Howes, William V. (Washington State University, Pullman) and Bruce A. McFadden. Isocitrate lyase and malate synthase in Pseudomonas indigofera. I. Suppression and stimulation during growth. J. Bacteriol. 84:1216–1221. 1962.—Specific activities of isocitrate lyase and malate synthase from Pseudomonas...

Bacillithiol (Cys-GlcN-malate, BSH) has recently been identified as a novel low-molecular weight thiol in Bacillus anthracis, Staphylococcus aureus, and several other Gram-positive bacteria lacking glutathione and mycothiol. We have now characterized the first two enzymes for the BSH biosynthetic pathway in B. anthracis, which combine to produce {alpha}-D-glucosaminyl L-malate (GlcN-malate) from UDP-GlcNAc and L-malate. The structure of the GlcNAc-malate intermediate has been determined, as have the kinetic parameters for the BaBshA glycosyltransferase ({yields}GlcNAc-malate) and the BaBshB deacetylase ({yields}GlcN-malate). BSH is one of only two natural products reported to contain a malyl glycoside, and the crystal structure of the BaBshA-UDP-malate ternary complex, determined in this work at 3.3 {angstrom} resolution, identifies several active-site interactions important for the specific recognition of L-malate, but not other {alpha}-hydroxy acids, as the acceptor substrate. In sharp contrast to the structures reported for the GlcNAc-1-D-myo-inositol-3-phosphate synthase (MshA) apo and ternary complex forms, there is no major conformational change observed in the structures of the corresponding BaBshA forms. A mutant strain of B. anthracis deficient in the BshA glycosyltransferase fails to produce BSH, as predicted. This B. anthracis bshA locus (BA1558) has been identified in a transposon-site hybridization study as required for growth, sporulation, or germination [Day, W. A., Jr., Rasmussen, S. L., Carpenter, B. M., Peterson, S. N., and Friedlander, A. M. (2007) J. Bacteriol. 189, 3296-3301], suggesting that the biosynthesis of BSH could represent a target for the development of novel antimicrobials with broad-spectrum activity against Gram-positive pathogens like B. anthracis. The metabolites that function in thiol redox buffering and homeostasis in Bacillus are not well understood, and we present a composite picture based on this and other recent work.

The effects of iron (Fe) deficiency on carboxylate metabolism were investigated in barley (Hordeum vulgare L.) using two cultivars, Steptoe and Morex, which differ in their Fe efficiency response. In both cultivars, root extracts of plants grown in Fe-deficient conditions showed higher activities of enzymes related to organic acid metabolism, including citrate synthase, malate dehydrogenase and phosphoenolpyruvate carboxylase, compared to activities measured in root extracts of Fe-sufficient plants. Accordingly, the concentration of total carboxylates was higher in Fe-deficient roots of both cultivars, with citrate concentration showing the greatest increase. In xylem sap, the concentration of total carboxylates was also higher with Fe deficiency in both cultivars, with citrate and malate ...

After exposure to a doubled CO{sub 2} concentration of 750 {mu}mol mol{sup -1} air for about 3 months, glucose and starch in the chlorenchyma of basal cladodes of Opuntia ficus-indica increased 175 and 57%, respectively, compared with the current CO{sub 2} concentration of 370 {mu}mol mol{sup -1}, but sucrose content was virtually unaffected. Doubling the CO{sub 2} concentration increased the noncturnal malate production in basal cladodes by 75%, inorganic phosphate (Pi) by 32% soluble starch synthase activity by 30%, and sucrose-Pi synthase activity by 146%, but did not affect the activity of hexokinase. Doubling CO{sub 2} accelerated phloem transport of sucrose out of the basal cladodes, resulting in a 73% higher dry weight for the daughter cladodes. Doubling CO{sub 2} increased the glucose content in 14-d-old daughter cladodes by 167%, increased nocturnal malate production by 22%, decreased total amino acid content by 61%, and increased soluble starch synthase activity by 30% and sucrose synthase activity by 62%. No downward acclimation of photosynthesis during long-term exposure to elevated CO{sub 2} concentrations occurs for O. ficus-indica, consistent with its higher source capacity and sink strength than under current CO{sub 2}. These changes apparently do not result in Pi limitation of photosynthesis or suppression of genes governing photosynthesis for this perennial Crassulacean acid metabolism species, as occur for some annual crops.

The Rhodobacter sphaeroides hemA gene codes for 5-aminolevulinate (ALA) synthase. This enzyme catalyzes the pyridoxal phosphate-dependent condensation of succinyl coenzyme A and glycine-forming ALA. The R. sphaeroides hemA gene in the pUC18/19 vector system was transformed into Escherichia coli. The effects of both genetic and physiological factors on the expression of ALA synthase and the production of ALA were studied. ALA synthase activity levels were maximal when hemA had the same transcription direction as the lac promoter. The distance between the lac promoter and hemA affected the expression of ALA synthase on different growth substrates. The E. coli host strain used had an enormous effect on the ALA synthase activity level and on the production of ALA, with E. coli DH1 being best suited. The ALA synthase activity level was also dependent on the carbon source. Succinate, L-malate, fumarate, and L-aspartate gave the highest levels of ALA synthase activity, while the use of lactose as a carbon source resulted in a repression of ALA synthase. After growth on succinate, ALA synthase represented {approx}5% of total cellular protein. The ALA synthase activity level was also dependent on the pH of the medium, with maximal activity occurring at pH 6.5. ALA production by whole cells was limited by the availability of glycine, and the addition of 2 g of glycine per liter to the growth medium increased the production of ALA fivefold, to 2.25 mM. In recombinant E. coli extracts, up to 22 mM ALA was produced from succinate, glycine, and ATP. 58 refs., 4 figs., 7 tabs.

The locus glc (min 64.5), associated with the glycolate utilization trait in Escherichia coli, is known to contain glcB, encoding malate synthase G, and the gene(s) needed for glycolate oxidase activity. Subcloning, sequencing, insertion mutagenesis, and expression studies showed five additional genes: glcC and in the other direction glcD, glcE, glcF, and glcG followed by glcB. The gene glcC may encode the glc regulator protein. Consistently a chloramphenicol acetyltransferase insertion mutation abolished both glycolate oxidase and malate synthase G activities. The proteins encoded from glcD and glcE displayed similarity to several flavoenzymes, the one from glcF was found to be similar to iron-sulfur proteins, and that from glcG had no significant similarity to any group of proteins. The insertional mutation by a chloramphenicol acetyltransferase cassette in either glcD, glcE, or glcF abolished glycolate oxidase activity, indicating that presumably these proteins are subunits of this enzyme. No effect on glycolate metabolism was detected by insertional mutation in glcG. Northern (RNA) blot experiments showed constitutive expression of glcC but induced expression for the structural genes and provided no evidence for a single polycistronic transcript. PMID:8606183

The effects of iron (Fe) deficiency on carboxylate metabolism were investigated in barley (Hordeum vulgare L.) using two cultivars, Steptoe and Morex, which differ in their Fe efficiency response. In both cultivars, root extracts of plants grown in Fe-deficient conditions showed higher activities of enzymes related to organic acid metabolism, including citrate synthase, malate dehydrogenase and phosphoenolpyruvate carboxylase, compared to activities measured in root extracts of Fe-sufficient plants. Accordingly, the concentration of total carboxylates was higher in Fe-deficient roots of both cultivars, with citrate concentration showing the greatest increase. In xylem sap, the concentration of total carboxylates was also higher with Fe deficiency in both cultivars, with citrate and malate being the major organic acids. Leaf extracts of Fe-deficient plants also showed increases in citric acid concentration and in the activities of glucose-6-phosphate dehydrogenase and fumarase activities, and decreases in aconitase activity. Our results indicate that changes in root carboxylate metabolism previously reported in Strategy I species also occur in barley, a Strategy II plant species, supporting the existence of anaplerotic carbon fixation via increases in the root activities of these enzymes, with citrate playing a major role. However, these changes occur less intensively than in Strategy I plants. Activities of the anaerobic metabolism enzymes pyruvate decarboxylase and lactate dehydrogenase did not change in barley roots with Fe deficiency, in contrast to what occurs in Strategy I plants, suggesting that these changes may be Strategy I-specific. No significant differences were observed in overall carboxylate metabolism between cultivars, for plants challenged with high or low Fe treatments, suggesting that carboxylate metabolism changes are not behind the Fe-efficiency differences between these cultivars. Citrate synthase was the only measured enzyme with constitutively higher activity in Steptoe relative to Morex leaf extracts. PMID:22709961

Rockfish are commercially and recreationally important, yet due to the depths they inhabit (3-500m), little is known about their ecology. The present study examined 19 different species of Sebastes from the Southern California Bight over four seasons (late summer, fall, early winter, and spring) using metabolic enzyme assays. Enzymes used were lactate dehydrogenase (LDH), malate dehydrogenase (MDH), pyruvate kinase (PK), and citrate synthase (CS). Muscle proximate composition data (protein, water and lipid content) were also used to help interpret the enzyme data. Enzyme activity was lowest in the summer when expressed as activity per gram wet weight but when it was expressed per gram protein the trend was reversed. The rockfish have the highest protein as a percentage of wet mass (P%WM) i...

Manganese (Mn) toxicity is partially mediated by reduced ATP production. We have used oxidation rate assays-a measure of ATP production-under rapid phosphorylation conditions to explore sites of Mn{sup 2+} inhibition of ATP production in isolated liver, brain, and heart mitochondria. This approach has several advantages. First, the target tissue for Mn toxicity in the basal ganglia is energetically active and should be studied under rapid phosphorylation conditions. Second, Mn may inhibit metabolic steps which do not affect ATP production rate. This approach allows identification of inhibitions that decrease this rate. Third, mitochondria from different tissues contain different amounts of the components of the metabolic pathways potentially resulting in different patterns of ATP inhibition. Our results indicate that Mn{sup 2+} inhibits ATP production with very different patterns in liver, brain, and heart mitochondria. The primary Mn{sup 2+} inhibition site in liver and heart mitochondria, but not in brain mitochondria, is the F{sub 1}F{sub 0} ATP synthase. In mitochondria fueled by either succinate or glutamate + malate, ATP production is much more strongly inhibited in brain than in liver or heart mitochondria; moreover, Mn{sup 2+} inhibits two independent sites in brain mitochondria. The primary site of Mn-induced inhibition of ATP production in brain mitochondria when succinate is substrate is either fumarase or complex II, while the likely site of the primary inhibition when glutamate plus malate are the substrates is either the glutamate/aspartate exchanger or aspartate aminotransferase.

Howes, William V. (Washington State University, Pullman) and Bruce A. McFadden. Isocitrate lyase and malate synthase in Pseudomonas indigofera. II. Enzyme changes during the phase of adjustment and the early exponential phase. J. Bacteriol.84:1222–1227. 1962.—The differential rates of synthesis (DRS...

For Escherichia coli, growth on acetate requires the induction of the enzymes of the glyoxylate bypass, isocitrate lyase and malate synthase. The branch point between the glyoxylate bypass and the Krebs cycle is controlled by phosphorylation of isocitrate dehydrogenase (IDH), inhibiting that enzyme'...

A gram-negative bacterium, Mesorhizobium loti, contains a NADP+-malic enzyme (mlr5329) and a malate oxidoreductase (mlr0809) in the genome. We have screened transposon-induced mutants from the signature-tagged mutant library to survey their roles in nodule nitrogenase activity. The nodules induced by malate oxidoreductase mutants failed to fix N2, although NADP+-malic enzyme (NADP+-ME) mutants induced nodules exhibiting no change in nodule nitrogenase activity. When malate-degrading enzyme activities were compared between malate oxidoreductase mutants and wild-type M. loti, NAD+-malic enzyme (NAD+-ME) activity was decreased significantly in malate oxidoreductase mutants, suggesting it is an NAD+-ME mutant. We found that NADP+-ME was not required for N2 fixation. The fact that significant accumulations of sucrose, starch granules and malate were observed in the nodules induced by malate oxidoreductase mutant suggests that low nitrogenase activity of the nodules resulted in photosynthate accumulation. These data suggest that this malate oxidoreductase has a similar function to NAD+-ME in both Bradyrhizobium japonicum and Sinorhizobium meliloti.   

Mice with juvenile visceral steatosis (JVS) develop remarkable cardiac hypertrophy and exhibit an increased number of mitochondria in their heart. However, the biochemical characteristics and physiological functions of these mitochondria cardiac are little known. Here we show that the respiratory activities at state 3 with glutamate plus malate or succinate in the heart mitochondria of JVS mice were greatly decreased to 47% or 77%, respectively, compared with those of control mice. The contents of cytochromes a+a3, b, and c+c1 in the heart mitochondria of these mice were also decreased, to 51%, 45%, and 79%, respectively, of those of the control mice. Oligomycin-sensitive ATPase activitiy in these mitochondria, however, was increased to about 2 times over that of the control mice. Surprisingly, the ATP-Pi exchange activity of the heart mitochondria of JVS mice was greatly decreased, to 35% of that of control mice. On the other hand, the expression levels of 2 subunits of H+-ATP synthase, i.e., coupling factor 6 and ? subunit, in heart mitochondria from control and JVS mice were almost the same. These results indicate that the coordinate regulation of mitochondrial proliferation and gene expression for components of the oxidative phosphorylation system was markedly defective in the heart of JVS mice. Our current results also suggest the presence of a novel regulatory mechanisms of ATP synthase activities in the heart.   

Monospecific antibodies to glyoxysomal, mitochondrial, and cytosolic I malate dehydrogenase were used for the fluorescence immunohistochemical localization of these isoenzymes in dark-grown watermelon (Citrullus vulgaris Schrad.) cotyledons. It was demonstrated that, with cell organelles isolated by sucrose density gradient centrifugation, antibodies to glyoxysomal malate dehydrogenase were specific markers for glyoxysomes, and similarly, antibodies to mitochondrial malate dehydrogenase were markers for mitochondria. The time course of the glyoxysomal malate dehydrogenase appearance and decline was not synchronous for the individual tissues and differed completely from that of the mitochondria. The cytosolic malate dehydrogenase I was confined to restricted regions of the lower epidermis. The activity which was definitively localized outside the cell organelles decreased during the first days of germination. PMID:16662632

Based on the presence and absence of enzyme activities, the biochemical pathways for the fermentation of inulin by Clostridium thermosuccinogenes DSM 5809 are proposed. Activities of nine enzymes (lactate dehydrogenase, phosphoenolpyruvate carboxylase, malate dehydrogenase, fumarase, fumarate reduct...

Incorporation of /sup 32/P/sub i/ into the adenine nucleotide pool of intact bovine spermatozoa utilizing endogenous substrates results in a specific activity (S.A.) ratio ATP/ADP of 0.3 to 0.5, suggesting compartmentation of nucleotide pools or a pathway for phosphorylation of AMP in addition to the myokinase reaction. Incubation of filipin-permeabilized cells with pyruvate, acetylcarnitine, or ..cap alpha..-ketoglutarate (..cap alpha..KG) resulted in ATP-ADP S.A. ratios of 0.5, 0.8, and 1.6, respectively, for mitochondrial nucleotides. However, when malate was included with pyruvate or acetylcarnitine, the ATP/ADP S.A. ratio increased by 400% to 2.0 for pyruvate/malate and by 290% to 2.8 for acetylcarnitine/malate, while the ATP/ADP ratio increased by less than 100% in both cases. These results may indicate that under conditions of limited flux through the citric acid cycle a pathway for phosphorylation of AMP from a precursor other than ATP exists or that ATP is compartmented within the mitochondrion. In the presence of uncoupler and oligomycin with ..cap alpha..KG, pyruvate/malate, or acetylcarnitine/malate, /sup 32/P/sub i/ is incorporated primarily into ATP, resulting in an ATP/ADP S.A. ratio of 4.0 for ..cap alpha..KG, 2.7 for pyruvate/malate, and 2.8 for acetylcarnitine/malate. These data are consistent with phosphorylation of ADP during substrate level phosphorylation in the citric acid cycle.

