Sir2-Related Protein 1 from Leishmania amazonensis is a glycosylated NAD(+)-dependent deacetylase

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

Crystallographic studies on the binding of isonicotinyl-NAD adduct to wild-type and isoniazid resistant 2-trans-enoyl-ACP (CoA) reductase from Mycobacterium tuberculosis

The resumption of tuberculosis led to an increased need to understand the molecular mechanisms of drug action and drug resistance, which should provide significant insight into the development of newer compounds. Isoniazid (INH), the most prescribed drug to treat TB, inhibits an NADH-dependent enoyl-acyl carrier protein reductase (InhA) that provides precursors of mycolic acids, which are components of the mycobacterial cell wall. InhA is the major target of the mode of action of isoniazid. INH is a pro-drug that needs activation to form the inhibitory INH-NAD adduct. Missense mutations in the inhA structural gene have been identified in clinical isolates of Mycobacterium tuberculosis resistant to INH. To understand the mechanism of resistance to INH, we have solved the structure of two InhA mutants (121V and S94A), identified in INH-resistant clinical isolates, and compare them to INH-sensitive WT InhA structure in complex with the INH-NAD adduct. We also solved the structure of unliganded INH-resistant S94A protein, which is the first report on apo form of InhA. The salient features of these structures are discussed and should provide structural information to improve our understanding of the mechanism of action of, and resistance to, INH in M. tuberculosis. The unliganded structure of InhA allows identification of conformational changes upon ligand binding and should help structure-based drug design of more potent antimycobacterial agents. (c) 2007 Elsevier B.V. All rights reserved.

Upregulation of gp91(phox) Subunit of NAD(P)H Oxidase Contributes to Erectile Dysfunction Caused by Long-term Nitric Oxide Inhibition in Rats: Reversion by Regular Physical Training

OBJECTIVES To test the hypothesis that glyco protein 91phox (gp91(phox)) subunit of nicotinamide adenine dinucleotide phosphate [NAD(P) H] oxidase is a fundamental target for physical activity to ameliorate erectile dysfunction (ED). Vascular risk factors are reported to contribute to ED. Regular physical exercise prevents cardiovascular diseases by increasing nitric oxide (NO) production and/or decreasing NO inactivation.METHODS Male Wistar rats received the NO synthesis inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) for 4 weeks, after which animals were submitted to a run training program for another 4 weeks. Erectile functions were evaluated by in vitro cavernosal relaxations and intracavernous pressure measurements. Expressions of gp91(phox) subunit and neuronal nitric oxidase synthase in erectile tissue, as well as superoxide dismutase activity and nitrite/nitrate (NO(x)) levels were determined.RESULTS The in vitro acetylcholine-and electrical field stimulation-induced cavernosal relaxations, as well as the increases in intracavernous pressure were markedly reduced in sedentary rats treated with L-NAME. Run training significantly restored the impaired cavernosal relaxations. No alterations in the neuronal nitric oxidase synthase protein expression (and its variant penile neuronal nitric oxidase synthase) were detected. A reduction of NO(x) levels and superoxide dismutase activity was observed in L-NAME-treated animals, which was significantly reversed by physical training. Gene expression of subunit gp91(phox) was enhanced by approximately 2-fold in erectile tissue of L-NAME-treated rats, and that was restored to basal levels by run training.CONCLUSIONS Our study shows that ED seen after long-term L-NAME treatment is associated with gp91(phox) subunit upregulation and decreased NO bioavailability. Exercise training reverses the increased oxidative stress in NO-deficient rats, ameliorating the ED. UROLOGY 75: 961-967, 2010. (C) 2009 Elsevier B.V.

