the local charge and hydrophobicity of the interfacial face. Structural and sequence comparisons confirm the evolutionary relationship between sphingomyelinases D and the glicerophosphodiester phosphoesterases which utilize a similar catalytic mechanism. (c) 2006 Elsevier B.V. All rights reserved.

The interaction between heparin and Lys49 phospholipase A(2) reveals the natural binding of heparin on the enzyme

We have studied at a molecular level the interaction of heparins on bothropstoxin-1 (BthTx-1), a phospholipase A(2) toxin. The protein was monitored using gel filtration chromatography, dynamic light scattering (DLS), circular dichroism (CD), attenuated total reflectance Fourier transform infrared (ATR-FTIR) and intrinsic tryptophan fluorescence emission (ITFE) spectroscopy. The elution profile of the protein presents a displacement of the protein peak to larger complexes when interacting with higher concentration of heparin. The DLS results shows two R-h at a molar ratio of 1, one to the distribution of the protein and the second for the action of heparin on BthTx-I structures, and a large distribution with the increase of protein. The interaction is accompanied by significant changes in the CD spectra, showing two common features: a decrease in signal at 208 nm (3 and 6 kDa heparins) and an isodichroic point near 226 nm (3 kDa heparin). FTIR spectra indicate that only a few amino acid residues are involved in this interaction. Alterations in the ITFE by binding heparins suggest that the initial binding occurs on the ventral face of BthTx-1. Together, these results add an experimental and structural basis on the action mechanism of the heparins over the phospholipases A(2) and provide a molecular model to elucidate the interaction of the enzyme-heparin complex at a molecular level. (c) 2005 Elsevier B.V. All rights reserved.

Structural basis for branching-enzyme activity of glycoside hydrolase family 57: Structure and stability studies of a novel branching enzyme from the hyperthermophilic archaeon Thermococcus Kodakaraensis KOD1

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

MECHANISM OF ACTION OF COBALT IONS ON RAT OSSEOUS PLATE ALKALINE-PHOSPHATASE

Polidocanol-solubilized osseous plate alkaline phosphatase was modulated by cobalt ions in a similar way as by magnesium ions. For concentrations up to 1 mu M, the Chelex-treated enzyme was stimulated by cobalt ions, showing K-d = 6.0 mu M, V = 977.5 U/mg, and site-site interactions (n = 2.5). Cobalt-enzyme was highly unstable at 37 degrees C, following a biphasic inactivation process with inactivation constants of about 0.0625 and 0.0015 min(-1). Cobalt ions stimulated the enzyme synergistically in the presence of magnesium ions (K-d = 5.0 mu M; V = 883.0 U/mg) or in the presence of zinc ions (K-d = 75.0 mu M; V = 1102 U/mg). A steady-state kinetic model for the modulation of enzyme activity by cobalt ions is proposed.

Structure and catalytic mechanism of glucosamine 6-phosphate deaminase from Escherichia coli at 2.1 Å resolution

Background: Glucosamine 6-phosphate deaminase from Escherichia coli is an allosteric hexameric enzyme which catalyzes the reversible conversion of D-glucosamine 6-phosphate into D-fructose 6-phosphate and ammonium ion and is activated by N-acetyl-D-glucosamine 6-phosphate. Mechanistically, it belongs to the group of aldose-ketose isomerases, but its reaction also accomplishes a simultaneous amination/deamination. The determination of the structure of this protein provides fundamental knowledge for understanding its mode of action and the nature of allosteric conformational changes that regulate its function. Results: The crystal structure of glucosamine 6-phosphate deaminase with bound phosphate ions is presented at 2.1 Å resolution together with the refined structures of the enzyme in complexes with its allosteric activator and with a competitive inhibitor. The protein fold can be described as a modified NAD-binding domain. Conclusions: From the similarities between the three presented structures, it is concluded that these represent the enzymatically active R state conformer. A mechanism for the deaminase reaction is proposed. It comprises steps to open the pyranose ring of the substrate and a sequence of general base-catalyzed reactions to bring about isomerization and deamination, with Asp72 playing a key role as a proton exchanger.

Toxic mechanism of nickel exposure on cardiac tissue

The incidence of cardiovascular disease has increased in the general population, and cardiac damage is indicated as one important cause of mortality. In addition, pollution and metal exposure have increased in recent years. For this reason, toxic effects of metals, such as nickel, and their relation to cardiac damage should be urgently established. Although free radical-mediated cellular damage and reactive oxygen species have been theorized as contributing to the nickel mechanism of toxicity, recent investigations have established that free radicals may be important contributors to cardiac dysfunction. However, there is little information on the effect of nickel exposure on markers of oxidative stress in cardiac tissue. Nickel exposure (Ni2+ 100 mg L-1 from NiSO4) significantly increased lipoperoxide and total lipid concentrations in cardiac tissue. We also observed increased serum levels of cholesterol (59%), lactate dehydrogenase (LDH-64%), and alanine transaminase (ALT-30%) in study animals. The biochemical parameters recovered to the control values with tocopherol intake (0.2 mg 200 g-1). Vitamin E alone significantly decreased the lipoperoxide concentration and increased superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities in the heart. Since no alterations were observed in catalase and GSH-Px activities by nickel exposure while SOD activities were decreased, we conclude that superoxide radical (O2 -) generated by nickel exposure is of primary importance in the pathogenesis of cardiac damage. Tocopherol, by its antioxidant activity, decreased the toxic effects of nickel exposure on heart of rats.

Toxic mechanism of cadmium exposure on cardiac tissue

The presence of toxic substances in the workplace environment requires systematic evaluation of exposure and health status in exposed subjects. Cadmium is a highly toxic element found in water. Although free mediated cellular damage and reactive oxygen species (ROS), had been theorized as contributing to the cadmium mechanism of toxicity, and recent investigations have established that free radicals may be important contributors to cardiac dysfunction, there is little information on the effect of cadmium exposure on markers of oxidative stress in cardiac tissue. Cadmium exposure (Cd2+ - 100 mg/1-from CdCl2) in drinking water, during 15 days, significantly increased lipoperoxide and decreased the activities of superoxide dismutase and glutathione peroxidase. No alterations were observed in catalase activity in heart of rats with cadmium exposure. We also observed decreased glycogen and glucose concentration and increased total lipid content in cardiac tissue of rats with cadmium exposure. The decreased activities of alanine transaminase and aspartate transaminase reflected decreased metabolic protein degradation, and increased lactate dehydrogenase activity was related with increases in capacity of glycolysis. Since the metabolic pathways were altered by cadmium exposure, we can conclude that Cd2+ exposure induced ROS and initiate some series of events that occur in the heart and resulted in metabolic pathways alterations.