Changes in malate concentration and activity of NADP-dependent malic enzyme were observed as the effect of Botrytis cinerea infection of C3 or CAM-performing Mesembryanthemum crystallinum plants. Biotic stress applied on C3 plants led to increase in malate concentration during the night and in consequence it led to increase in ?????-malate (day/night fluctuations) in infected leaves on the 2nd day post infection (dpi). It corresponded with induction of additional isoform of NADP-malic enzyme (NADP-ME3). On the contrary, CAM-performing M. crystallinum plants exhibited decrease in malate concentration and decay in its diurnal fluctuations as a reaction to B. cinerea infection. This correlated with significant decrease in activities of NADP-malic enzyme isoforms on the 2nd dpi as well as no f...

  Malate synthase (EC 4.1.3.2), the key enzyme of the glyoxylate cycle, was purified to a homogeneous protein from the wood-rotting basidiomycete Fomitopsis palustris grown on glucose. The purified enzyme, with a molecular mass of 520 kDa, was found to consist of eight 65-kDa subunits, and to have Km of 45 and 2.2 ?M for glyoxylate and acetyl-CoA, respectively. The enzyme activity was competitively inhibited by oxalate (Ki, 8.5 ?M) and glycolate (Ki, 17 ?M), and uncompetitively by coenzyme A (Ki, 100 ?M). The potent inhibition of the activity by p-chloromercuribenzoate suggests that the enzyme has a sulfhydryl group at the active center. However, the enzyme was inhibited moderately by adenine nucleotides and weakly by some of the metabolic intermediates of glycolysis and tricarboxylic acid cycle. The enzyme was completely inactive in the absence of metal ions and was maximally activated by Mg2+ (Km, 0.4 ?M), which also served to significantly prevent enzyme inactivation during storage.   

Despite the fact that the organic acid content of a fruit is regarded as one of its most commercially important quality traits when assessed by the consumer, relatively little is known concerning the physiological importance of organic acid metabolism for the fruit itself. Here, we evaluate the effect of modifying malate metabolism in a fruit-specific manner, by reduction of the activities of either mitochondrial malate dehydrogenase or fumarase, via targeted antisense approaches in tomato (Solanum lycopersicum). While these genetic perturbations had relatively little effect on the total fruit yield, they had dramatic consequences for fruit metabolism, as well as unanticipated changes in postharvest shelf life and susceptibility to bacterial infection. Detailed characterization suggested that the rate of ripening was essentially unaltered but that lines containing higher malate were characterized by lower levels of transitory starch and a lower soluble sugars content at harvest, whereas those with lower malate contained higher levels of these carbohydrates. Analysis of the activation state of ADP-glucose pyrophosphorylase revealed that it correlated with the accumulation of transitory starch. Taken together with the altered activation state of the plastidial malate dehydrogenase and the modified pigment biosynthesis of the transgenic lines, these results suggest that the phenotypes are due to an altered cellular redox status. The combined data reveal the importance of malate metabolism in tomato fruit metabolism and development and confirm the importance of transitory starch in the determination of agronomic yield in this species. PMID:21239646

Isocitrate lyase (ICL) and malate synthase (MS) of a psychrophilic marine bacterium, Colwellia maris, were purified to electrophoretically homogeneous state. The molecular mass of the ICL was found to be 240 kDa, composed of four identical subunits of 64.7 kDa. MS was a dimeric enzyme composed of 76.3 kDa subunits. N-Terminal amino acid sequences of the ICL and MS were analyzed. Purified ICL had its maximum activity at 20°C and was rapidly inactivated at the temperatures above 30°C, but the optimum temperature for the activity of MS was 45°C. NaCl was found to protect ICL from heat inactivation above 30°C, but the salt did not stabilize MS. Effects of temperatures on the kinetic parameters of both the enzymes were examined. The Km for the substrate (isocitrate) of ICL was decreased with decreasing temperature. On the other hand, the Km for the substrate (glyoxylate) of MS was increased with decreasing temperature. The calculated value of free energy of activation of ICL was on the same level as that of MS.   

The glyoxylate cycle, catalysed by two unique enzymes: isocitrate lyase (ICL; EC 4.1.3.1) and malate synthase (MS; EC 4.1.3.2), is necessary for the net conversion of acetate into glucose. This metabolic pathway operates in microorganisms, higher plants and nematodes. Two bacterial genes, encoding ICL and MS, were modified in order to introduce them into the mouse germ line. The ovine metallothionein-Ia (MT-Ia) promoter-ace B gene-ovine growth hormone (GH) gene (3' GH sequence) construct was fused to the ovine, MT-Ia promoter-ace A gene-ovine GH gene (3' GH sequence). Therefore, in this single DNA sequence, both ace A and ace B are under independent MT-Ia promoter control and can be induced by zinc. Transgenic mice were generated by pronuclear microinjection of the ace B-ace A gene construct. We now report the establishment of four mouse lines carying these two transgenes. Studies on the progeny of these lines indicate that one line (No. 91) is expressing both genes at the mRNA and enzyme levels in the liver and intestine, whereas another line (No. 66) has a much lower expression. Both enzyme activities were detected in the liver and intestine at levels up to 25% of those measured in fully derepressed Escherichia coli cells. PMID:8840530

The metabolic pathways specified by the glc and ace operons in Escherichia coli yield glyoxylate as a common intermediate, which is acted on by two malate synthase isoenzymes: one encoded by glcB and the other by aceB. Null mutations in either gene exhibit no phenotype, because of cross-induction of the ace operon by glycolate and the glc operon by acetate. In this study, the regulation of the glc operon, comprising the structural genes glcDEFGB, was analyzed at the molecular level. This operon, activated by growth on glycolate, is transcribed as a single message and is under the positive control of GlcC encoded by a divergent gene. Expression of the glc operon is strongly dependent on the integration host factor (IHF) and is repressed by the global respiratory regulator ArcA-P. In vitro gel-shift experiments demonstrated direct binding of the promoter DNA to IHF and ArcA-P. Mutant analysis indicated that cross-induction of the glc operon by acetate is mediated by the GlcC protein that recognizes the compound as an alternative effector. The similar pattern of regulation of the Glc and Ace systems by IHF and ArcA-P ensures their effective cross-induction. PMID:9880556

Mitochondrial Complex II (succinate:ubiquinoneoxidoreductase) is purified in a partially innactivated state, which canbe activated by removal of tightly bound oxaloacetate (Kearney, E.B. etal. Biochem Biophys Res Commun 49, 1115-1121). We crystallized Complex IIin the presence of oxaloacetate or with the endogenous inhibitor bound.The structure showed a ligand essentially identical to the "malate-likeintermediate" found in Shewanella Flavocytochrome c crystallized withfumarate (Taylor, P., et al. Nat Struct Biol 6, 1108-1112.)Crystallization of Complex II in the presence of excess fumarate alsogave the malate-like intermediate or a mixture of that and fumarate atthe active site. In order to more conveniently monitor the occupationstate of the dicarboxylate site, we are developing a library of UV/Visspectral effects induced by binding different ligands to the site.Treatment with fumarate results in rapid development of the fumaratedifference spectrum and then a very slow conversion into a speciesspectrally similar to the OAA liganded complex. Complex II is known to becapable of oxidizing malate to the enol form of oxaloacetate (Belikova,Y.O., et al. Biochim Biophys Acta 936, 1-9). The observations abovesuggest it may also be capable of interconverting fumarate and malate. Itmay be useful for understanding the mechanism and regulation of theenzyme to identify the malate-like intermediate and its pathway offormation from oxaloacetate or fumarate.

Underwater photosynthesis by aquatic plants is often limited by low availability of CO2, and photorespiration can be high. Some aquatic plants utilize crassulacean acid metabolism (CAM) photosynthesis. The benefits of CAM for increased underwater photosynthesis and suppression of photorespiration were evaluated for Isoetes australis, a submerged plant that inhabits shallow temporary rock pools. • Leaves high or low in malate were evaluated for underwater net photosynthesis and apparent photorespiration at a range of CO2 and O2 concentrations. • CAM activity was indicated by 9.7-fold higher leaf malate at dawn, compared with at dusk, and also by changes in the titratable acidity (lmol H+ equivalents) of leaves. Leaves high in malate showed not only higher underwater net photosynthesis at low external CO2 concentrations but also lower apparent photorespiration. Suppression by CAM of apparent photorespiration was evident at a range of O2 concentrations, including values below air equilibrium. At a high O2 concentration of 2.2-fold the atmospheric equilibrium concentration, net photosynthesis was reduced substantially and, although it remained positive in leaves containing high malate concentrations, it became negative in those low in malate. • CAM in aquatic plants enables higher rates of underwater net photosynthesis over large O2 and CO2 concentration ranges in floodwaters, via increased CO2 fixation and suppression of photorespiration.

Metabolic plasticity of Mycobacterium renders high degree of adaptive advantages in the persistence through the upregulation of glyoxylate shunt. The malate synthase (MS), an important enzyme of the shunt belongs to the G isoform and expressed predominantly as monomer. Here we did a comparative unfolding studies of two homologous MS from Mycobacterium tuberculosis (MtbMS) and Escherichia coli (ecMS) using various biophysical techniques. Despite having high sequence identities, they show different structural, stability and functional properties. The study suggests that the differences in the stability and unfolding of the two enzymes are by virtue of differential electrostatic modulation unique to their respective molecular assembly.

Abstract Streptococcus mutans and certain other oral lactic-acid bacteria were found to have the ability to carry out malolactic fermentation involving decarboxylation ofl-malate to yieldl-lactic acid and concomitant reduction in acidity. The activity was inducible byl-malate in S. mutans UA159 growing in suspensions or biofilms. The optimal pH for the fermentation was c. 4.0 for both suspensions and biofilms, although the pH optimum for malolactic enzyme in permeabilized cells of S. mutans UA159 was close to 5.5. Although malate did not serve as a catabolite for growth of S. mutans, it did serve to protect the organism against acid killing and to maintain ATP pool levels during starvation. Alkalinization associated with malolactic fermentation resulted in pH rise or increased need to add ...

Pitaya is a crassulacean acid metabolism (CAM) plant that is normally grown in subtropical regions, but it would be useful to cultivate this crop in temperate regions. In this study, we measured concentrations of organic acids and carbohydrates, and activities of the enzymes phosphoenolpyruvate carboxylase (PEPC), malic enzyme (ME), and malate dehydrogenase (MDH) in pitaya grown in a temperate zone, and compared the diurnal changes in these components between winter and summer. In summer, the diurnal changes in malate, citrate, and starch in pitaya were typical of starch-using CAM plants. Activities of PEPC and ME also showed typical CAM-type diurnal patterns in summer. In particular, changes in ME activity were closely associated with changes in malate content. In winter, changes in malate content showed a typical CAM pattern, although the amount accumulated was only half of that accumulated in summer, however, the citrate content in winter remained at an almost constant low level throughout the day. PEPC and ME activities were almost constant through the day in winter, however, PEPC activity in winter was similar to its minimum level in summer, whereas ME activity in winter was similar to its maximum level in summer. MDH activity was higher in summer than in winter, but there was no distinct diurnal pattern observed in either summer or winter. These results suggest that pitaya shows normal photosynthesis and metabolism in summer. In winter, however, malate accumulation is restricted, result in decreased concentrations of downstream products and metabolites. This may be because of decreased PEPC activity and increased ME activity during the night in winter. Our results suggest that temperature is not the only factor that affects CAM in this plant, because the summer temperature in this study was similar to the winter temperature in our previous study, which was carried out in a subtropical region (Ishigaki Island). Furthermore, diurnal profiles of metabolites and enzyme activities in both regions were similar in summer and winter; therefore, daylength may also be an important environmental factor.   

nitric oxide synthase (Types I and II) require calcium/calmodulin for activity; Type II| ... targetsincludephosphodiesterases,ion channels,anda cyclicGMP-dependent ... III nitric oxide synthase has been identified in LLC-PK1 kidney epithelial cells ...

An automatic flow-injection method for the determination of malate dehydrogenase activity is proposed. The manifold used includes a selecting valve for closing the circuit along which the reacting plug is continuously circulated, and passed through the flow-cell of a conventional spectrophotometer, ...

The relationship between the O2 input rate into a suspension of Rhizobium leguminosarum bacteroids, the cellular ATP and ADP pools, and the whole-cell nitrogenase activity during L-malate oxidation has been studied. It was observed that inhibition of nitrogenase by excess O2 coincided with an increa...

Aerial parts (shoots) of maize seedlings fed hydroponically with 300 muM sodium arsenate [As(V)] or 250 muM sodium arsenite [As(III)] for 24 h were analyzed for differentially expressed proteins by 2-DE and digital image analysis. About 15% of total detected proteins (74 out of 500) were up- or, mainly, down-regulated by arsenic, among which 14 were selected as being those most affected by the metalloid. These proteins were analyzed by MALDI-TOF MS and 7 of them were identified: translation initiation factor eIF-5A, ATP synthase, cysteine synthase, malate dehydrogenase, protein kinase C inhibitor, Tn10 transposase-like protein, and guanine nucleotide binding protein. Each of these proteins was completely repressed by As(V) and/or As(III), except protein kinase C inhibitor, which was newly detected after exposure to As(V). PMID:16534746

Undecaprenyl pyrophosphate synthase is a cis-prenyltransferase enzyme, which is required for cell wall biosynthesis in bacteria. Undecaprenyl pyrophosphate synthase is an attractive target for antimicrobial therapy. We performed long molecular dynamics simulations and docking studies on undecaprenyl pyrophosphate synthase to investigate its dynamic behavior and the influence of protein flexibility on the design of undecaprenyl pyrophosphate synthase inhibitors. We also describe the first X-ray crystallographic structure of Escherichia coli apo-undecaprenyl pyrophosphate synthase. The molecular dynamics simulations indicate that undecaprenyl pyrophosphate synthase is a highly flexible protein, with mobile binding pockets in the active site. By carrying out docking studies with experimentall...

We identified and characterized a malate dehydrogenase from Streptomyces coelicolor A3(2) (ScMDH). The molecular mass of ScMDH was 73,353.5 Da with two 36,675.0 Da subunits as analyzed by matrix-assisted laser-desorption ionization–time-of-flight mass spectrometry (MALDI-TOF-MS). The detailed kinetic parameters of recombinant ScMDH are reported here. Heat inactivation studies showed that ScMDH was more thermostable than most MDHs from other organisms, except for a few extremely thermophile bacteria. Recombinant ScMDH was highly NAD+-specific and displayed about 400-fold (kcat) and 1,050-fold (kcat?Km) preferences for oxaloacetate reduction over malate oxidation. Substrate inhibition studies showed that ScMDH activity was inhibited by excess oxaloacetate (Ki=5.8 mM) and excess L-malate (Ki=12.8 mM). Moreover, ScMDH activity was not affected by most metal ions, but was strongly inhibited by Fe2+ and Zn2+. Taken together, our findings indicate that ScMDH is significantly thermostable and presents a remarkably high catalytic efficiency for malate synthesis.   

The growths of Saccharomyces cerevisiae wild-type strain and another strain containing a disrupted structural gene for chitin synthase (chs1::URA3), defective in chitin synthase 1 (Chs1) but showing a new chitin synthase activity (Chs2), were affected by Calcofluor. To be effective, the interaction ...

