Chemotactic Interactions of Scenedesmus sp. and Azospirillum brasilense Investigated by Microfluidic Methods.

The use of algae for industrial, biotechnological, and agricultural purposes is spreading globally. Scenedesmus species can play an essential role in the food industry and agriculture due to their favorable nutrient content and plant-stimulating properties. Previous research and the development of Scenedesmus-based foliar fertilizers raised several questions about the effectiveness of large-scale algal cultivation and the potential effects of algae on associative rhizobacteria. In the microbiological practice applied in agriculture, bacteria from the genus Azospirillum are one of the most studied plant growth-promoting, associative, nitrogen-fixing bacteria. Co-cultivation with Azospirillum species may be a new way of optimizing Scenedesmus culturing, but the functioning of the co-culture system still needs to be fully understood. It is known that Azospirillum brasilense can produce indole-3-acetic acid, which could stimulate algae growth as a plant hormone. However, the effect of microalgae on Azospirillum bacteria is unclear. In this study, we investigated the behavior of Azospirillum brasilense bacteria in the vicinity of Scenedesmus sp. or its supernatant using a microfluidic device consisting of physically separated but chemically coupled microchambers. Following the spatial distribution of bacteria within the device, we detected a positive chemotactic response toward the microalgae culture. To identify the metabolites responsible for this behavior, we tested the chemoeffector potential of citric acid and oxaloacetic acid, which, according to our HPLC analysis, were present in the algae supernatant in 0.074 mg/ml and 0.116 mg/ml concentrations, respectively. We found that oxaloacetic acid acts as a chemoattractant for Azospirillum brasilense.

In vitro nitazoxanide exposure affects energetic metabolism of Taenia crassiceps.

Nitazoxanide (NTZ) is a broad-spectrum drug used in intestinal infections, but still poorly explored in the treatment of parasitic tissular infections. This study aimed to evaluate the in vitro responses of the energetic metabolism of T. crassiceps cysticerci induced by NTZ. The organic acids of the tricarboxylic acid cycle, products derived from fatty acids oxidation and protein catabolism were analyzed. These acids were quantified after 24 h of in vitro exposure to different NTZ concentrations. A positive control group was performed with albendazole sulfoxide (ABZSO). The significant alterations in citrate, fumarate and malate concentrations showed the NTZ influence in the tricarboxylic acid (TCA) cycle. The non-detection of acetate confirmed that the main mode of action of NTZ is effective against T. crassiceps cysticerci. The statistical differences in fumarate, urea and beta-hydroxybutyrate concentrations showed the NTZ effect on protein catabolism and fatty acid oxidation. Therefore, the main energetic pathways such as the TCA cycle, protein catabolism and fatty acids oxidation were altered after in vitro NTZ exposure. In conclusion, NTZ induced a significant metabolic stress in the parasite indicating that it may be used as an alternative therapeutic choice for cysticercosis treatment. The use of metabolic approaches to establish comparisons between anti parasitic drugs mode of actions is proposed.

Biochemical characterization of phosphoenolpyruvate carboxykinases from Arabidopsis thaliana.

ATP-dependent phosphoenolpyruvate carboxykinases (PEPCKs, EC 4.1.1.49) from C4 and CAM plants have been widely studied due to their crucial role in photosynthetic CO2 fixation. However, our knowledge on the structural, kinetic and regulatory properties of the enzymes from C3 species is still limited. In this work, we report the recombinant production and biochemical characterization of two PEPCKs identified in Arabidopsis thaliana: AthPEPCK1 and AthPEPCK2. We found that both enzymes exhibited high affinity for oxaloacetate and ATP, reinforcing their role as decarboxylases. We employed a high-throughput screening for putative allosteric regulators using differential scanning fluorometry and confirmed their effect on enzyme activity by performing enzyme kinetics. AthPEPCK1 and AthPEPCK2 are allosterically modulated by key intermediates of plant metabolism, namely succinate, fumarate, citrate and α-ketoglutarate. Interestingly, malate activated and glucose 6-phosphate inhibited AthPEPCK1 but had no effect on AthPEPCK2. Overall, our results demonstrate that the enzymes involved in the critical metabolic node constituted by phosphoenolpyruvate are targets of fine allosteric regulation.

Conformational changes on substrate binding revealed by structures of Methylobacterium extorquens malate dehydrogenase.