Efeito da temperatura de estocagem de leveduras de panificação sobre a atividade da glicerol-3-fosfato desidrogenase

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

Pancreatic islets from dexamethasone-treated rats show alterations in global gene expression and mitochondrial pathways

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

Dihydrolipoyl dehydrogenase as a source of reactive oxygen species inhibited by caloric restriction and involved in Saccharomyces cerevisiae aging

Replicative life span in Saccharomyces cerevisiae is increased by glucose (G1c) limitation [ calorie restriction (CR)] and by augmented NAD(+). Increased survival promoted by CR was attributed previously to the NAD(+)-dependent histone deacetylase activity of sirtuin family protein Sir2p but not to changes in redox state. Here we show that strains defective in NAD(+) synthesis and salvage pathways (pnc1 Delta, npt1 Delta, and bna6 Delta) exhibit decreased oxygen consumption and increased mitochondrial H2O2 release, reversed over time by CR. These null mutant strains also present decreased chronological longevity in a manner rescued by CR. Furthermore, we observed that changes in mitochondrial H2O2 release alter cellular redox state, as attested by measurements of total, oxidized, and reduced glutathione. Surprisingly, our results indicate that matrix-soluble dihydrolipoyl-dehydrogenases are an important source of CR-preventable mitochondrial reactive oxygen species (ROS). Indeed, deletion of the LPD1 gene prevented oxidative stress in npt1 Delta and bna6 Delta mutants. Furthermore, pyruvate and alpha-ketoglutarate, substrates for dihydrolipoyl dehydrogenase-containing enzymes, promoted pronounced reactive oxygen release in permeabilized wild-type mitochondria. Altogether, these results substantiate the concept that mitochondrial ROS can be limited by caloric restriction and play an important role in S. cerevisiae senescence. Furthermore, these findings uncover dihydrolipoyl dehydrogenase as an important and novel source of ROS leading to life span limitation.

SIRT1 Deacetylase Activity and the Maintenance of Protein Homeostasis in Response to Stress: An Overview

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Kinetic aspects of rat (Rattus-norvegicus albinus) submandibular glands lactic-dehydrogenase

Submandibular glands of male rats were homogenized with 33 mM sodium potassium phosphate buffer, pH 6.5, containing 1 mM MgCl2 and 0.1 mM DTT and purified with ammonium sulphate, phosphocellulose chromatography, eluted with KC1 0.5 M, followed by Blue Sepharose CL-6B chromatography, eluted with NADH 0.5 mM. The enzyme kepts stable for 60 days when stored at -15-degrees-C in 33 mM phosphate buffer. In other experiment the enzyme was purified by oxamate-agarose chromatography from a crude extract of submandibular gland and the results obtained were better than by phosphocellulose and Sepharose CL-6B chromatography. The Km values for pyruvate. NADH, lactate and NAD+ were established. Sodium oxamate at 0.1 and 0.9 mM concentrations inhibited the LDH activity by 40 and 85%, respectively (competitive); with sodium oxalate the inhibition was of 30% (uncompetitive) and with 3-acetyl pyridine adenine dinucleotide was 80%.

Variable influence of ferric and cupric ions on Saccharomyces cerevisiae strains used in asymmetric organic synthesis.

Fe (III) and Cu (II) each at 50 mu M in four commercial strains of Saccharomyces cerevisiae induced an increase of NAD(P)(+) reduction in one strain (Turkish), but two others (Chilean and Brazilian), the presence of Fe(III) and/or Cu(II) diminished NAD(P)(+) reduction presumably due to free radicals formation inside these living cells. Suprisingly, in the American strain, Fe(III) induced a decrease and Cu (II) an increase of NAD(P)(+) reduction.

The effect of alpha-tocopherol on the oxidative cleavage of beta-carotene

Two cleavage pathways of beta-carotene have been proposed, one by central cleavage and the other by random (excentric) cleavage. The central cleavage pathway involves the metabolism of beta-carotene at the central double bond (15, 15') to produce retinal by beta-carotene 15, 15'-dioxygenase (E.C.888990988). The random cleavage of beta-carotene produces beta-apo-carotenoids, but the mechanism is not clear. To understand the various mechanisms of beta-carotene cleavage, beta-carotene was incubated with the intestinal postmitochondrial fractions of 10-week-old male rats for 1 h and cleavage products of beta-carotene were analyzed using reverse-phase, high-performance liquid chromatography (HPLC). We also studied the effects of alpha-tocopherol and NAD(+)/NADH on beta-carotene cleavage. In addition to beta-carotene, we used retinal and beta-apo-14'-carotenoic acid as substrates in these incubations. Beta-apo-14'-carotenoic acid is the two-carbon longer homologue of retinoic acid. In the presence of alpha-tocopherol, beta-carotene was converted exclusively to retinal, whereas in the absence of alpha-tocopherol, both retinal and beta-apo-carotenoids were formed. Retinoic acid was produced from both retinal and beta-apo-14'-carotenoic acid incubations only in the presence of NAD(+). Our data suggest that in the presence of an antioxidant such as alpha-tocopherol, beta-carotene is converted exclusively to retinal by central cleavage. In the absence of an antioxidant, beta-carotene is cleaved randomly by enzyme-related radicals to produce beta-apo-carotenoids, and these beta-apo-carotenoids can be oxidized further to retinoic acid via retinal. (C) 2000 Elsevier B.V.