Two high mountain plants Soldanella alpina (L.) and Ranunculus glacialis (L.) were transferred from their natural environment to two different growth conditions (22 degrees C and 6 degrees C) at low elevation in order to investigate the possibility of de-acclimation to light and cold and the importance of antioxidants and metabolite levels. The results were compared with the lowland crop plant Pisum sativum (L.) as a control. Leaves of R. glacialis grown for 3 weeks at 22 degrees C were more sensitive to light-stress (defined as damage to photosynthesis, reduction of catalase activity (EC 1.11.1.6) and bleaching of chlorophyll) than leaves collected in high mountains or grown at 6 degrees C. Light-stress tolerance of S. alpina leaves was not markedly changed. Therefore, acclimation is reversible in R. glacialis leaves, but constitutive or long-lasting in S. alpina leaves. The different growth conditions induced significant changes in non-photochemical fluorescence quenching (qN) and the contents of antioxidants and xanthophyll cycle pigments. These changes did not correlate with light-stress tolerance, questioning their role for light- and cold-acclimation of both alpine species. However, ascorbate contents remained very high in leaves of S. alpina under all growth conditions (12-19% of total soluble carbon). In cold-acclimated leaves of R. glacialis, malate represented one of the most abundant compounds of total soluble carbon (22%). Malate contents declined significantly in de-acclimated leaves, suggesting a possible involvement of malate, or malate metabolism, in light-stress tolerance. Leaves of the lowland plant P. sativum were more sensitive to light-stress than the alpine species, and contained only low amounts of malate and ascorbate. PMID:12493869

When Pichia pinus was cultivated in a glucose mineral medium in a series of 3 fermentors (the 1st fermentor being a turbidostat), the biomass yield in the 1st, 2nd, and 3rd fermentors was 2.12, 6.23, 11.31 g/L (dry wt.), respectively. The activities of hexokinase and pyruvate kinase gradually decreased whereas the activities of glucose 6-phosphate dehydrogenase, malate dehydrogenase, and phosphorylase progressively increased. The size of the cells increased during sequential cultivation.

Tartrate oxidation activity was found in the crude extract of an aerobic hyperthermophilic archaeon Aeropyrum pernix, and the enzyme was identified as (S)-malate dehydrogenase (MDH), which, when produced in Escherichia coli, was mainly obtained as an inactive inclusion body. The inclusion body was dissolved in 6?M guanidine?HCl and gradually refolded to the active enzyme through dilution of the denaturant. The purified recombinant enzyme consisted of four identical subunits with a molecular mass of about 110?kDa. NADP was preferred as a coenzyme over NAD for (S)-malate oxidation and, unlike MDHs from other sources, this enzyme readily catalyzed the oxidation of (2S,3S)-tartrate and (2S,3R)-tartrate. The tartrate oxidation activity was also observed in MDHs from the hyperthermophilic archae...

The malate dehydrogenase (MDH) (EC 1.1.1.37) from Corynebacterium glutamicum (Brevibacterium flavum) ATCC14067 was purified to homogeneity. Its amino-terminal sequence (residues 1 to 8) matched the sequence (residues 2 to 9) of the MDH from C. glutamicum (GenBank accession no. CAC83073). The molecular mass of the native enzyme was 130 kDa. The protein was a homotetramer, with a 33-kDa subunit molecular mass. The enzyme was almost equally active both for NADH andNADPH as coenzyme on the bases of the kcat values at pH 6.5 which is the optimum pH for the both coenzymes. Plotting of the logarithms of the 1/Km, kcat, and kcat/Km values with respect to oxalacetate against pH lead to speculation that imidazolium is possibly a functional group in the active center of the enzyme. Citrate activated the enzyme in the oxidation of malate to oxalacetate and inhibited it in the reverse reaction.   

Pyruvate dehydrogenase (PDH) is a key regulator of cardiac substrate selection and is regulated by both pyruvate dehydrogenase kinase (PDK)-mediated phosphorylation and feedback inhibition. The extent to which chronic upregulation of PDK protein levels, acutely increased PDK activity and acute feedback inhibition limit PDH flux remains unclear because existing in vitro assessment methods inherently disrupt the regulation of the enzyme complex. We have demonstrated previously that hyperpolarised (13)C-labelled metabolic tracers coupled with MRS can monitor flux through PDH in vivo. The aim of this study was to determine the relative contributions of acute and chronic changes in PDK and PDH activities to in vivo myocardial PDH flux. We examined both fed and fasted rats with either hyperpolarised [1-(13)C]pyruvate alone or hyperpolarised [1-(13)C]pyruvate co-infused with malate [to modulate mitochondrial nicotinamide adenine dinucleotide (NADH/NAD(+)) and acetyl-coenzyme A (acetyl-CoA)/CoA ratios, which alter both PDH activity and flux]. To confirm the metabolic fate of infused malate, we performed in vitro (1)H NMR spectroscopy on cardiac tissue extracts. We observed that, in fed rats, where PDH activity was high, the presence of malate increased PDH flux by 27%, whereas, in the fasted state, malate infusion had no effect on PDH flux. These observations suggest that pyruvate oxidation is limited by feedback inhibition from acetyl-CoA only when PDH activity is high. Therefore, in the case of PDH, and potentially other enzymes, hyperpolarised (13)C MRI can be used to assess noninvasively enzymatic regulation. PMID:21387444

Two malate dehydrogenases (MDH-M1 and MDH-M2) were found in a methanol-using yeast, Candida sp. N-16. MDH-M2 was induced with methanol. These enzymes were purified as electrophoretically and isoelectrophoretically homogeneous proteins. The molecular weights of MDH-M1 and MDH-M2 were estimated to be about 78,000 (homodimer) and 160,000 (homotetramer). Several kinetic properties were significantly different between the two enzymes. The value (2.07) of Vmax(oxaloacetate)/Vmax(malate) and Kcats (555 s-1 for oxaloacetate, 481 s-1 for NADH) of MDH-M2 were higher than the ratio (1.37) of Vmax and Kcats(241 s-1 for oxaloacetate, 271 s-1 for NADH) of MDH-M1, respectively. The activity of MDH-M2 was inhibited by a high concentration of NAD+ and the activity of MDH-M1 by oxaloacetate.   

We detected mouse liver malate, sorbitol and aldehyde dehydrogenases by negative staining, analysis of malate and sorbitol dehydrogenase activities using each substrate, and electron transfers including nicotinamide adenine dinucleotide (NAD) and nitroblue tetrazolium in non-denaturing two-dimensional electrophoresis (2-DE) gel. Dehydrogenases were also identified by electrospray ionization tandem mass spectrometry (ESI-MS/MS) after 2-DE separation and protein detection by negative staining. Spots of dehydrogenases separated by 2-DE were excised, and simultaneously transferred and immobilized on polyvinylidene difuoride (PVDF) resin by electrophoresis. The dehydrogenase activities remained intact after immobilization. In conclusion, resin-immobilized dehydrogenases can be simultaneously obtained after separation by non-denaturing 2-DE, detection by negative staining and transferring to resins.

In Rhodobacter capsulatus, the hupL gene encoding the large subunit of the uptake-hydrogenase (Hup) enzyme complex was mutated by insertion of an interposon. The mutant neither synthesized an active hydrogenase nor grew photoautotrophically. Under conditions of nitrogen (N) limitation, photoheterotrophic cultures of the wild type and the mutant evolved H[sub 2] by activity of the nitrogenase enzyme complex. When grown with glutamate as an N source and either D,L-malate or L-lactate as carbon sources, the efficiency of H[sub 2] production by the HupL mutant was higher than 90%, whereas wild-type cultures exhibit efficiencies of 54% (with D,L-malate) and 64% (with L-lactate), respectively. With NH[sub 4][sup +] as the N source, efficiencies of H[sub 2] production were 70% (mutant) and 52% (wild type). (orig.)

Wild-type Acetobacter aceti NBRC 14818 possesses genes encoding isocitrate lyase (aceA) and malate synthase (glcB), which constitute the glyoxylate pathway. In contrast, several acetic acid bacteria that are utilized for vinegar production lack these genes. Here, an aceA-glcB knockout mutant of NBRC 14818 was constructed and used for investigating the role of the glyoxylate pathway in acetate productivity. In medium containing ethanol as a carbon source, the mutant grew normally during ethanol oxidation to acetate, but exhibited slower growth than that of the wild-type strain as the accumulated acetate was oxidized. The mutant grew similarly to that of the wild-type strain in medium containing glucose as a carbon source, indicating that the glyoxylate pathway was not necessary for glucose ...

Efforts were exerted to achieve efficient expression of proteins of hyperthermophilic bacteria, hyperthermophilic archaeabacteria in particular, using a heterogene expression system in which Escherichia coli was the host. In an effort to search for genes related to protein folding and to elucidate the mechanism of folding, chaperonin and prefoldin subunit genes, out of various factors participating in protein folding in hyperthermophilic archaeabacteria, were cloned, and expressed in Escherichia coli. As a system for analyzing protein folding reaction, an experimental system was established on a substrate comprising isopropyl malate dehydrogenase, citrate synthase, glucose dehydrogenase, and a green fluorescent protein. Studies were further conducted to elucidate the mechanism of expression of enzyme genes in Escherichia coli for the establishment of a mass production method for useful enzymes. Also carried out was the research and development of an element technology evaluation system involving protein expression. (NEDO)

In an aim to elucidate the structure-function relationship of NAD-linked malic enzyme [EC 1.1.1.38] from Escherichia coli W, the effect of chemical modification on the catalytic and regulatory properties of the enzyme was studied. Upon photooxidation of the enzyme in the presence of methylene blue, a time-dependent inactivation occurred following pseudo-first order kinetics. The pH-dependence of the inactivation rate exhibited a pK value of 6.1. L-Malate, NAD+, and Mn2+ markedly protected the enzyme against the inactivation. Prior masking of the catalytically essential sulfhydryl groups with p-mercuribenzoate did not result in a retardation of the rate of photoinactivation. This excluded the possibility of an involvement of sulfhydryl group modification in the photoinactivation. Although the Km values for L-malate and NAD+ were not affected by photooxidation, the S0.5 value and the Hill coefficient for Mn2+ were considerably altered, and the cooperative nature of the saturation profile for Mn2+ in the native enzyme was completely abolished. The activating effect of L-aspartate on the native enzyme was completely abolished upon photooxidation, and the inhibitory effect of CoA was also diminished to a marked extent upon the treatment. The oxaloacetate decarboxylating activity of the enzyme was lost in parallel with the loss of the activity for oxidative decarboxylation of L-malate. These results suggest a possible involvement of histidyl residue(s) in the catalytic and regulatory functions of the enzyme. PMID:3898156

Sucrose synthase, which catalyzes uridine diphosphate (UDP)-dependent cleavage of sucrose into fructose and UDP-glucose, is induced by oxygen deficiency in rice seedlings and is considered to play an important role in energy production under hypoxic conditions. In this study, we analyzed the relationship between coleoptile elongation and sucrose synthase activity in rice (Oryza sativa L.) cultivars under submerged conditions. We also analyzed the activity of ?-amylase, which digests starch reserves in the endosperm and is considered to be important for energy production in young seedlings. The results indicated that different rice cultivars had different sucrose synthase and ?-amylase activities under submerged conditions. Moreover, sucrose synthase activity in whole seedlings was significantly correlated with coleoptile length under submerged conditions, whereas the correlation between ?-amylase activity and coleoptile length was low. Sugar content of shoots differed with the cultivar. Correlation analysis demonstrated that sucrose content was highly correlated with coleoptile length and sucrose synthase activity, but not with ?-amylase activity.   

The effects of chronic treatment with testosterone propionate (TP) on compensatory muscle hypertropy in female rats are examined. The 48 female rats were placed in one of four test groups: (1) no overload (synergist removal), no TP, (2) overload, no TP, (3) no overload + TP, and (4) overload + TP. The technique used to administer the TP is described. The preparation of the plantaris muscle, the analysis of pyruvate oxidation and the determination of malate and lactate dehydrogenases and the noncollogen protein are explained. The results which reveal the effect of overload and TP on body weight, noncollogen protein concentration, lactate and malate dehydrogenase activities, and pyruvate oxidation are presented and discussed. It is concluded that in terms of body weight, protein content, pyruvate, glycolysis, and oxidative metabolisms chronic TP treatments do not change compensatory muscle hypertropy.

  Membrane-bound NAD(P)-independent malate dehydrogenase (EC 1.1.99.16) was purified to homogeneity from the membrane of thermotolerant Acetobacter sp. SKU 14, an isolate from Thailand. The enzyme was solubilized from the membrane fraction of glycerol-grown cells with 1% Triton X-100 in the presence of 0.1 M KCl, and purified to homogeneity through steps of column chromatographies on DEAE-Sephadex A-50 and DEAE-Toyopearl in the presence of 0.1% Triton X-100. The purified enzyme showed a single protein band in both native-PAGE and SDS-PAGE. The enzyme was a homodimer with a molecular mass of 60 kDa subunit and had noncovalently bound FAD as the cofactor. The enzyme was stable over pH 5 and had its maximum activity at pH 11.0 when ferricyanide was used as an electron acceptor. The enzyme activity was elevated by the addition of ammonium ions. The substrate specificity was very strict to only L-malate, of which the apparent Km was 10 mM and over 20 compounds involving D-malate were not oxidized by the enzyme.   

The flavor and taste of fruits are often determined by terpenes. We identified three cDNAs encoding putative terpene synthases from olive fruits of cv. Frantoio and Grignano. Heterologous expression in a bacterial system demonstrated that one of the terpene synthases, OeGES1, was an active monoterpene synthase that converted geranyl diphosphate to the monoterpene alcohol geraniol. The transcript accumulation pattern of this gene showed a peak during fruit ripening in both genotypes, indicating that the enzyme may be involved in the production of monoterpene flavor compounds in olive fruit. Although the putative terpene synthases OeTPS2 and OeTPS3 clustered with a-farnesene synthases and angiosperm monoterpene synthases, no detectable in vitro activity was found after expression in a bacter...

The aim of this study was to investigate the secondary fermentation of alcoholic green cider by Lactobacillus brevis and Oenococcus oeni in a membrane bioreactor so as to compare the performance of the two organisms to rapidly carry out the malolactic fermentation (MLF), an important step in reducing acidity and enhancing the flavor characteristics of the beverages. First, the growth of both organisms was intensified by using perfusion culture in a membrane bioreactor (MBR). O. oeni and L. brevis were grown up to 12.8 g dry cell weight (DCW) l(-1) and 15.5 g DCW l(-1) in the MBR. Secondly, the resultant cells were then used for the malolactic transformation of green cider in the MBR. The influences of the residence time in the MBR and the ethanol concentration of the green cider on the organic acid transformation were investigated. Both organisms showed a good tolerance against the acidic conditions (pH 3.0-4.0) and ethanol (90 g l(-1)). Good levels of malate removal in the MBR were achieved by both organisms but O. oeni was more tolerant to high ethanol concentrations and was capable of growth and malate removal in 130 g ethanol l(-1) green cider. L. brevis malate removal was significantly inhibited above 110 g ethanol l(-1). The MBR allowed the development of high concentrations of active cells capable of rapid MLF and could be achieved over a prolonged period and over a wide range of conditions thus allowing the control of malate transformation rate. Organism selection for the transformation will be governed by the desired beverage characteristics. There is considerable scope to optimize the process further both with the choice of organisms and the design and operation of the reactor. Rapid beverage maturation on a commercial scale may be possible using MBR and pure cultures of MLF lactic acid bacteria. PMID:20386953

A cDNA encoding a novel plant type III polyketide synthase was cloned and sequenced from the Chinese club moss Huperzia serrata (Huperziaceae). The deduced amino acid sequence of Hu. serrata polyketide synthase 1 showed 44-66% identity to those of other chalcone synthase superfamily enzymes of plant origin. Further, phylogenetic tree analysis revealed that Hu. serrata polyketide synthase 1 groups with other nonchalcone-producing type III polyketide synthases. Indeed, a recombinant enzyme expressed in Escherichia coli showed unusually versatile catalytic potential to produce various aromatic tetraketides, including chalcones, benzophenones, phloroglucinols, and acridones. In particular, it is remarkable that the enzyme accepted bulky starter substrates such as 4-methoxycinnamoyl-CoA and N-methylanthraniloyl-CoA, and carried out three condensations with malonyl-CoA to produce 4-methoxy-2',4',6'-trihydroxychalcone and 1,3-dihydroxy-N-methylacridone, respectively. In contrast, regular chalcone synthase does not accept these bulky substrates, suggesting that the enzyme has a larger starter substrate-binding pocket at the active site. Although acridone alkaloids have not been isolated from Hu. serrata, this is the first demonstration of the enzymatic production of acridone by a type III polyketide synthase from a non-Rutaceae plant. Interestingly, Hu. serrata polyketide synthase 1 lacks most of the consensus active site sequences with acridone synthase from Ruta graveolens (Rutaceae). PMID:17250741

Oxygen consumption, content of several carbohydrate metabolites, and activities of their coupled enzymes were studied in bivalve molluscs with different tolerance to oxygen deficit: Mytilus galloprovincialis Lam. (black morpha) and Anadara Inaequivalvis Br. It has been shown that under conditions of external normoxia the anadara resistance to hypoxia preserves anaerobic orientation of metabolism. Its tissues are distinguished by high activities of malate and lactate dehydrogenases with the decreased content of glucose and the increased level of lactate. In several organs the succinate thiokinase and fumarate reductase reactions are realized, which is indicated by elevated activities of the alanine and aspartate aminotransferases. The anaerobic orientation of protein metabolism is added by ...