Three high-resolution X-ray crystal structures of malate dehydrogenase (MDH; EC 1.1.1.37) from the methylotroph Methylobacterium extorquens AM1 are presented. By comparing the structures of apo MDH, a binary complex of MDH and NAD+, and a ternary complex of MDH and oxaloacetate with ADP-ribose occupying the pyridine nucleotide-binding site, conformational changes associated with the formation of the catalytic complex were characterized. While the substrate-binding site is accessible in the enzyme resting state or NAD+-bound forms, the substrate-bound form exhibits a closed conformation. This conformational change involves the transition of an α-helix to a 310-helix, which causes the adjacent loop to close the active site following coenzyme and substrate binding. In the ternary complex, His284 forms a hydrogen bond to the C2 carbonyl of oxaloacetate, placing it in a position to donate a proton in the formation of (2S)-malate.

The complex allosteric and redox regulation of the fumarate hydratase and malate dehydratase reactions of Arabidopsis thaliana Fumarase 1 and 2 gives clues for understanding the massive accumulation of fumarate.

Arabidopsis thaliana possesses two fumarase genes (FUM), AtFUM1 (At2g47510) encoding for the mitochondrial Krebs cycle-associated enzyme and AtFUM2 (At5g50950) for the cytosolic isoform required for fumarate massive accumulation. Here, the comprehensive biochemical studies of AtFUM1 and AtFUM2 shows that they are active enzymes with similar kinetic parameters but differential regulation. For both enzymes, fumarate hydratase (FH) activity is favored over the malate dehydratase (MD) activity; however, MD is the most regulated activity with several allosteric activators. Oxalacetate, glutamine, and/or asparagine are modulators causing the MD reaction to become preferred over the FH reaction. Activity profiles as a function of pH suggest a suboptimal FUM activity in Arabidopsis cells; moreover, the direction of the FUM reaction is sensitive to pH changes. Under mild oxidation conditions, AtFUMs form high mass molecular aggregates, which present both FUM activities decreased to a different extent. The biochemical properties of oxidized AtFUMs (oxAtFUMs) were completely reversed by NADPH-supplied Arabidopsis leaf extracts, suggesting that the AtFUMs redox regulation can be accomplished in vivo. Mass spectrometry analyses indicate the presence of an active site-associated intermolecular disulfide bridge in oxAtFUMs. Finally, a phylogenetic approach points out that other plant species may also possess cytosolic FUM2 enzymes mainly encoded by paralogous genes, indicating that the evolutionary history of this trait has been drawn through a process of parallel evolution. Overall, according to our results, a multilevel regulatory pattern of FUM activities emerges, supporting the role of this enzyme as a carbon flow monitoring point through the organic acid metabolism in plants.

Biochemical and genetic characterization of the Enterococcus faecalis oxaloacetate decarboxylase complex.

Enterococcus faecalis encodes a biotin-dependent oxaloacetate decarboxylase (OAD), which is constituted by four subunits: E. faecalis carboxyltransferase subunit OadA (termed Ef-A), membrane pump Ef-B, biotin acceptor protein Ef-D, and the novel subunit Ef-H. Our results show that in E. faecalis, subunits Ef-A, Ef-D, and Ef-H form a cytoplasmic soluble complex (termed Ef-AHD) which is also associated with the membrane. In order to characterize the role of the novel Ef-H subunit, coexpression of oad genes was performed in Escherichia coli, showing that this subunit is vital for Ef-A and Ef-D interaction. Diminished growth of the oadA and oadD single deletion mutants in citrate-supplemented medium indicated that the activity of the complex is essential for citrate utilization. Remarkably, the oadB-deficient strain was still capable of growing to wild-type levels but with a delay during the citrate-consuming phase, suggesting that the soluble Ef-AHD complex is functional in E. faecalis. These results suggest that the Ef-AHD complex is active in its soluble form, and that it is capable of interacting in a dynamic way with the membrane-bound Ef-B subunit to achieve its maximal alkalinization capacity during citrate fermentation.

Two phosphoenolpyruvate carboxykinases coexist in the Crassulacean Acid Metabolism plant Ananas comosus. Isolation and characterization of the smaller 65 kDa form.

Two phosphoenolpyruvate carboxykinase (PEPCK, EC 4.1.1.49) isoforms of 74 and 65 kDa were found to coexist in vivo in pineapple leaves, a constitutive Crassulacean Acid Metabolism plant. The 65 kDa form was not the result of proteolytic cleavage of the larger form since extraction methods reported to prevent PEPCK proteolysis in other plant tissues failed to yield a single immunoreactive PEPCK polypeptide in leaf extracts. In this work, the smaller form of 65 kDa was purified to homogeneity and physically and kinetically characterized and showed parameters compatible with a fully active enzyme. The specific activity was nearly twice higher for decarboxylation of oxaloacetate when compared to carboxylation of phosphoenolpyruvate. Kinetic parameters fell within the range of those estimated for other plant PEPCKs. Its activity was affected by several metabolites, as shown by inhibition by 3-phosphoglycerate, citrate, malate, fructose-1,6-bisphosphate, l-asparagine and activation of the decarboxylating activity by succinate. A break in the Arrhenius plot at about 30°C indicates that PEPCK structure is responsive to changes in temperature. The results indicate that pineapple leaves contain two PEPCK forms. The biochemical characterization of the smaller isoform performed in this work suggests that it could participate in both carbon and nitrogen metabolism in vivo by acting as a decarboxylase.

