Biomolecules produced in liquid-state fermentation by a marine-derived fungus, Penicillium roqueforti

Screening of biomass of a new marine-derived strain of Penicillium roqueforti, as produced by liquid-state fermentation, led to the identification of several volatile organic compounds active in the fatty acid pathway as well as fragments produced by their catabolism, terpenoids, and metabolites from the shikimic acid pathway. In addition, five non-volatile organic compounds, triolein, ergosterol peroxide, 9(11)-dehydroergosterol peroxide, 4-hydroxybenzaldehyde, and d-mannitol, were isolated and identified by spectroscopy. The results showed that this fungal strain did not produce any mycotoxin in the culture conditions applied, and thus is useful for industrial applications, where high value-added biomolecules are generated.

Chemical indexes calculated for 8,11,13-trien-abietane diterpenoids isolated from Swartzia species

The disparity found in the molecular structures of compounds isolated from nine plants of the Swartzia genus indicates that the Swartzia species that furnished cassane diterpenoids and triterpenoidal saponins are more recent, since these metabolites have adopted the mevalonic acid route of formation, abandoning the shikimic acid/acetate route that produces the isoflavonoids found in the remaining species. Chemical indexes calculated from the molecular structure diversities of sixteen 8,11,13-trien-abietane diterpenoids isolated from Swartzia langsdorffii and S. arborescens indicate that S. arborescens is more recent than S. langsdorffii. The results suggest a more evolved position in Swartzia species of the section Possira.

Glyphosate influence on the physiological parameters of Conyza bonariensis biotypes

The objective of this study was to determine changes in gas exchange and inhibition of EPSPs, based on the accumulation of shikimic acid in horseweed biotypes resistant and sensitive to glyphosate. Two experiments were conducted in a factorial model. The first one evaluated horseweed biotypes (one resistant and one susceptible to glyphosate), and herbicide rates (0 and 1,080 g a.e. ha ¹) applied on the weed. In the second experiment, the horseweed biotypes (susceptible and resistant to glyphosate) were evaluated in five periods as following: 0, 3, 7, 10, and 14 days after herbicide application (DAH). The photosynthetic rate, transpiration, carboxylation efficiency, and water efficiency were determined using an infrared gas analyzer (IRGA), and shikimic acid concentration by HPLC. The application of glyphosate damaged the photosynthetic parameters of the susceptible biotype, causing complete inhibition of the photosynthetic rate, transpiration rate, carboxylation efficiency and water use efficiency, starting from the 7 DAH. On the other hand, total inhibition of the photosynthetic parameters was not observed for the resistant biotype. Shikimic acid accumulation occurred in both biotypes after glyphosate application but the susceptible biotype had the highest concentrations, indicating greater sensitivity of the enzyme EPSPs. The accumulation of shikimic acid in the resistant biotype indicates that the mechanism of resistance is not related to the total insensitivity of the enzyme EPSPs to glyphosate and/or that other resistance mechanisms may be involved.

Shikimate accumulation, glyphosate absorption and translocation in horseweed biotypes

In Brazil, few research works on mechanisms of weed resistance to glyphosate have been conducted so far. Therefore, this research aimed to study analytical procedures determining the relation between the concentration of plant shikimate after glyphosate application and the plant resistance to this herbicide; and evaluate the glyphosate absorption and translocation into two resistant ® and susceptible (S) horseweed biotypes to glyphosate. Horseweed plants with nine true leaves received glyphosate (720 g a.e. ha-1), and 2, 3, 4, 7 and 10 days after application (DAA) the concentration of shikimic acid was measured by HPLC. In another experiment, plants were treated with radiolabeled glyphosate (14C) (1.456 MBq mmol-1 specific activity) and radioactivity was measured 4, 8, 24, 48 and 72 hours after treatment (HAT) by liquid scintillation spectrometry. The shikimate concentration in plants increased 16,351.14 and 7,892.25 mg kg-1 of dry weight, for R and S plants respectively, at seven DAA. Therefore, the procedure for quantification of shikimic acid was suitable for R and S plants differentiation to glyphosate, indicating that the R population is actually resistant to glyphosate. On average, 98% of glyphosate applied was absorbed by the studied biotypes, at 72 HAT. Around 68% of the absorbed radioactivity remained on the biotypes leaves treated, the S biotype showing the highest translocation. Therefore, the R biotype resistance mechanism studied is associated to the differential translocation.

Approaches to the synthesis of 3-fluoroshikimic acids

Exploitation of the dual dehydrating and fluorodeoxygenating properties of the dialkylaminosulfurtrifluorides has allowed access to the C3-fluorinated analogues of (-)-shikimic acid. (C) 2004 Elsevier Ltd. All rights reserved.