The activities of cystathionine synthase [L-serine hydro-lyase (adding homocysteine), EC 4.2.1.22], uroporphyrinogen I synthase [porphobilinogen ammonia-lyase (polymerizing), EC 4.3.1.8], and glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NADP+ 1-oxidoreductase, EC 1.1.1.49) have been meas...

We describe the subcellular location of chitin synthase 1 (CHS-1), one of seven chitin synthases in Neurospora crassa. Laser scanning confocal microscopy of growing hyphae showed CHS-1–green fluorescent protein (GFP) localized conspicuously in regions of active wall synthesis, namely, the core of th...

Previous work led to the puzzling conclusion that chitin synthase 1, the major chitin synthase activity in Saccharomyces cerevisiae, is not required for synthesis of the chitinous primary septum. The mechanism of in vivo synthesis of chitin has now been clarified by cloning the structural gene for t...

(Dihydro)ceramide synthase 2 (cers2, formerly called lass2) is the most abundantly expressed member of the ceramide synthase gene family, which includes six isoforms in mice. CERS2 activity has been reported to be specific toward very long fatty acid residues (C22–C24). In order to study the biologi...

The alpha- and beta-subunits of membrane-bound ATP synthase complex bind ATP and ADP: beta contributes to catalytic sites, and alpha may be involved in regulation of ATP synthase activity. The sequences of beta-subunits are highly conserved in Escherichia coli and bovine mitochondria. Also alpha and...

Cyclopropane fatty acids (CFAs) are generally synthesized as bacterial cultures enter stationary phase. In Escherichia coli, the onset of CFA synthesis results from increased transcription of cfa, the gene encoding CFA synthase. However, the increased level of CFA synthase activity is transient; the...

An HPLC assay is described for the enzyme strictosidine synthase in which the formation of strictosidine and the decrease of tryptamine can be followed at the same time. In cell cultures of Catharanthus roseus significant amounts of strictosidine glucosidase activity were detected. In crude preparations, the strictosidine synthase reaction is therefore best measured by the secologanin-dependent decrease of tryptamine. In this way, the specific synthase activity in a cell free extract was found to be 56 pkat/mg of protein. Inclusion of 100 mM D(+)-gluconic acid-delta-lactone in the incubation mixture inhibited 75% of the glucosidase activity, without inhibiting the synthase activity. The synthase activity was readily separated from the glucosidase activity by gel filtration on Sephadex G-75 or Ultrogel AcA-44. Cell cultures of Tabernaemontana orientalis did not contain measurable amounts of strictosidine glucosidine activity. The specific strictosidine synthase activity was 130-200 pkat/mg of protein during the growth of this cell culture. Strictosidine synthase is stable at -20 degrees C for at least 2 months. PMID:2742131

It is noteworthy that in spite of the similarity of the reactions catalyzed by these prenyltransferases, the modes of expression of catalytic function are surprisingly different, varying according to the chain length and stereochemistry of reaction products. These enzymes are summarized and classified into four groups, as shown in Figure 13. Short-chain prenyl diphosphates synthases such as FPP and GGPP synthases require no cofactor except divalent metal ions, Mg2+ or Mn2+, which are commonly required by all prenyl diphosphate synthases. Medium-chain prenyl diphosphate synthases, including the enzymes for the synthesis of all-E-HexPP and all-E-HepPP, are unusual because they each consist of two dissociable dissimilar protein components, neither of which has catalytic activity. The enzymes for the synthesis of long-chain all-E-prenyl diphosphates, including octaprenyl (C40), nonaprenyl-(C45), and decaprenyl (C50) diphosphates, require polyprenyl carrier proteins that remove polyprenyl products from the active sites of the enzymes to maintain efficient turnovers of catalysis. The enzymes responsible for Z-chain elongation include Z,E-nonaprenyl-(C45) and Z,E-undecaprenyl (C55) diphosphate synthases, which require a phospholipid. The classification of mammalian synthases seems to be fundamentally similar to that of bacterial synthases except that no medium-chain prenyl diphosphate synthases are included. The Z-prenyl diphosphate synthase in mammalian cells is dehydrodolichyl PP synthase, which catalyzes much longer chain elongations than do bacterial enzymes. Dehydrodolichyl PP synthase will be a major target of future studies in this field in view of its involvement in glycoprotein biosynthesis. PMID:9090291

The object of this work was to study the activity and the isozyme spectra of hexokinase (the triggering enzyme of glycolysis), glucose-6-phosphate dehydrogenase (the key enzyme of the pentose-phosphate shunt), malate dehydrogenase and isocitrate dehydrogenase (the enzymes of the citric acid cycle) and alcohol dehydrogenase (the enzyme involved in the first steps of ethanol oxidation) in Saccharomyces cerevisiae, race Ya, S. carlsbergensis, race 4228, and their hybrid 67. The parent organisms and their hybrid were shown to differ from one another in the qualitative composition and the activity of the isozyme spectra of the above enzymes. PMID:6363888

Summary Infections with the microaerophilic parasite Trichomonas vaginalis are treated with the 5-nitroimidazole drug metronidazole, which is also in use against Entamoeba histolytica, Giardia intestinalis and microaerophilic/anaerobic bacteria. Here we report that in T. vaginalis the flavin enzyme thioredoxin reductase displays nitroreductase activity with nitroimidazoles, including metronidazole, and with the nitrofuran drug furazolidone. Reactive metabolites of metronidazole and other nitroimidazoles form covalent adducts with several proteins that are known or assumed to be associated with thioredoxin-mediated redox regulation, including thioredoxin reductase itself, ribonucleotide reductase, thioredoxin peroxidase and cytosolic malate dehydrogenase. Disulphide reducing activity of thi...

Polygalacturonic acid (PGA) synthase successively transfers galacturonic acid to oligogalacturonic acid by an ?1,4-linkage to synthesize PGA, the backbone of plant pectic homogalacturonan. PGA synthase has not been purified to date due to its instability in vitro. In this study, we found stable conditions in vitro and separated a minimum active component of the enzymes from pea and azuki bean epicotyls. The PGA synthase lost its activity in 500 mM of sodium chloride or potassium chloride, while it was relatively stable at low salt concentrations. Under low salt concentrations, three peaks bearing PGA synthase activity were separated, by gel filtration and sucrose density gradient centrifugation. The molecular masses of these enzymes solubilized with 3-[(3-cholamidopropyl)dimethyl-ammonio]propanesulfonic acid were estimated to be 21,000, 5,000, and 590 kDa. The two higher molecular mass PGA synthases converted to smaller PGA synthase proteins when treated with high salt concentrations, while retaining their activity, indicating that PGA synthase has a minimum active component for its activity.   

Bacillus subtilis cells grown in yeast extract medium accumulated 3-fluoro-l-erythro-[1,2-14C2]malate more than 30-fold from the surrounding medium. No metabolic products derived from 3-fluoro-l-erythro-malate could be detected in these cells. l-Malate competitively inhibited transport of 3-fluoro-l...

We recently engineered Corynebacterium glutamicum for aerobic production of 2-ketoisovalerate by inactivation of the pyruvate dehydrogenase complex, pyruvate:quinone oxidoreductase, transaminase B, and additional overexpression of the ilvBNCD genes, encoding acetohydroxyacid synthase, acetohydroxyacid isomeroreductase, and dihydroxyacid dehydratase. Based on this strain, we engineered C. glutamicum for the production of isobutanol from glucose under oxygen deprivation conditions by inactivation of l-lactate and malate dehydrogenases, implementation of ketoacid decarboxylase from Lactococcus lactis, alcohol dehydrogenase 2 (ADH2) from Saccharomyces cerevisiae, and expression of the pntAB transhydrogenase genes from Escherichia coli. The resulting strain produced isobutanol with a substrate-specific yield (YP/S) of 0.60 ± 0.02 mol per mol of glucose. Interestingly, a chromosomally encoded alcohol dehydrogenase rather than the plasmid-encoded ADH2 from S. cerevisiae was involved in isobutanol formation with C. glutamicum, and overexpression of the corresponding adhA gene increased the YP/S to 0.77 ± 0.01 mol of isobutanol per mol of glucose. Inactivation of the malic enzyme significantly reduced the YP/S, indicating that the metabolic cycle consisting of pyruvate and/or phosphoenolpyruvate carboxylase, malate dehydrogenase, and malic enzyme is responsible for the conversion of NADH+H+ to NADPH+H+. In fed-batch fermentations with an aerobic growth phase and an oxygen-depleted production phase, the most promising strain, C. glutamicum ?aceE ?pqo ?ilvE ?ldhA ?mdh(pJC4ilvBNCD-pntAB)(pBB1kivd-adhA), produced about 175 mM isobutanol, with a volumetric productivity of 4.4 mM h?1, and showed an overall YP/S of about 0.48 mol per mol of glucose in the production phase.

Crassulacean acid metabolism (CAM) plants fix carbon dioxide at night by the carboxylation of phosphoenolpyruvate. If CO2 fixation is conducted with TC YO2, then in the absence of carbonic anhydrase, the malate formed by dark CO2 fixation should also contain high levels of carbon-13 and oxygen-18. Conversely, if carbonic anhydrase is present and highly active, oxygen exchange between CO2 and cellular H2O will occur more rapidly than carboxylation, and the ( TC) malate formed will contain little or no oxygen-18 above the natural abundance level. The presence of oxygen-18 in these molecules can be detected either by nuclear magnetic resonance or by mass spectrometry. Studies of phosphoenolpyruvate carboxylase in the presence and absence of carbonic anhydrase in vitro confirm the validity of the method. When CAM plants are studied by this method, we find that most species show incorporation of a significant amount of oxygen-18. Comparison of these results with results of isotope fractionation and gas exchange studies permits calculation of the in vivo activity of carbonic anhydrase toward HCO3 compared with that of phosphoenolpyruvate carboxylase. The ratio (carbonic anhydrase activity/phosphoenolpyruvate carboxylase activity) is species dependent and varies from a low of about 7 for Ananas comosus to values near 20 for Hoya carnosa and Bryophyllum pinnatum, 40 for Kalanchoee daigremontiana, and 100 or greater for Bryophyllum tubiflorum, Kalanchoee serrata, and Kalanchoae tomentosa. Carbonic anhydrase activity increases relative to phosphoenolpyruvate carboxylase activity at higher temperature. 37 references, 2 figures, 8 tables.

Mature wheat endosperm contains A-, B-, C-type starch granules, and each class has unique physiochemical properties which determine the quality of starch. The dynamics of the starch granule size distribution, activities of starch synthases and expression of starch synthase encoding genes were studied in superior and inferior grains during grain filling. Compared with inferior grains, superior grains showed higher grain weight, contents of starch, amylose and amylopectin. The formation of A-, B-, C-type starch granules initiated at 4, 8, 20 DAF, respectively, and was well consistent with the temporally change patterns of starch synthase activities and relative gene expression levels. For instance, activities of soluble and granule-bound starch synthases (designated SSS and GBSS) peaked at 20 and 24 DAF. Genes encoding isoforms of starch synthases expressed at different grain filling periods. In addition, SS I was generally expressed over the grain filling stage; the SS II and SS III were expressed over the early and mid grain filling stage, and the GBSS I was expressed during the mid to late grain filling stage. In addition, the time-course changes in activities of starch synthases and expression of starch synthase encoding genes explained well the dynamics of the starch granule size distribution.

The enzyme glycogen synthase (UDP glocose:glycogen 4-[alpha]-D-glucosyltransferase, EC 2.4.1.11) catalyzes the formation of glycogen from uridine diphosphate glucose (UPDG). Impaired activation of muscle glycogen synthase by insulin has been noted in patients with genetic risk of developing non-insulin-dependent diabets mellitus (NIDDM) and this may represent an early defect in the pathogenesis of this disorder. As such, glycogen synthase represents a candidate gene for contributing to genetic susceptibility. As a first step in studying the role of glycogen synthase in the genetics of NIDDM, we have isolated a cosmid encoding the human glycogen synthase gene (gene symbol GYS) and determined its chromosomal localization by fluorescence in situ hybridization. 4 refs., 1 fig.

During fruit development, the concentration of main polyphenols (flavonols, flavanols, dihydrochalcones, hydroxycinnamic acids, anthocyanins) and the activities of related enzymes (phenylalanine ammonia lyase, chalcone synthase/chalcone isomerase, flavanone 3-hydroxylase, dihydroflavonol 4-reductase, flavonol synthase, peroxidase) were monitored in apple (Malus domestica Borkh.). The seasonal survey was performed at five different sampling dates and included the healthy peel of the resistant cultivar ?Florina? and healthy peel, scab symptomatic spot and the tissue around the infected spot of the susceptible cultivar ?Golden Delicious?. From all enzymes tested, chalcone synthase/chalcone isomerase had the highest activity in both cultivars, while phenylalanine ammonia lyase had the lowest. ...

Rabbit antibodies have been raised to pig heart citrate synthase. Using purified IgG, competitive enzyme-linked immunoassays and assays of citrate synthase activity indicate the presence of antibodies to a number of antigenic sites on the enzyme, only some of which are essential for catalytic activity. From a comparison of citrate synthases from prokaryotic and eukaryotic organisms, the degree of interaction between antibody and enzyme was in the order: pig heart greater than pigeon breast greater than Bacillus megaterium greater than Escherichia coli. These findings are discussed in terms of the known interspecies diversity of the enzyme. PMID:2578989

We have studied the fate of the watermelon (Citrullus vulgaris Schrad.) glyoxysomal enzyme, malate dehydrogenase (gMDH), after synthesis in the methylotrophic yeast, Hansenula polymorpha. The gene encoding the precursor form of gMDH (pre-gMDH) was cloned in an H. polymorpha expression vector downstream of the inducible H. polymorpha alcohol oxidase promoter. During methylotrophic growth, pre-gMDH was synthesized and imported into peroxisomes, where it was enzymatically active. The apparent molecular mass of the protein located in H. polymorpha peroxisomes was equal to that of pre-gMDH (41 kDa), indicating that N-terminal processing of the transit peptide had not occurred in the yeast. PMID:8224215

Titanium dioxide thin films were prepared by using four water-soluble titanium complexes of titanium-lactate, tartalate, malate and salicylate complex solutions. The crystalline phases detected in the films were anatase. The surface microstructures of the four film samples were different in their grain sizes. Photocatalytic decomposition activity of the four films was almost the same, but their photoinduced hydrophilicities were different. The film prepared using titanium-salicylate complex exhibited lower hydrophilic conversion rate than the other films. Grain size and stress yielded to the film are considered to be important factors on the photoinduced hydrophilicity. PMID:21615742

rv1098c, an essential gene in Mycobacterium tuberculosis, codes for a class II fumarase. We describe here the crystal structure of Rv1098c in complex with l-malate, fumarate or the competitive inhibitor meso-tartrate. The models reveal that substrate binding promotes the closure of the active site through conformational changes involving the catalytic SS-loop and the C-terminal domain, which likely represents a general feature of this enzyme superfamily. Analysis of ligand-enzyme interactions as well as site-directed mutagenesis suggest Ser318 as one of the two acid-base catalysts. PMID:22561013

Abstract Methylated L-arginine analogs are involved in nitric oxide synthase activity regulation. Methods based on high-performance liquid chromatography with fluorescence, capillary electrophoresis, or ion exchange chromatography with absorbance detection were first applied for the quantita...