Dehydromonocrotaline induces cyclosporine A-insensitive mitochondrial permeability transition/cytochrome c release

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

Application of methylotrophic yeast Pichia pastoris in the field of food industry - A review

Since ancient times, the utilization of yeasts by the man has a great impact on the socio-economic development. After the advent of the technology of recombinant DNA, great advances have occurred due to the acquisition of strains of mutant yeasts in the field of applied research, and Saccharomyces cerevisiae has soon been outstanding as an interesting candidate for the expression of heterologous proteins of biotechnological interest. As the time goes by other alternative systems of expression have been shown because they have advantages over Saccharomyces cerevisiae. Among those new systems, Pichia pastoris is outstanding as methylotrophic yeast capable of growing in a culture medium containing methanol as the only source of carbon and energy. The induction of production of glycerol-3-phosphate dehydrogenase (GPD, NAD(+): oxido-redutase EC 1.1. 1.8) by Pichia pastoris was accomplished in the medium containing methanol. One of the most important key parameters in Pichia pastoris expression system is the methanol concentration. Bibliographic reviews on the Pichia pastoris production system have shown that the best culture conditions vary according to the strain used and/or kind of heterologous protein desired to be expressed. Therefore, we have sought to develop a system, involving expression of glycerol-3-phosphate dehydrogenase in the yeast Pichia pastoris, for generating sufficient quantities of the enzyme in order to asses its potential value for use in various food bioanalytical determination. Dehydrogenases have been widely used in the enzymatic assays of diverse composites of industrial interest, being enclosed among them glycerol and a number of important bioanalytical applications.

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.

Enzymatic and oxidative metabolites of lycopene.

Using the post-mitochondrial fraction of rat intestinal mucosa, we have investigated lycopene metabolism. The incubation media was composed of NAD+, KCI, and DTT with or without added lipoxygenase. The addition of lipoxygenase into the incubation significantly increased the production of lycopene metabolites. The enzymatic incubation products of 2H10 lycopene were separated using high-performance liquid chromatography and analyzed by UV/Vis spectrophotometer and atmospheric pressure chemical ionization-mass spectroscopy. We have identified two types of products: cleavage products and oxidation products. The cleavage products are likely: (1) 3-keto-apo-13-lycopenone (C18H24O2 or 6,10,14-trimethyl-12-one-3,5,7,9,13-pentadecapentaen-2-one) with lambdamax = 365 nm and m/z =272 and (2) 3,4-dehydro-5,6-dihydro-15-apo-lycopenal (C20H28O or 3,7,11,15-tetramethyl-2,4,6,8,12,14-hexadecahexaen-l-al) with lambdamax= 380 nm and m/z = 284. The oxidative metabolites are likely: (3) 2-ene-5,8-lycopenal-furanoxide (C37H50O) with lambdamax = 415 nm, 435 nm, and 470 nm, and m/z = 510; (4) lycopene-5, 6, 5', 6'-diepoxide (C40H56O2) with lambdamax = 415 nm, 440 nm, and 470 nm, and m/z =568; (5) lycopene-5,8-furanoxide isomer (I) (C40H56O2) with lambdamax = 410 nm, 440 nm, and 470 nm, and m/z = 552; (6) lycopene-5,8-epoxide isomer (II) (C40H56O) with lambdamax = 410, 440, 470 nm, and m/z = 552; and (7) 3-keto-lycopene-5',8'-furanoxide (C40H54O2) with lambdamax = 400 nm, 420 nm, and 450 nm, and m/z = 566. These results demonstrate that both central and excentric cleavage of lycopene occurs in the rat intestinal mucosa in the presence of soy lipoxygenase.

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.