Neuroprotective effect of pyruvate and oxaloacetate during pilocarpine induced status epilepticus in rats.

Recent research data have shown that systemic administration of pyruvate and oxaloacetate causes an increased brain-to-blood glutamate efflux. Since increased release of glutamate during epileptic seizures can lead to excitotoxicity and neuronal cell death, we tested the hypothesis that glutamate scavenging mediated by pyruvate and oxaloacetate systemic administration could have a neuroprotective effect in rats subjected to status epilepticus (SE). SE was induced by a single dose of pilocarpine (350mg/kgi.p.). Thirty minutes after SE onset, a single dose of pyruvate (250mg/kgi.p.), oxaloacetate (1.4mg/kgi.p.), or both substances was administrated. Acute neuronal loss in hippocampal regions CA1 and hilus was quantitatively determined five hours after SE onset, using the optical fractionator method for stereological cell counting. Apoptotic cascade in the hippocampus was also investigated seven days after SE using caspase-1 and -3 activity assays. SE-induced neuronal loss in CA1 was completely prevented in rats treated with pyruvate plus oxaloacetate. The SE-induced caspase-1 activation was significantly reduced when rats were treated with oxaloacetate or pyruvate plus oxaloacetate. The treatment with pyruvate and oxaloacetate caused a neuroprotective effect in rats subjected to pilocarpine-induced SE.

Electrostatic interactions play a significant role in the affinity of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase for Mn2+.

Phosphoenolpyruvate (PEP) carboxykinases catalyse the reversible formation of oxaloacetate (OAA) and ATP (or GTP) from PEP, ADP (or GDP) and CO(2). They are activated by Mn(2+), a metal ion that coordinates to the protein through the epsilon-amino group of a lysine residue, the N(epsilon-2)-imidazole of a histidine residue, and the carboxylate from an aspartic acid residue. Neutrality in the epsilon-amino group of Lys213 of Saccharomyces cerevisiae PEP carboxykinase is expected to be favoured by the vicinity of ionised Lys212. Glu272 and Glu284, located close to Lys212, should, in turn, electrostatically stabilise its positive charge and hence assist in keeping the epsilon-amino group of Lys213 in a neutral state. The mutations Glu272Gln, Glu284Gln, and Lys212Met increased the activation constant for Mn(2+) in the main reaction of the enzyme up to seven-fold. The control mutation Lys213Gln increased this constant by ten-fold, as opposed to control mutation Lys212Arg, which did not affect the Mn(2+) affinity of the enzyme. These observations indicate a role for Glu272, Glu284, and Lys212 in assisting Lys213 to properly bind Mn(2+). In an unexpected result, the mutations Glu284Gln, Lys212Met and Lys213Gln changed the nucleotide-independent OAA decarboxylase activity of S. cerevisiae PEP carboxykinase into an ADP-requiring activity, implying an effect on the OAA binding characteristics of PEP carboxykinase.

Purification, properties, and kinetic studies of cytoplasmic malate dehydrogenase from Taenia solium cysticerci.

Malate dehydrogenase (L: -malate: NAD oxidoreductase, EC 1.1.1.37) from the cytoplasm of Taenia solium cysticerci (cMDHTs) was purified 48-fold through a four-step procedure involving salt fractionation, ionic exchange, and dye affinity chromatography. cMDHTs had a native M (r) of 64,000, while the corresponding value per subunit, obtained under denaturing conditions, was 32,000. The enzyme is partially positive, with an isoelectric point of 8.7, and had a specific activity of 2,615 U mg(-1) in the reduction of oxaloacetate. The second to the 21st amino acids from cMDHTs N-terminal group were P G P L R V L I T G A A G Q I A Y N L S. This sequence is 100% identical to that of Echinococcus granulosus. Basic kinetic parameters were determined for this enzyme. The optimum pH for enzyme reaction was at 7.6 for oxaloacetate reduction and at 9.6 for malate oxidation. K (m) values for oxaloacetate, malate, NAD, and NADH were 2.4, 215, 50, and 48 microM, respectively. V (max) values for the substrates and cosubstrates as described above were 1,490, 87.8, 104, and 1,714 micromol min(-1) mg(-1). Several NAD analogs, structurally altered in either the pyridine or purine moiety, were observed to function as coenzymes in the reaction catalyzed by the purified malate dehydrogenase. cMDHTs activity was uncompetitive inhibited by arsenate for both the forward (Ki = 8.2 mM) and reverse (Ki = 77 mM) reactions. The mechanism of the cMDHTs reactivity was investigated kinetically by the product inhibition approach. The results of this study are qualitatively consistent with an Ordered Bi Bi reaction mechanism, in which only the coenzymes can react with the free enzyme.