Following successful chemotherapy in canine visceral leishmaniasis, monocyte-derived macrophages can induce antileishmanial activity via a gamma interferon-dependent mechanism in the presence of autologous lymphocytes. The killing of leishmania correlated with the induction of the NO synthase pathwa...

Aluminum (Al) toxicity, which is caused by the solubilization of Al3+ in acid soils resulting in inhibition of root growth and nutrient/water acquisition, is a serious limitation to crop production, because up to one-half of the world's potentially arable land is acidic. To date, however, no Al tolerance genes have yet been cloned. The physiological mechanisms of tolerance are somewhat better understood; the major documented mechanism involves the Al-activated release of Al-binding organic acids from the root tip, preventing uptake into the primary site of toxicity. In this study, a quantitative trait loci analysis of Al tolerance in Arabidopsis was conducted, which also correlated Al tolerance quantitative trait locus (QTL) with physiological mechanisms of tolerance. The analysis identified two major loci, which explain approximately 40% of the variance in Al tolerance observed among recombinant inbred lines derived from Landsberg erecta (sensitive) and Columbia (tolerant). We characterized the mechanism by which tolerance is achieved, and we found that the two QTL cosegregate with an Al-activated release of malate from Arabidopsis roots. Although only two of the QTL have been identified, malate release explains nearly all (95%) of the variation in Al tolerance in this population. Al tolerance in Landsberg erecta x Columbia is more complex genetically than physiologically, in that a number of genes underlie a single physiological mechanism involving root malate release. These findings have set the stage for the subsequent cloning of the genes responsible for the Al tolerance QTL, and a genomics-based cloning strategy and initial progress on this are also discussed. PMID:12805622

A new biological activity of 6-(methylsulfinyl)hexyl isothiocyanate derived from Wasabia japonica was discovered as an inhibitor of glycogen synthase kinase-3?. The most potent isothiocyanate, 9-(methylsulfinyl)hexyl isothiocyanate, inhibited glycogen synthase kinase-3? at a Ki value of 10.5 ?M and showed ATP competitive inhibition. The structure-activity relationship revealed an inhibitory potency of methylsulfinyl isothiocyanate dependent on the alkyl chain length and the sulfoxide, sulfone, and/or the isothiocyanate moiety.   

Nitrogen-fixing activity and populations of nitrogen-fixing bacteria associated with two varieties of rice grown in dryland and wetland conditions were measured at various growth stages during the dry season. Acetylene reduction activities were measured both in the field and for the hydroponically grown rice, which was transferred from the field to water culture 1 day before assay. The activities measured by both methods were higher in wetland than in dryland rice. The population of nitrogen-fixing heterotrophic bacteria associated with rhizosphere soil, root, and basal shoots was determined by the most probable number method with semisolid glucose-yeast extract and semisolid malate-yeast extract media. The number of nitrogen-fixing bacteria was higher in wetland conditions than in dryland conditions. The difference between two conditions was most pronounced in the population associated with the basal shoot. The glucose medium gave higher counts than did the malate medium. Colonies were picked from tryptic soy agar plates, and their nitrogen-fixing activity was tested on a semisolid glucose-yeast extract medium. The incidence of nitrogen-fixing bacteria among aerobic heterotrophic bacteria in association with rhizosphere soil, root, and basal shoots was much lower in dryland rice than in wetland rice. (Refs. 11).

The enzymatic incorporation of sn-glycerol 3-phosphate into lipid by extracts of cucumber (Cucumis sativus) cotyledons showed an absolute requirement for ATP (saturation 2 mM). The incorporation was stimulated 4-fold by 0.2 mM oleate. Ethyldiaminetetraacetate stimulated the incorporation at concentrations below 1 mM and inhibited at higher concentrations. Mg2+ did not affect the reaction. Triton X-100 and Cutscum inhibited the reaction, while a third detergent, Span 80, was stimulatory. p-Mercuribenzoate was inhibitory. The enzymatic reaction has a pH optimum in the range of 8.8 to 9.6. The Michaelis constant was 112 ?M for sn-glycerol 3-phosphate. The major amount of product was phosphatidic acid, the remainder was diacylglycerol, monoacylglycerol, and an unknown phospholipid. The activity profiles for two glyoxysmal enzymes, malate synthetase and catalase, were compared to the activities of four enzymes involved in phospholipid synthesis. Phosphatidylcholine and phosphatidylethanolamine synthesis paralleled the activity profiles of catalase and malate synthetase, as well as the levels of endogenous diglycerides. sn-Glycerol 3-phosphate incorporation peaked at a later stage of cucumber cotyledon growth than the glyoxysomal enzymes and seemed to be the major pathway of phosphatidic acid synthesis. Diglyceride phosphokinase activity did not reach appreciable levels during the first 11 days of cucumber cotyledon growth. Images

Nitrate reductase (NR) activity is necessary for the synthesis of nitric oxide (NO), a key signaling molecule in plants. Here, we investigated the effect of NR deficiency on NO production and phenylpropanoid metabolism of Arabidopsis thaliana leaves. HPLC-mass spectrometry analysis showed that the NR double mutant (nia1 nia2) is deficient in the synthesis of sinapoylmalate (SM), the main phenylpropanoid end-product in wild-type leaves, resulting in accumulation of its precursor sinapoylglucose (SG). While real-time PCR analysis revealed no significant difference at the transcript level, sinapoylglucose:malate sinapoyltransferase (SMT) activity in leaf extracts was reduced in the mutant compared with the wild type. The low levels of SM in nia1 nia2 leaves do not result from the deficient nitrogen incorporation into amino acids, since the recovery of the amino acid content of nia1 nia2 by irrigating the plants with glutamine did not change the metabolic profile of this mutant. In contrast, an increased supply of nitrate stimulated NR activity and NO production, and enhanced SM and decreased SG levels in both genotypes. Nevertheless, sinapic acid esters in nia1 nia2 were not recovered when compared with those detected in the leaves of the wild-type plant. Mutant plants grown in medium supplemented with malate and an NO donor recovered SM to the levels of wild-type leaves. Overall, the results suggest that SMT activity is dependent on the NR-dependent steady-state levels of NO during plant development. PMID:22833666

An enzyme assay for bacterial undecaprenyl pyrophosphate (UPP) synthase was performed to screen microbial culture broths for inhibitors of UPP synthase. During the course of this screening program, an EtOH extract of a rice culture of Penicillium brasilianum FKI-3368 was found to inhibit UPP synthase activity. From activity-guided purification, a new compound-designated spirohexaline was isolated together with the structurally related and known viridicatumtoxin by ethyl acetate extraction silica gel and octadecylsilane column chromatographies and high-performance liquid chromatography. The structure of spirohexaline was elucidated by spectroscopic analysis, including NMR. Spirohexaline and viridicatumtoxin have a common hexacycline structure produced by fusion of a tetracycline-type ring with a spiro-type ring. They inhibited UPP synthase activity with IC(50) values of 9.0 and 4.0??M, respectively.The Journal of Antibiotics advance online publication, 21 November 2012; doi:10.1038/ja.2012.83. PMID:23168407

Mentha citrata Ehrh. (bergamot mint; Lamiaceae) produces an essential oil containing only the acyclic monoterpenol (-)-3R-linalool and its acetate ester. A cloning strategy based upon the assumption that the responsible monoterpene synthase would resemble, in sequence, monoterpene cyclases from this plant family yielded a cDNA encoding the (--)-3R-linalool synthase. The nucleotide sequence of this monoterpene synthase is similar to those of several monoterpene cyclases from the mint (Lamiaceae) family (62-72% identity), but differs substantially from that of 3S-linalool synthase from Clarkia (41% identity; this composite gene appears to be of recent origin) and from that of 3R-linalool synthase from Artemisia (52% identity; the functional role of this gene is uncertain). Heterologous expression in Escherichia coli of a truncated version of the cDNA (in which the plastidial transit peptide was deleted) allowed purification and characterization of the enzyme, which was shown to possess most properties similar to other known monoterpene cyclases, but with a K(m) value for the natural substrate, geranyl diphosphate, of 56 microM with k(cat) of 0.83 s(-1). These kinetic constants for this 3R-linalool synthase are higher than those of any defined monoterpene cyclase, but the kinetic efficiency does not approach that reported for the 3S-linalool synthase from Clarkia. Although linalyl diphosphate is an enzyme-bound intermediate of monoterpene cyclase reactions, this tertiary allylic isomer of the geranyl substrate is not an efficient precursor of linalool with the M. citrata synthase. Modeling of the active site of this linalool synthase from Mentha and comparison to the modeled active sites of phylogenetically related monoterpene cyclases revealed structural differences in the binding of the diphosphate moiety which initiates the ionization step of the electrophilic reaction sequence and in the access of water to the active site to permit stereoselective quenching of the initially formed carbocationic intermediate to produce 3R-linalool. PMID:12176064

The Pereskia are morphologically primitive, leafed members of the Cactaceae. Gas exchange characteristics using a dual isotope porometer to monitor (14)CO(2) and tritiated water uptake, diurnal malic acid fluctuations, phosphoenolpyruvate carboxylase, and malate dehydrogenase activities were examined in two species of the genus Pereskia, Pereskia grandifolia and Pereskia aculeata. Investigations were done on well watered (control) and water-stressed plants. Nonstressed plants showed a CO(2) uptake pattern indicating C(3) carbon metabolism. However, diurnal fluctuations in titratable acidity were observed similar to Crassulacean acid metabolism. Plants exposed to 10 days of water stress exhibited stomatal opening only during an early morning period. Titratable acidity, phosphoenolpyruvate carboxylase activity, and malate dehydrogenase activity fluctuations were magnified in the stressed plants, but showed the same diurnal pattern as controls. Water stress causes these cacti to shift to an internal CO(2) recycling ("idling") that has all attributes of Crassulacean acid metabolism except nocturnal stomata opening and CO(2) uptake. The consequences of this shift, which has been observed in other succulents, are unknown, and some possibilities are suggested. PMID:16661857

Carbohydrate metabolism changes during cellular senescence. Cytosolic malate dehydrogenase (MDH1) catalyzes the reversible reduction of oxaloacetate to malate at the expense of reduced nicotinamide adenine dinucleotide (NADH). Here, we show that MDH1 plays a critical role in the cellular senescence of human fibroblasts. We observed that the activity of MDH1 was reduced in old human dermal fibroblasts (HDFs) [population doublings (PD) 56], suggesting a link between decreased MDH1 protein levels and aging. Knockdown of MDH1 in young HDFs (PD 20) and the IMR90 human fibroblast cell line resulted in the appearance of significant cellular senescence features, including senescence-associated ?-galactosidase staining, flattened and enlarged morphology, increased population doubling time, and elevated p16(INK4A) and p21(CIP1) protein levels. Cytosolic NAD/NADH ratios were decreased in old HDFs to the same extent as in MDH1 knockdown HDFs, suggesting that cytosolic NAD depletion is related to cellular senescence. We found that AMP-activated protein kinase, a sensor of cellular energy, was activated in MDH1 knockdown cells. We also found that sirtuin 1 (SIRT1) deacetylase, a controller of cellular senescence, was decreased in MDH1 knockdown cells. These results indicate that the decrease in MDH1 and subsequent reduction in NAD/NADH ratio, which causes SIRT1 inhibition, is a likely carbohydrate metabolism-controlled cellular senescence mechanism. PMID:22971926

Genetic suppression was studied in the purple mutant of Drosophila melanogaster and in suppressed purple by measurement of sepiapterin synthase activity. The addition of ammonium sulfate fractions from adult Drosophila that contain one, two, three or four doses of su(s)/sup +/ to the suppressed purple sepiapterin synthase resulted in an inhibition that increased progressively as the dosage of su(s)/sup +/ increased; the wild-type sepiapterin synthase was not inhibited. This inhibition is caused by a heat-labile macromolecule. The authors suggest that the mechanism of suppression is neither transcriptional nor translational but is the result of decreased amounts, or altered properties, of the normal product of the su(s)/sup +/ locus.

Summary We previously isolated two putative monoterpene synthase genes, RlemTPS1 and RlemTPS2, from rough lemon (Citrus jambhiri) and showed that gene expression of RlemTPS2 was induced by microbial attack. The protein product of RlemTPS2 was obtained using a prokaryotic expression system, and GC and GC-MS of monoterpene synthesis by RlemTPS2 determined that RlemTPS2 encodes a sabinene synthase. Sabinene has antifungal activity toward Alternaria alternata. Furthermore, site-directed mutagenesis identified one amino acid, Ile, located at the front of the metal ion binding motif as an important residue for the product specificity of sabinene synthase.

Abstract in spanish El uso de malation para el control de mosquitos en Cuba durante 7 años hasta 1986 seleccionó 2 mecanismos de resistencia, el de elevada actividad de esterasas no específicas y la acetilcolinesterasa alterada (Ache) en Culex quinquefasciatus (Say). En La Habana, específicamente en el área de estudio (Río Quibú), el malation fue reemplazado por cipermetrina en 1987, y ciclos de tratamiento con cipermetrina han sido usados desde 1987 hasta la fecha en forma de radioba (more) tida cuando se incrementan las poblaciones de Aedes o Culex. En Culex quinquefasciatus (Say) del Río Quibú, los niveles de resistencia declinaron significativamente desde 1986 hasta 1997, sobre todo a malation, no resultó así para los piretroides, donde se observó un incremento de la resistencia durante este período de 11 años. El mecanismo de esterasas elevadas se incrementó a una frecuencia de 1 al igual que hubo un incremento en la frecuencia del mecanismo de Ache. Hasta 1986 se seleccionó en esta población la esterasa B1, responsable de la resistencia a malation, pero no a piretroides. A partir del uso de piretroides para el control en esta área se seleccionaron 2 nuevos fenotipos de esterasas, nombradas A6 y B6, en apariencia relacionadas con la resistencia a piretroides. Abstract in english The use of malathion to control mosquitoes in Cuba during 7 years until 1986 selected 2 resistance mechanisms: that of elevated activity of nonspecific esterases and that of altered acetylcholinesterase (Ache) in Culex quinquefasciatus (Say). In Havana, specifically in the area under study (Quibú River), malathion was replaced by cypermethrin in 1987 and cycles of treatment with cypermethrin have been intensively used since l987 up to now when the populations of Aedes or (more) Culex increase. In Culex quin-quefasciatus (Say) from the Quibú River the resistance levels, mainly to malathion, declined significantly from 1986 to 1997. An increase of resistance to pyrethroid was observed during that period of 11 years. The mechanism of elevated esterases rose to a frequency of 1 and there was also an increase in the frequency of the mechanism of Ache. The esterase B1, responsible for the resistance to malathion, but not to pyretrhroid, was selected in this population until 1986. Starting from the use of pyrethroid for the control in this area, 2 new phenotypes of esterases named A6 and B6, apparently related to pyrethroid resistance, were selected.