Low glyphosate rates do not affect Citrus limonia (L.) Osbeck seedlings

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Glyphosate applied at low doses can stimulate plant growth

BACKGROUND: Glyphosate blocks the shikimic acid pathway, inhibiting the production of aromatic amino acids and several secondary compounds derived from these amino acids. Non-target plants can be exposed to low doses of glyphosate by herbicide drift of spray droplets and contact with treated weeds. Previous studies have reported that low doses of glyphosate stimulate growth, although these data are very limited. The objective of this study was to determine the effects of low glyphosate doses on growth of a range of plant species.RESULTS: Growth of maize, conventional soybean, Eucalyptus grandis Hill ex Maiden, Pinus caribea L. and Commelia benghalensis L. was enhanced by 1.8-36 g glyphosate ha(-1). Growth of glyphosate-resistant soybean was unaffected by any glyphosate dose from 1.8 to 720 g AE ha(-1). The optimum doses for growth stimulation were distinct for plant species and tissue evaluated. The greatest stimulation of growth was observed for C. benghalensis and P. caribea. Shikimic acid levels in tissues of glyphosate-treated soybean and maize were measured and found to be elevated at growth-stimulating doses.CONCLUSION: Subtoxic doses of glyphosate stimulate the growth of a range of plant species, as measured in several plant organs. This hormesis effect is likely to be related to the molecular target of glyphosate, since the effect was not seen in glyphosate-resistant plants, and shikimate levels were enhanced in plants with stimulated growth. (c) 2008 Society of Chemical Industry.

Alterações no metabolismo da cana-de-açúcar em função da aplicação de maturadores

O objetivo deste trabalho foi avaliar as alterações nos níveis de ácido chiquímico e ácido salicílico em plantas de cana-de-açúcar submetidas à aplicação de maturadores. Aplicou-se glyphosate nas doses de 400 e 200 mL ha-1 e na dose de 150 mL ha-1 em mistura com sulfumeturon-methyl a 12 e 20 g ha-1 e sulfumeturon-methyl a 20 g ha-1. As avaliações foram realizadas aos 15 e 30 dias após a aplicação (DAA) e aos 30, 60, 90, 120 e 150 dias após a colheita da cana-de-açúcar. Os teores de ácido chiquímico e salicílico nas plantas de canade-açúcar foram determinados por cromatografia líquida e espectrometria de massas. Os resultados mostraram que as doses de glyphosate correlacionaram-se diretamente com as concentrações de ácido chiquímico na planta, sendo superiores à da testemunha. Aos 30 DAA, houve aumento na concentração de ácido salicílico em todos os tratamentos estudados, revelando um processo de senescência da planta. Maiores doses de glyphosate promoveram aumento na concentração de ácido chiquímico e ácido salicílico antes da colheita da canade-açúcar. No período de crescimento da planta, aumentos nos teores dos ácidos chiquímico e salicílico revelaram dependência da aplicação dos produtos e também dos fatores abióticos e bióticos a que a cultura foi exposta.

Shikimate Kinase (EC 2.7.1.71) from Mycobacterium tuberculosis: Kinetics and Structural Dynamics of a Potential Molecular Target for Drug Development

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

PURIFICATION AND PROPERTIES OF SHIKIMATE DEHYDROGENASE FROM CUCUMBER (CUCUMIS-SATIVUS L)

Shikimate dehydrogenase (SDH, EC 1.1.1.25) extracted from cucumber pulp (Cucumis sativus L.) was purified 7-fold by precipitation with ammonium sulfate and elution from columns of Sephadex G-25, DEAE-cellulose, and hydroxyapatite. Two activity bands were detected on polyacrylamide gel electrophoresis at the last purification step. pH optimum was 8.7, and molecular weight of 45 000 was estimated on a Sephadex G-100 column. SDH was inhibited competitively by protocatechuic acid with a K(i) value of 2 x 10-4 M. K(m) values of 6 x 10-5 and 1 x 10-5 M were determined for shikimic acid and NADP+, respectively. The enzyme was completely inhibited by HgCl2 and p-(chloromercuri)benzoate (PCMB). NaCl and KCl showed partial protection against inhibition by PCMB. Heat inactivation between 50 and 55-degrees-C was biphasic, and the enzyme was completely inactivated after 10 min at 60-degrees-C. Incubation of SDH with either NADP+ or shikimic acid protected the enzyme against heat inactivation.

Shikimate kinase: A potential target for development of novel antitubercular agents

Tuberculosis (TB) remains the leading cause of mortality due to a bacterial pathogen, Mycobacterium tuberculosis. However, no new classes of drugs for TB have been developed in the past 30 years. Therefore there is an urgent need to develop faster acting and effective new antitubercular agents, preferably belonging to new structural classes, to better combat TB, including MDR-TB, to shorten the duration of current treatment to improve patient compliance, and to provide effective treatment of latent tuberculosis infection. The enzymes in the shikimate pathway are potential targets for development of a new generation of antitubercular drugs. The shikimate pathway has been shown by disruption of aroK gene to be essential for the Mycobacterium tuberculosis. The shikimate kinase (SK) catalyses the phosphorylation of the 3-hydroxyl group of shikimic acid (shikimate) using ATP as a co-substrate. SK belongs to family of nucleoside monophosphate (NMP) kinases. The enzyme is an alpha/beta protein consisting of a central sheet of five parallel beta-strands flanked by alpha-helices. The shikimate kinases are composed of three domains: Core domain, Lid domain and Shikimate-binding domain. The Lid and Shikimate-binding domains are responsible for large conformational changes during catalysis. More recently, the precise interactions between SK and substrate have been elucidated, showing the binding of shikimate with three charged residues conserved among the SK sequences. The elucidation of interactions between MtSK and their substrates is crucial for the development of a new generation of drugs against tuberculosis through rational drug design.

Detection of Sourgrass (Digitaria insularis) Biotypes Resistant to Glyphosate in Brazil

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

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.