The malate dehydrogenase isoforms from Trypanosoma brucei: subcellular localization and differential expression in bloodstream and procyclic forms.

Trypanosoma brucei procyclic forms possess three different malate dehydrogenase isozymes that could be separated by hydrophobic interaction chromatography and were recognized as the mitochondrial, glycosomal and cytosolic malate dehydrogenase isozymes. The latter is the only malate dehydrogenase expressed in the bloodstream forms, thus confirming that the expression of malate dehydrogenase isozymes is regulated during the T. brucei life cycle. To achieve further biochemical characterization, the genes encoding mitochondrial and glycosomal malate dehydrogenase were cloned on the basis of previously reported nucleotide sequences and the recombinant enzymes were functionally expressed in Escherichia coli cultures. Mitochondrial malate dehydrogenase showed to be more active than glycosomal malate dehydrogenase in the reduction of oxaloacetate; nearly 80% of the total activity in procyclic crude extracts corresponds to the former isozyme which also catalyzes, although less efficiently, the reduction of p-hydroxyphenyl-pyruvate. The rabbit antisera raised against each of the recombinant isozymes showed that the three malate dehydrogenases do not cross-react immunologically. Immunofluorescence experiments using these antisera confirmed the glycosomal and mitochondrial localization of glycosomal and mitochondrial malate dehydrogenase, as well as a cytosolic localization for the third malate dehydrogenase isozyme. These results clearly distinguish Trypanosoma brucei from Trypanosoma cruzi, since in the latter parasite a cytosolic malate dehydrogenase is not present and mitochondrial malate dehydrogenase specifically reduces oxaloacetate.

Characterization and regulation of NADP+-isocitrate dehydrogenase from Saccharopolyspora erythraea.

NADP+-Isocitrate dehydrogenase (ICDH) activity was detected in cell-free extracts of Saccharopolyspora erythraea CA340, an erythromycin producer. Apparent Km values for DL-isocitrate and NADP+ were 0.14 microM and 0.026 microM, respectively. ATP, ADP, GTP, citric acid, oxaloacetate, alpha-ketoglutarate, glyoxalate and glyoxalate plus oxaloacetate, each at 1 mM concentration, caused 50, 20 10, 50, 25, 60, 20 and 50% inhibition of ICDH activity, respectively. Phosphoenolpyruvate, fructose 1,6-diphosphate and pyruvate had no effect. ICDH specific activity profile was growth-associated and activity with dextrose or fructose as sole carbon source, was twice of that obtained with lactose.

Metabolic flux redistribution in Corynebacterium glutamicum in response to osmotic stress.

Osmotic stress constitutes a major bacterial stress factor in the soil and during industrial fermentation. In this paper, we quantified the metabolic response, in terms of metabolic flux redistribution, of a lysine-overproducing strain of Corynebacterium glutamicum grown under continuous culture, to gradually increasing osmolality. Oxygen and carbon dioxide evolution rates, and the changes in concentration of extracellular, as well as intracellular, metabolites were measured throughout the osmotic gradient. The metabolic fluxes were estimated from these measurements and from the mass balance constraints at each metabolite-node of the assumed metabolic reaction network. Our results show that formation rates of compatible solutes--trehalose first and proline at a later stage of the gradient--increased with osmotic stress to equilibrate the external osmotic pressure. Estimated flux distributions indicate that the observed increase in the glucose specific uptake rate with osmotic stress is channeled through the main energy generating pathways-- glycolysis and the tricarboxylic acid cycle--while the flux through the pentose phosphate pathway remains constant throughout the gradient. This results in a significant increase in the net specific ATP production rate, which may possibly be used to support the higher energy requirements required for cellular maintenance at high osmolalities. Finally, nodal analysis confirmed that the PEP/pyruvate node is essentially rigid and that the glucose-6-phosphate, oxaloacetate and alpha-ketoglutarate nodes are flexible and therefore adaptable to changes in osmotic pressure in C. glutamicum.