Tomato ACC synthase is inactivated by its substrate SAM, with the moiety of aminobutyrate being covalently linked to ACC synthase during the catalytic reactions. A partial purified ACC synthase (the catalytic activity 100 {mu}mol/h{center dot}mg protein) from pellets of apple extract was incubated with (3,4{sup 14}C) SAM. Only one radioactive peak was revealed in a C-4 reverse phase HPLC and one radioactive band on SDS-PAGE with an M.W. of 48 kDa. Apple ACC synthase in native form is resistant to V8, {alpha}-chromtrypsin and carboxylpeptidase A digestion, but effectively inactivated by trypsin and ficin, as demonstrated by both the activity assay and SAM labeling. The radioactive protein cut from the SDS-PAGE was injected to three mice, two of the mice showed responses to the protein in western blot analysis. The antibodies from mice is currently under characterization.

Sphingolipids are ubiquitous and essential components of eukaryotic membranes, particularly the plasma membrane. The biosynthetic pathway for the formation of these lipid species is conserved up to the formation of sphinganine. However, a divergence is apparent in the synthesis of complex sphingolipids. In animal cells, ceramide is a substrate for sphingomyelin (SM) production via the enzyme SM synthase. In contrast, fungi utilise phytoceramide in the synthesis of inositol phosphorylceramide (IPC) catalysed by IPC synthase. Due to the absence of a mammalian equivalent, this essential enzyme represents an attractive target for anti-fungal compounds. In common with the fungi, the kinetoplastid protozoa (and higher plants) synthesize IPC rather than SM. However, orthologues of the gene believed to encode the fungal IPC synthase (AUR1) are not readily identified in the complete genome databases of these species. By utilising bioinformatic and functional genetic approaches, we have isolated a functional orthologue of AUR1 in the kinetoplastids, causative agents of a range of important human diseases. Expression of this gene in a mammalian cell line led to the synthesis of an IPC-like species strongly indicating that IPC synthase activity is reconstituted. Furthermore the gene product can be specifically inhibited by an anti-fungal targeting IPC synthase. We propose that the kinetoplastid AUR1 functional orthologue encodes an enzyme which defines a new class of protozoan sphingolipid synthase. The identification and characterisation of the protozoan IPC synthase, an enzyme with no mammalian equivalent, will raise the possibility of developing anti-protozoal drugs with minimal toxic side-affects.

Class II diterpene cyclases catalyze bicyclization of geranylgeranyl diphosphate. While this reaction typically is terminated via methyl deprotonation to yield copalyl diphosphate, in rare cases hydroxylated bicycles are produced instead. Abietadiene synthase is a bifunctional diterpene cyclase that usually produces a copalyl diphosphate intermediate. Here it is shown that substitution of aspartate for a conserved histidine in the class II active site of abietadiene synthase leads to selective production of 8?-hydroxy-CPP instead, demonstrating striking plasticity. PMID:23167845

Intermittent hypoxia (IH) markedly enhances cardiac tolerance against ischemia/reperfusion injury, but its mechanism and molecular basis remain unclear. For exploring the expression of mitochondrial proteins induced by IH, two-dimensional electrophoresis and Thermo Finnigan LTQ mass spectrometer (MS) were applied. After comparing the protein profiles of myocardial mitochondria between IH and normoxic hearts, 14 protein spots were found to be altered more than threefold between the two groups, 11 of which were identified by Finnigan LTQ MS. Among these 11 proteins, 9 were involved in energy metabolism, including 7 that were increased after IH. The latter were identified as aldehyde dehydrogenase, methylmalonate-semialdehyde dehydrogenase, ATP synthase ? chain, mitochondrial aconitase, malate dehydrogenase, electron transfer flavoprotein ? subunit and sirtuin 5. Two other proteins, ubiquinol-cytochrome C reductase iron-sulfur subunit and aspartate aminotransferase, were decreased after IH. Biochemical tests for energy metabolism in mitochondria supported the proteomic results. IH exposure also increased the expression of a molecular chaperone-heat shock protein 60 and an antioxidant protein, peroxiredoxin 5. These findings will provide clues for understanding the mechanism of IH-induced cardiac protection and may lead to the development of interventional strategies designed to utilize the advantages of IH clinically. PMID:21735218

Determination of the accurate three-dimensional structure of large proteins by NMR remains challenging due to a loss in the density of experimental restraints resulting from the often prerequisite perdeuteration. Solution small-angle scattering, which carries long-range translational information, presents an opportunity to enhance the structural accuracy of derived models when used in combination with global orientational NMR restraints such as residual dipolar couplings (RDCs) and residual chemical shift anisotropies (RCSAs). We have quantified the improvements in accuracy that can be obtained using this strategy for the 82 kDa enzyme Malate Synthase G (MSG), currently the largest single chain protein solved by solution NMR. Joint refinement against NMR and scattering data leads to an improvement in structural accuracy as evidenced by a decrease from {approx}4.5 to {approx}3.3 A of the backbone rmsd between the derived model and the high-resolution X-ray structure, PDB code 1D8C. This improvement results primarily from medium-angle scattering data, which encode the overall molecular shape, rather than the lowest angle data that principally determine the radius of gyration and the maximum particle dimension. The effect of the higher angle data, which are dominated by internal density fluctuations, while beneficial, is also found to be relatively small. Our results demonstrate that joint NMR/SAXS refinement can yield significantly improved accuracy in solution structure determination and will be especially well suited for the study of systems with limited NMR restraints such as large proteins, oligonucleotides, or their complexes.

Ralstonia eutropha H16 is capable of growth and polyhydroxyalkanoate production on plant oils and fatty acids. However, little is known about the triacylglycerol and fatty acid degradation pathways of this bacterium. We compare whole-cell gene expression levels of R. eutropha H16 during growth and polyhydroxyalkanoate production on trioleate and fructose. Trioleate is a triacylglycerol that serves as a model for plant oils. Among the genes of note, two potential fatty acid ?-oxidation operons and two putative lipase genes were shown to be upregulated in trioleate cultures. The genes of the glyoxylate bypass also exhibit increased expression during growth on trioleate. We observed that single ?-oxidation operon deletion mutants of R. eutropha could grow using palm oil or crude palm kernel oil as the sole carbon source, regardless of which operon was present in the genome, but a double mutant was unable to grow under these conditions. A lipase deletion mutant did not exhibit a growth defect in emulsified oil cultures but did exhibit a phenotype in cultures containing nonemulsified oil. Mutants of the glyoxylate shunt gene for isocitrate lyase were able to grow in the presence of oils, while a malate synthase (aceB) deletion mutant grew more slowly than wild type. Gene expression under polyhydroxyalkanoate storage conditions was also examined. Many findings of this analysis confirm results from previous studies by our group and others. This work represents the first examination of global gene expression involving triacylglycerol and fatty acid catabolism genes in R. eutropha. PMID:10458604

Mycobacterium tuberculosis is the etiological agent for tuberculosis in humans. The studies related to survival of this pathogen in the human host and development of drugs against reveal that the organism uses a complex physiology to adapt to the host environment. Many studies were targeted to key enzymes that allow this pathogen to either survive or remain latent within the host. Most of the models, which address the survival of pathogen, have evaluated limited dissolved oxygen and prevailing stress conditions. Hence, the truncated citric acid cycle, with the glyoxylate shunt was suggested as an option for survival of the pathogen and pathogenesis. We propose that the precursors to support this pathway could also be generated via enzymatic conversion involving poly-beta-hydroxybutyrate (PHB). We have used available genome sequence data and analyzed for the possible enzymatic conversions that can generate glyoxylate, acetyl CoA, and other enolases that can also be useful for various fatty acid transformations. The enzymes for accumulation and further hydrolysis of PHB were examined in sequence data analysis. The target enzymes were searched for in the genome using identified conserved domains. Using M. tuberculosis H37Rv as a model bacterium a supportive pathway has been envisaged and integrated with glyoxylate cycle to provide a complete option to pathogen for sustainable consumption of available carbon source(s). The study proposes that the enzymes of PHB synthesis and hydrolysis are possible targets for drug design, and that this should be considered when evaluating isocitrate lyase and malate synthase as targets. PMID:17897060

To elucidate the mechanism underlying the regulation of human GD3 synthase gene expression in human melanoma SK-MEL-2 cells, we identified the promoter region of the human GD3 synthase gene. The 5'-rapid amplification of cDNA end (5'-RACE) using mRNA prepared from SK-MEL-2 cells revealed the presence of multiple transcription start sites of human GD3 synthase gene. Promoter analyses of the 5'-flanking region of the human GD3 synthase gene using luciferase gene reporter system showed the strong promoter activity in SK-MEL-2 cells. Deletion study revealed that the region as the core promoter from -1146 to -646 (A of the translational start ATG as position +1) was indispensable for endogenous expression of human GD3 synthase gene. This region lacks apparent TATA and CAAT boxes but contains putative binding sites for transcription factors c-Ets-1, CREB, AP-1 and NF-kappaB. Electrophoretic mobility shift assays using specific competitors, chromatin immunoprecipitation assay and site-directed mutagenesis demonstrated that only NF-kappaB element in this region is required for the promoter activity in SK-MEL-2 cells. These results indicate that NF-kappaB plays an essential role in the transcriptional activity of human GD3 synthase gene essential for GD3 synthesis in SK-MEL-2 cells. PMID:17913261

A comparative study was made of the effects of biphenyl and polychlorinated biphenyls (Kanechlors, KCs) on the respiratory and energy linked activities of rat liver mitochondria. With ..cap alpha..-ketoglutarate/malate as the substrate, biphenyl acted as the strongest inhibitor of state 3 respiration. The inhibition of state 3 respiration was decreased as chlorine content increased. The stimulation of state 4 respiration was greatest in KC-200, 300, and 400; intermediate in biphenyl and KC-500; least in KC-600. When succinate was the substrate, from the results of this and a previous study the action of biphenyl as an inhibitor of state 3 respiration was least effective. The inhibitory action on state 3 respiration was strengthened by the chlorination of aromatic rings (KC-200), reaching a peak where maximum inhibition was observed (KC-300, 400, and 500), further increases in chlorine content (KC-600) repressed the inhibitory action. The stimulating ability of state 4 respiration was greatly diminished with increases in chlorination. Biphenyl and KCs induced the K/sup +/-release from mitochondria, thereby dissipating membrane potential across the mitochondrial membranes in the order: biphenyl > KC-200 > KC-300 > KC-400 > KC-500 > KC-600. It is concluded that with ..cap alpha..-ketoglutarate/malate (NAD/sup +/-linked substrate), KCs act as uncouplers by increasing the permeability of mitochondrial inner membranes to ions, and that when succinate is the substrate, KCs act as inhibitors rather than uncouplers of oxidative phosphorylation of mitochondria by inhibiting the electron transport chain.

Modulation of the malate content of tomato fruit by altering the expression of mitochondrially localized enzymes of the tricarboxylic acid (TCA) cycle resulted in enhanced transitory starch accumulation and subsequent effects on post-harvest fruit physiology. In the current study we assessed whether such a manipulation would similarly affect starch biosynthesis in an organ which displays a linear, as opposed to a transient, kinetic of starch accumulation. For this purpose we used RNA interference to downregulate the expression of fumarase in potato under the control of the tuber specific B33 promoter. Despite displaying similar reductions in both fumarase activity and malate content as observed in tomato fruit expressing the same construct the resultant transformants were neither characterized by an increased flux to, or accumulation of, starch nor by alteration in yield parameters. Since the effect in tomato was mechanistically linked to de-repression of the reaction catalyzed by ADP glucose pyrophosphorylase we evaluated whether the lack of effect on starch biosynthesis was due to differences in enzymatic properties of the enzyme from potato and tomato or rather due to differential subcellular compartmentation of reductant in the different organs. The results are discussed in the context both of current models of metabolic compartmentation and engineering. PMID:23064409

During symbiotic nitrogen fixation (SNF), the nodule becomes a strong sink for photosynthetic carbon. Here, it was studied whether nodule dark CO(2) fixation could participate in a mechanism for CO(2) recycling through C(4)-type photosynthesis. Differences in the natural ?(13)C abundance between Lotus japonicus inoculated or not with the N-fixing Mesorhizobium loti were assessed. (13)C labelling and gene expression of key enzymes of CO(2) metabolism were applied in plants inoculated with wild-type or mutant fix(-) (deficient in N fixation) strains of M. loti, and in non-inoculated plants. Compared with non-inoculated legumes, inoculated legumes had higher natural ?(13)C abundance and total C in their hypergeous organs and nodules. In stems, (13)C accumulation and expression of genes coding for enzymes of malate metabolism were greater in inoculated compared with non-inoculated plants. Malate-oxidizing activity was localized in stem xylem parenchyma, sieve tubes, and photosynthetic outer cortex parenchyma of inoculated plants. In stems of plants inoculated with fix(-) M. loti strains, (13)C accumulation remained high, while accumulation of transcripts coding for malic enzyme isoforms increased. A potential mechanism is proposed for reducing carbon losses during SNF by the direct reincorporation of CO(2) respired by nodules and the transport and metabolism of C-containing metabolites in hypergeous organs. PMID:21307384

Photoaffinity labeling of purified cellulose synthase with (beta-32P)5-azidouridine 5'-diphosphoglucose (UDP-Glc) has been used to identify the UDP-Glc binding subunit of the cellulose synthase from Acetobacter xylinum strain ATCC 53582. The results showed exclusive labeling of an 83-kDa polypeptide. Photoinsertion of (beta-32P)5-azido-UDP-Glc is stimulated by the cellulose synthase activator, bis-(3'----5') cyclic diguanylic acid. Addition of increasing amounts of UDP-Glc prevents photolabeling of the 83-kDa polypeptide. The reversible and photocatalyzed binding of this photoprobe also showed saturation kinetics. These studies demonstrate that the 83-kDa polypeptide is the catalytic subunit of the cellulose synthase in A. xylinum strain ATCC 53582.

Several GM3 derivatives have been synthesized. Among them were lyso-GM3 derivatives and GM3 analogues with modifications in the sialic acid moiety. They were used as glycolipid acceptors in assays for GM2 and GD3 synthase of rat liver Golgi. Analysis of the resulting enzyme activities and of the reaction products revealed different substrate specificities for GM2 and GD3 synthase although the normal glycolipid acceptor for both transferases is ganglioside GM3. Specificity of GD3 synthase is strongly determined by the substrate's negative charge and the acyl residue in amide bond to the amino group of neuraminic acid, while GM2 synthase reacts quite indifferently to these changes in the sialic moiety of the substrate. Both enzymes seem to be sensitive to the spatial extension at the neuraminic acid's carboxylic group. PMID:3115774

Cofactors play key roles in metabolic pathways. Among them F(420) has proved to be a very attractive target for the selective inhibition of archaea and actinobacteria. Its biosynthesis, in a unique manner, involves a key enzyme, F(0)-synthase. This enzyme is a large monomer in actinobacteria, while it is constituted of two subunits in archaea and cyanobacteria. We report here the purification of both types of F(0)-synthase and their in vitro activities. Our study allows us to establish that F(0)-synthase, from both types, uses 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and tyrosine as substrates but not 4-hydroxylphenylpyruvate as previously suggested. Furthermore, our data support the fact that F(0)-synthase generates two 5'-deoxyadenosyl radicals for catalysis which is unprecedented in reaction catalyzed by radical SAM enzymes. PMID:23072415

Delta(1)-tetrahydrocannabinolic acid (THCA) synthase is the enzyme that catalyzes oxidative cyclization of cannabigerolic acid into THCA, the precursor of Delta(1)-tetrahydrocannabinol. We cloned a novel cDNA (GenBank trade mark accession number AB057805) encoding THCA synthase by reverse transcription and polymerase chain reactions from rapidly expanding leaves of Cannabis sativa. This gene consists of a 1635-nucleotide open reading frame, encoding a 545-amino acid polypeptide of which the first 28 amino acid residues constitute the signal peptide. The predicted molecular weight of the 517-amino acid mature polypeptide is 58,597 Da. Interestingly, the deduced amino acid sequence exhibited high homology to berberine bridge enzyme from Eschscholtzia californica, which is involved in alkaloid biosynthesis. The liquid culture of transgenic tobacco hairy roots harboring the cDNA produced THCA upon feeding of cannabigerolic acid, demonstrating unequivocally that this gene encodes an active THCA synthase. Overexpression of the recombinant THCA synthase was achieved using a baculovirus-insect expression system. The purified recombinant enzyme contained covalently attached FAD cofactor at a molar ratio of FAD to protein of 1:1. The mutant enzyme constructed by changing His-114 of the wild-type enzyme to Ala-114 exhibited neither absorption characteristics of flavoproteins nor THCA synthase activity. Thus, we concluded that the FAD binding residue is His-114 and that the THCA synthase reaction is FAD-dependent. This is the first report on molecular characterization of an enzyme specific to cannabinoid biosynthesis. PMID:15190053

The cholesterol ozonolysis products secosterol-A and its aldolization product secosterol-B were recently detected in human atherosclerotic tissues and brain specimens, and have been postulated to play pivotal roles in the pathogenesis of atherosclerosis and neurodegenerative diseases. We examined several oxidized cholesterol metabolites including secosterol-A, secosterol-B, 25-hydroxycholesterol, 5?,6?-epoxycholesterol and 7-ketocholesterol for their effects on the activities of three nitric oxide synthases. In contrast to other oxidized metabolites, secosterol-A was found to be a potent inhibitor against the neuronal- and endothelial-type, but not the inducible-type nitric oxide synthase, with IC50 values of 22 ± 1 and 50 ± 5 ?M, respectively. The calmodulin-binding regions of the neuronal- and endothelial-nitric oxide synthases contain lysine residues which are not present in the inducible-type nitric oxide synthase. Secosterol-A modifies proteins through the formation of a Schiff base with the lysine epsilon-amino group. It is possible that secosterol-A modifies lysine residues of constitutive nitric oxide synthases, leading to the inhibition of enzymatic activities. As nitric oxide is a critical signaling molecule in vascular function and in long-term potentiation, its reduced production through inhibition of constitutive nitric oxide synthases by secosterol-A may contribute to the development of atherosclerosis and memory impairment in particular neurodegenerative diseases.   

Sialic acid-containing glycosphingolipids (gangliosides) have been implicated in the regulation of various biological phenomena such as atherosclerosis. Recent report suggests that exogenously supplied disialoganglioside (GD3) serves a dual role in vascular smooth muscle cells (VSMC) proliferation and apoptosis. However, the role of the GD3 synthase gene in VSMC responses has not yet been elucidated. To determine whether a ganglioside is able to modulate VSMC growth, the effect of overexpression of the GD3 synthase gene on DNA synthesis was examined. The results show that the overexpression of this gene has a potent inhibitory effect on DNA synthesis and ERK phosphorylation in cultured VSMC in the presence of PDGF. The suppression of the GD3 synthase gene was correlated with the down-regulation of cyclinE/CDK2, the up-regulation of the CDK inhibitor p21 and blocking of the p27 inhibition, whereas up-regulation of p53 as the result of GD3 synthase gene expression was not observed. Consistently, blockade of GD3 function with anti-GD3 antibody reversed VSMC proliferation and cell cycle proteins. The expression of the GD3 synthase gene also led to the inhibition of TNF-alpha-induced matrix metalloproteinase-9 (MMP-9) expression in VSMC as determined by zymography and immunoblot. Furthermore, GD3 synthase gene expression strongly decreased MMP-9 promoter activity in response to TNF-alpha. This inhibition was characterized by the down-regulation of MMP-9, which was transcriptionally regulated at NF-kappaB and activation protein-1 (AP-1) sites in the MMP-9 promoter. Finally, the overexpression of MMP-9 in GD3 synthase transfectant cells rescued VSMC proliferation. However, MMP-2 overexpression was not affected by cell proliferation. These findings suggest that the GD3 synthase gene represents a physiological modulator of VSMC responses that may contribute to plaque instability in atherosclerosis. PMID:15175338

A large number of diterpenes have been isolated from Euphorbiaceae plants, many of which are of interest due to toxicity or potential therapeutic activity. Specific Euphorbiaceae diterpenes of medical interest include the latent HIV-1 activator prostratin (and related 12-deoxyphorbol esters), the analgesic resiniferatoxin, and the anticancer drug candidate ingenol 3-angelate. In spite of the large number of diterpenes isolated from these plants and the similarity of their core structures, there is little known about their biosynthetic pathways. Other than the enzymes involved in gibberellin biosynthesis, the only diterpene synthase isolated to date from the Euphorbiaceae has been casbene synthase, responsible for biosynthesis of a macrocyclic diterpene in the castor bean (Ricinus communis). Here, we have selected five Euphorbiaceae species in which to investigate terpene biosynthesis and report on the distribution of diterpene synthases within this family. We have discovered genes encoding putative casbene synthases in all of our selected Euphorbiaceae species and have demonstrated high-level casbene production through expression of four of these genes in a metabolically engineered strain of Saccharomyces cerevisiae. The only other diterpene synthase found among the five plants was a neocembrene synthase from R. communis (this being the first report of a neocembrene synthase gene). Based on the prevalence of casbene synthases, the lack of other candidates, and the structure of the casbene skeleton, we consider it likely that casbene is the precursor to a large number of Euphorbiaceae diterpenes. Casbene production levels of 31 mg/L were achieved in S. cerevisiae and we discuss strategies to further increase production by maximizing flux through the mevalonate pathway. PMID:20594566

Recent studies by our group and others have disclosed the presence of ceramides in mitochondria, and the activities of ceramide synthase and reverse ceramidase in mitochondria have also been reported. Since a possible contamination with the ER (endoplasmic reticulum)-related compartment MAM (mitochondria-associated membrane) could not be ruled out in previous studies, we have re-investigated the presence of the enzymes of ceramide metabolism in mitochondria and MAM highly purified from rat liver. In the present paper, we show that purified mitochondria as well as MAM are indeed able to generate ceramide in vitro through both ceramide synthase or reverse ceramidase, whereas the latter enzyme activity is barely detectable in microsomes. Moreover, ceramide synthase activities were recovered in outer mitochondrial membranes as well as in inner mitochondrial membranes. Using radiolabelled sphingosine as a substrate, mitochondria could generate ceramide and phytoceramide. However, the in vitro sensitivity of ceramide synthase toward FB1 (fumonisin B1) in mitochondria as well as in MAM was found to depend upon the sphingoid base: whereas dihydrosphingosine N-acyltransferase was inhibited by FB1 in a concentration-dependent manner, FB1 actually activated the ceramide synthase when using sphingosine as a substrate. Acylation of sphingosine 1-phosphate and dihydrosphingosine 1-phosphate, generating ceramide 1-phosphate, was also shown with both subcellular fractions. Moreover, the same difference in sensitivity towards FB1 for the ceramide synthase activities was seen between the two phosphorylated sphingoid bases, raising the possibility that distinct base-specific enzymes may be involved as ceramide synthases. Collectively, these results demonstrate the involvement of mitochondria in the metabolism of ceramides through different pathways, thereby supporting the hypothesis that topology of ceramide formation could determine its function.

The aim of this study was to evaluate the effects of Artemia nauplii enriched with different concentrations of highly unsaturated fatty acids (HUFAs) on the lipid metabolic response, peroxidation, and antioxidant defence status of the lined seahorse (Hippocampus erectus) juveniles. Twenty-day-old juveniles were fed Artemia nauplii enriched with four different concentrations (0.0 L/L [control, A], 13.5 L/L [B], 27.0 L/L [C], and 54.0 L/L [D]) of HUFAs (two thirds DHA and one third EPA) for 30 d. The activities of lipase and lipoproteinlipase of the juveniles significantly increased with increasing HUFA concentration; however, the activities of malate dehydrogenase and lactate content decreased with increasing HUFA concentration. Alkaline phosphatase activity and pyruvic acid content were no...

The effect of common salt (NaCl) on ion contents, Krebs cycle intermediates and its regulatory enzymes was investigated in growing mungbean (Vigna radiata L. Wilczek, B 105) seedlings. Sodium and chloride ion contents increased in both root and shoot whereas potassium ion content decreased in shoot of test seedlings with increasing concentrations of NaCl. Organic acids like pyruvate and citrate levels increased whereas malate level decreased under stress in both roots and shoots. Salt stress also variedly affected the activities of different enzymes of respiratory chain. The activity of pyruvate dehydrogenase (E.C. 1.2.4.1) decreased in 50 mM NaCl but increased in 100 mM and 150 mM concentrations, in both root and shoot samples. Succinate dehydrogenase (E.C. 1.3.5.1) activity was reduced i...

Imazamox is a selective herbicide applied in a wide spectrum of crops which belongs to the imidazolinone family, which mode of action is related to the inhibition of acetohydroxyacid synthase activity. The effect of this herbicide on plant growth, amino acids balance as well as the rate of inhibition of acetohydroxyacid synthase activity in different organs of common bean was studied. Our results indicated that total plant dry weight was not significantly affected two and seven days after herbicide application. Acetohydroxyacid synthase activity decreased in young tissues in response to imazamox treatments, whereas in mature organs the herbicide reversed the natural decrease of activity linked to ripeness. In shoot apical meristem and fully expanded leaves isoleucine content increased, whi...

To comprehend the catalytic and regulatory mechanism of the cyclic diguanylic acid (c-di-GMP)-dependent cellulose synthase of Acetobacter xylinum and its relatedness to similar enzymes in other organisms, the structure of this enzyme was analyzed at the polypeptide level. The enzyme, purified 350-fold by enzyme-product entrapment, contains three major peptides (90, 67, and 54 kDa), which, based on direct photoaffinity and immunochemical labeling and amino acid sequence analysis, are constituents of the native cellulose synthase. Labeling of purified synthase with either ({sup 32}P)c-di-GMP or ({alpha}-{sup 32}P)UDP-glucose indicates that activator- and substrate-specific binding sites are most closely associated with the 67- and 54-kDa peptides, respectively, whereas marginal photolabeling is detected in the 90-k-Da peptide. However, antibodies raised against a protein derived from the cellulose synthase structural gene (bcsB) specifically label all three peptides. The authors suggest that the structurally related 67- and 54-kDa peptides are fragments proteolytically derived from the 90-kDa peptide encoded by bcsB. The anti-cellulose synthase antibodies crossreact with a similar set of peptides derived from other cellulose-producing microorganisms and plants such as Agrobacterium tumefaciens, Rhizobium leguminosarum, mung bean, peas, barley, and cotton. The occurrence of such cellulose synthase-like structures in plant species suggests that a common enzymatic mechanism for cellulose biogenesis is employed throughout nature.

Invasive aspergillosis is a leading cause of mortality in immunocompromised patients. The fungal cell wall is an attractive antifungal target, but it is dynamic and responsive to external stressors. The existence of multiple chitin synthases within Aspergilli is thought to reflect specialized functions in cell wall damage responses that facilitate continued growth and viability. We previously reported increased transcription of Aspergillus fumigatus chitin synthases chsA and chsC following echinocandin treatment, suggesting important roles for these chitin synthases in cell wall compensation. As only partial disruptions have been made of these genes, we generated deletion mutants of chsA and chsC singly (?chsA and ?chsC) and doubly (?chsA ?chsC). The ?chsA ?chsC strain displayed reduced total chitin synthase activity. Interestingly, deletion of these chitin synthase genes did not affect levels of chitin or ?-1,3-glucan.The ?chsA, ?chsC and ?chsA ?chsC strains produced wild-type echinocandin-mediated chitin increases, consistent with unaltered cell wall compensation. Furthermore, transcript levels of the remaining chitin synthase genes were unchanged in the mutant strains. Taken together, these results indicate that chsA and chsC do not play a direct role in the cell wall stress response. Our findings support the existence of complex post-transcriptional regulatory mechanisms controlling chitin biosynthetic machinery in response to cell wall damage. PMID:21763289

Incubation of Chaps or digitonin-solubilized membrane proteins from cotton fiber with Ca{sup 2+} in combination with Mg{sup 2+}, leads to formation of a complex which can be sedimented within 15 min at 15,000 g. The complex is enriched >10-fold in callose synthase activity and possesses a characteristic pattern of enriched polypeptides when analyzed by SDS-PAGE. Although cation dependent, formation of the complex is not dependent upon the presence of the callose synthase substrate, UDP-glc, indicating that complex formation is not due to entrapment of the enzyme by association with glucan product. The enriched polypeptides include: >200, 50, and 46 kD, all of which have been shown by direct photo-labeling to interact with {sup 92}P-UDP-glc in a Ca{sup 2+} or beta-glucoside dependent reaction are considered likely subunits of callose synthase; a 60-62 kD doublet which is recognized by our MAb 2-1 which can form an immune complex with callose synthase; 74 and 34 kD polypeptides which also interact with UDP-glc, but do not associate with callose synthase in the presence of EDTA. A similar phenomenon is also observed with solubilized membrane proteins from mung beans. Possible functions of each of the enriched polypeptides, the catalytic properties, and ultra-structure of this macro-glucan synthase complex are currently under investigation.

Polyhydroxyalkanoates (PHAs) are accumulated as intracellular carbon and energy storage polymers by various bacteria and a few haloarchaea. In this study, 28 strains belonging to 15 genera in the family Halobacteriaceae were investigated with respect to their ability to synthesize PHAs and the types of their PHA synthases. Fermentation results showed that 18 strains from 12 genera could synthesize polyhydroxybutyrate (PHB) or poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). For most of these haloarchaea, selected regions of the phaE and phaC genes encoding PHA synthases (type III) were cloned via PCR with consensus-degenerate hybrid oligonucleotide primers (CODEHOPs) and were sequenced. The PHA synthases were also examined by Western blotting using haloarchaeal Haloarcula marismortui PhaC (PhaCHm) antisera. Phylogenetic analysis showed that the type III PHA synthases from species of the Halobacteriaceae and the Bacteria domain clustered separately. Comparison of their amino acid sequences revealed that haloarchaeal PHA synthases differed greatly in both molecular weight and certain conserved motifs. The longer C terminus of haloarchaeal PhaC was found to be indispensable for its enzymatic activity, and two additional amino acid residues (C143 and C190) of PhaCHm were proved to be important for its in vivo function. Thus, we conclude that a novel subtype (IIIA) of type III PHA synthase with unique features that distinguish it from the bacterial subtype (IIIB) is widely distributed in haloarchaea and appears to be involved in PHA biosynthesis.

The metabolism and synthesis of an important mycobacterial lipid component, phosphatidylinositol (PI), and its metabolites, was studied in Mycobacterium smegmatis and M. smegmatis subcellular fractions. Little is known about the synthesis of PI in prokaryotic cells. Only a cell wall fraction (P60) in M. smegmatis was shown to possess PI synthase activity. Product was identified as PI by migration on TLC, treatment with phospholipase C and ion exchange chromatography. PI was the only major product (92.3%) when both cells and P60 fraction were labeled with [3H]inositol. Also, a neutral lipid inositol-containing product (4.1% of the total label) was identified in the P60 preparations. Strangely, PI synthase substrates, CDP-dipalmitoyl-DAG and CDP-NBD-DAG, added to the assay did not stimulate [3H]PI and NBD-PI yield by M. smegmatis. At the same time, addition of both substrates to rat liver and Saccharomyces cerevisiae PI synthase assays resulted in an increase in the product yield. Upon addition of CHAPS to the mycobacterial PI synthase assay, both substrates were utilized in a dose-dependent manner for the synthesis of NBD-PI and [3H]PI. These results demonstrate a strict substrate specificity of mycobacterial PI synthase toward endogenous substrates. K(m) of the enzyme toward inositol was shown to be 25 microM; Mg2+ stimulated the enzyme to a greater degree than Mn2+. Structural analogs of myo-inositol, epi-inositol and scyllo-inositol and Zn2+ were shown to be more potent inhibitors of mycobacterial PI synthase than of mammalian analogs. Lack of sequence homology with mammalian PI synthases, different kinetic characteristics, existence of selective inhibitors and an important physiological role in mycobacteria, suggest that PI synthase may be a good potential target for antituberculosis therapy. PMID:9989274

The green alga Chlamydomonas reinhardtii has a network of fermentation pathways that become active when cells acclimate to anoxia. Hydrogenase activity is an important component of this metabolism, and we have compared metabolic and regulatory responses that accompany anaerobiosis in wild-type C. reinhardtii cells and a null mutant strain for the HYDEF gene (hydEF-1 mutant), which encodes an [FeFe] hydrogenase maturation protein. This mutant has no hydrogenase activity and exhibits elevated accumulation of succinate and diminished production of CO2 relative to the parental strain during dark, anaerobic metabolism. In the absence of hydrogenase activity, increased succinate accumulation suggests that the cells activate alternative pathways for pyruvate metabolism, which contribute to NAD(P)H reoxidation, and continued glycolysis and fermentation in the absence of O2. Fermentative succinate production potentially proceeds via the formation of malate, and increases in the abundance of mRNAs encoding two malateforming enzymes, pyruvate carboxylase and malic enzyme, are observed in the mutant relative to the parental strain following transfer of cells from oxic to anoxic conditions. Although C. reinhardtii has a single gene encoding pyruvate carboxylase, it has six genes encoding putative malic enzymes. Only one of the malic enzyme genes, MME4, shows a dramatic increase in expression (mRNA abundance) in the hydEF-1 mutant during anaerobiosis. Furthermore, there are marked increases in transcripts encoding fumarase and fumarate reductase, enzymes putatively required to convert malate to succinate. These results illustrate the marked metabolic flexibility of C. reinhardtii and contribute to the development of an informed model of anaerobic metabolism in this and potentially other algae.

Abstract in english This is the first study of isoenzyme variability in the leaf-cutting ants (Myrmicinae, Attini) Acromyrmex heyeri (Forel, 1899) and A. striatus (Roger, 1863) which are common throughout the southern Brazilian state of Rio Grande do Sul. We studied the alloenzyme variability of malate dehydrogenase (MDH), alpha-glycerophosphate dehydrogenase (alpha-GPDH) and amylase (AMY) in 97 colonies of A. heyeri and 103 colonies of A. striatus. Five loci were found for these enzyme syst (more) ems, one locus (Amy-1) being monomorphic in both species and four loci (Mdh-1, alpha-Gpdh-1, Amy-2, and Amy-4) being polymorphic. For each species there were exclusive alleles for the Mdh-1 and Amy-2 loci and differences were also found in the allele frequencies for the other polymorphic loci. Ontogenetically different gene activity was detected for the MDH and alpha-GPDH systems, with between-caste differences, probably related to flight activity, also being found for alpha-GPDH.

The purpose of this study was to explore how the mitochondrial AOX (alternative oxidase) pathway alleviates photoinhibition in Rumex K-1 leaves. Inhibition of the AOX pathway decreased the initial activity of NADP-malate dehydrogenase (EC 1.1.1.82, NADP-MDH) and the pool size of photosynthetic end electron acceptors, resulting in an over-reduction of the photosystem I (PSI) acceptor side. The over-reduction of the PSI acceptor side further inhibited electron transport from the photosystem II (PSII) reaction centers to the PSII acceptor side as indicated by an increase in VJ (the relative variable fluorescence at J-step), causing an imbalance between photosynthetic light absorption and energy utilization per active reaction center (RC) under high light, which led to the over-excitation of t...

Soluble aluminum (Al3+) is a major constraint to plant growth in highly acidic soils, which comprise up to 50% of the world?s arable land. The primary mechanism of Al resistance described in plants is the chelation of Al3+ cations by release of organic acids into the rhizosphere. Candidate aluminum tolerance genes encoding organic acid transporter of the ALMT (aluminum-activated malate transporter) and MATE (multi-drug and toxic compound extrusion) families have been characterized in several plant species. In this study, we have isolated in five different cultivars the rye ScAACT1 gene, homolog to barley aluminum activated citrate transporter HvAACT1. This gene mapped to the 7RS chromosome arm, 25?cM away from the ScALMT1 aluminum tolerance gene. The gene consisted of 13 exons and 12 intro...

Aristolochene synthase from Aspergillus terreus catalyzes the cyclization of the universal sesquiterpene precursor, farnesyl diphosphate, to form the bicyclic hydrocarbon aristolochene. The 2.2 {angstrom} resolution X-ray crystal structure of aristolochene synthase reveals a tetrameric quaternary structure in which each subunit adopts the {alpha}-helical class I terpene synthase fold with the active site in the 'open', solvent-exposed conformation. Intriguingly, the 2.15 {angstrom} resolution crystal structure of the complex with Mg{sup 2+}{sub 3}-pyrophosphate reveals ligand binding only to tetramer subunit D, which is stabilized in the 'closed' conformation required for catalysis. Tetramer assembly may hinder conformational changes required for the transition from the inactive open conformation to the active closed conformation, thereby accounting for the attenuation of catalytic activity with an increase in enzyme concentration. In both conformations, but especially in the closed conformation, the active site contour is highly complementary in shape to that of aristolochene, and a catalytic function is proposed for the pyrophosphate anion based on its orientation with regard to the presumed binding mode of aristolochene. A similar active site contour is conserved in aristolochene synthase from Penicillium roqueforti despite the substantial divergent evolution of these two enzymes, while strikingly different active site contours are found in the sesquiterpene cyclases 5-epi-aristolochene synthase and trichodiene synthase. Thus, the terpenoid cyclase active site plays a critical role as a template in binding the flexible polyisoprenoid substrate in the proper conformation for catalysis. Across the greater family of terpenoid cyclases, this template is highly evolvable within a conserved {alpha}-helical fold for the synthesis of terpene natural products of diverse structure and stereochemistry.

Neutral racemic antifungal alcohols of 8.O.4'-neolignan type, were evaluated for inhibitory activity towards the fungal cell wall, using the whole cell Neurospora crassa hyphal growth inhibition assay. Results strongly suggested that these compounds could act by inhibiting cell wall polymer synthesis or assembly. Active compounds were tested for their inhibitory activities against (1,3)-beta-glucan synthase, an enzyme that catalyzes the synthesis of the major wall polymer (1,3)-beta-glucan. Although these compounds were found to be inhibitors of the enzyme (inhibition ranging between 2 and 72% at 250 micro/ml), comparison of these results with those from agar dilution assays, allow us to infer that these compounds do not act via the inhibition of glucan synthase. In addition, ketones with same pattern of substitution as alcohols, which have no antifungal properties in agar dilution assays, still displayed similar glucan synthase inhibition. PMID:9720609

The effect of common salt (NaCl) on ion contents, Krebs cycle intermediates and its regulatory enzymes was investigated in growing mungbean (Vigna radiata L. Wilczek, B 105) seedlings. Sodium and chloride ion contents increased in both root and shoot whereas potassium ion content decreased in shoot of test seedlings with increasing concentrations of NaCl. Organic acids like pyruvate and citrate levels increased whereas malate level decreased under stress in both roots and shoots. Salt stress also variedly affected the activities of different enzymes of respiratory chain. The activity of pyruvate dehydrogenase (E.C. 1.2.4.1) decreased in 50 mM NaCl but increased in 100 mM and 150 mM concentrations, in both root and shoot samples. Succinate dehydrogenase (E.C. 1.3.5.1) activity was reduced in root whereas stimulated in shoot under increasing concentrations of salt. The activity of isocitrate dehydrogenase (E.C. 1.1.1.41) and malate dehydrogenase (E.C. 1.1.1.37) decreased in both root and shoot samples under salt stress. On the contrary, pretreatment of mungbean seeds with sublethal dose of NaCl was able to overcome the adverse effects of stress imposed by NaCl to variable extents with significant alterations of all the tested parameters, resulting in better growth and efficient respiration in mungbean seedlings. Thus, plants can acclimate to lethal level of salinity by pretreatment of seeds with sublethal level of NaCl, which serves to improve their health and production under saline condition, but the sublethal concentration of NaCl should be carefully chosen. PMID:23000814

A novel, simple, and rapid preparative method for purification of rat liver H{sup +}-ATP synthase by anion-exchange HPLC was developed. The H{sup +}-ATP synthase purified had higher ATPase activity in the absence of added phospholipids than any preparation reported previously, and this activity was completely inhibited by oligomycin. When reconstituted into proteoliposomes, the H{sup +}-ATP synthase showed an ATP-dependent 8-anilinonaphthalene-1-sulfonate response and ATP-P{sub i} exchange activity, both of which were also completely inhibited by oligomycin and an uncoupler, indicating the intactness of the H{sup +}-ATP synthase. An immunochemical study and a labeling experiment with N,N{prime}-({sup 14}C)dicyclohexylcarbondiimide (({sup 14}C)DCCD) demonstrated the presence of chargerin II (a product of mitochondrial A6L DNA) and DCCD-binding protein (subunit c) in the complex. The subunits of the complex were separated into 11 main fractions by reverse-phase HPLC, and 3 of them and the {sigma} subunit in F{sub 1} were partially sequenced. A search for sequence homologies indicated that these components were subunit b, coupling factor 6, subunit {sigma}, and subunit e. This is the first report of the existence of subunit b, factor 6, and chargerin II in K{sup +}-ATP synthase purified from rat liver mitochondria.

Glutamate synthase from Escherichia coli K-12 exhibits NH3-dependent activity. NH3-dependent activity is increased approximately 5-fold in apoglutamate synthase lacking flavin and non-heme iron. Whereas glutamine plus 2-oxoglutarate have the capacity to reoxidize the chemically reduced flavoenzyme, no such reoxidation is obtained with 2-oxoglutarate plus NH3. These results establish that the glutamine- and NH3-dependent syntheses of glutamate occur by different pathways of electron transfer from NADPH. The NH3-dependent activity of native and apoglutamate synthase exhibits similar catalytic properties. Some properties of apoglutamate synthase are similar to those of glutamate dehydrogenase. These properties include pH optima for synthesis and oxidative deamination of glutamate, inactivation by alkylating reagents and p-mercuribenzoate, an enhanced rate of inactivation by alkylating reagents and p-mercuribenzoate at low pH, 2-oxoglutarate protection against inactivation by p-mercuribenzoate, and reactivation of p-mercuribenzoate-treated enzyme by 2-mercaptoethanol. 2-Oxoglutarate protects against alkylation of glutamate synthase by iodo [1-14C]acetamide and reduces incorporation of methyl [1-14C]carboxamide into the small subunit of the enzyme. PMID:6450

Strictosidine synthase was partially purified from Catharanthus roseus cell suspension cultures and immobilized on CNBr-activated Sepharose. The immobilized enzyme exhibits a thermostability increased 300 fold over that of the soluble enzyme and catalyses exclusively the formation of the 3alpha(S)-isomer, strictosidine. Gram quantities of this biologically active glucoalkaloid, which hitherto had been difficult to synthesize and purify, were prepared. PMID:17396930

Nitric oxide synthases (NOS) are homodimeric enzymes that NADPH-dependently convert L-arginine to nitric oxide and L-citrulline. Interestingly, all NOS also require (6R)-5,6,7,8-tetrahydro-L-biopterin (H4Bip) for maximal activity although the mechanism is not fully understood. Basal NOS activity, i....

The treatment of Candida albicans (yeast form) with digitonin or dimethyl sulfoxide permeabilized cells and caused the activation of chitin synthase in situ. Endogenous activation was completely prevented by the sulfhydryl reagents N-ethylmaleimide, p-chloromercuribenzoate, and 5,5'-dithiobis(2-nitr...

The effects of a cell wall fraction of the fungus Phytophthora megasperma on the enzymatic activities of 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase (EC 4.1.2.15) in extracts of cultured parsley cells (Petroselinum crispum) were examined. The specific activity of a plastidic form of ...

We report that the activity of glycogen synthase kinase-3 (GSK-3) is necessary for the maintenance of the epithelial architecture. Pharmacological inhibition of its activity or reducing its expression using small interfering RNAs in normal breast and skin epithelial cells results in a reduction of E...

Krebs cycle enzyme activity in Bacillus subtilis was examined under aerobic and anaerobic conditions. Citrate synthase and aconitase activities in cells grown anaerobically in the presence of nitrate were reduced by as much as 10- and 30-fold, respectively, from levels observed under aerobic culture...

We have studied the significance of the N-terminal presequence of watermelon (Citrullus vulgaris) glyoxysomal malate dehydrogenase [gMDH; (S)-malate:NAD+ oxidoreductase; EC 1.1.1.37] in microbody targeting. The yeast Hansenula polymorpha was used as heterologous host for the in vivo expression of va...

The citric acid cycle enzyme malate dehydrogenase was purified to homogeneity from the nonsulfur purple bacteria Rhodobacter capsulatus, Rhodospirillum rubrum, Rhodomicrobium vannielii, and Rhodocyclus purpureus. Malate dehydrogenase was purified from each species by either a single- or a two-step p...

The estuarine crab Neohelice granulata was exposed (96h) to a sublethal copper concentration under two different physiological conditions (hyperosmoregulating crabs: 2ppt salinity, 1mg Cu/L; isosmotic crabs: 30ppt salinity, 5mg Cu/L). After exposure, gills (anterior and posterior) were dissected and activities of enzymes involved in glycolysis (hexokinase, phosphofructokinase, pyruvate kinase, lactate dehydrogenase), Krebs cycle (citrate synthase), and mitochondrial electron transport chain (cytochrome c oxidase) were analyzed. Membrane potential of mitochondria isolated from anterior and posterior gill cells was also evaluated. In anterior gills of crabs acclimated to 2ppt salinity, copper exposure inhibited hexokinase, phosphofructokinase, pyruvate kinase, and citrate synthase activity, ...

Anaplerosis, the synthesis of citric acid cycle intermediates, by pancreatic beta cell mitochondria has been proposed to be as important for insulin secretion as mitochondrial energy production. However, studies designed to lower the rate of anaplerosis in the beta cell have been inconclusive. To test the hypothesis that anaplerosis is important for insulin secretion, we lowered the activity of pyruvate carboxylase (PC), the major enzyme of anaplerosis in the beta cell. Stable transfection of short hairpin RNA was used to generate a number of INS-1 832/13-derived cell lines with various levels of PC enzyme activity that retained normal levels of control enzymes, insulin content, and glucose oxidation. Glucose-induced insulin release was decreased in proportion to the decrease in PC activity. Insulin release in response to pyruvate alone, 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) plus glutamine, or methyl succinate plus beta-hydroxybutyrate was also decreased in the PC knockdown cells. Consistent with a block at PC, the most PC-deficient cells showed a metabolic crossover point at PC with increased basal and/or glucose-stimulated pyruvate plus lactate and decreased malate and citrate. In addition, in BCH plus glutamine-stimulated PC knockdown cells, pyruvate plus lactate was increased, whereas citrate was severely decreased, and malate and aspartate were slightly decreased. The incorporation of 14C into lipid from [U-14C]glucose was decreased in the PC knockdown cells. The results confirm the central importance of PC and anaplerosis to generate metabolites from glucose that support insulin secretion and even suggest PC is important for insulin secretion stimulated by noncarbohydrate insulin secretagogues. PMID:18697738

The glutamine amidotransferase (GATase) family comprises enzyme complexes which consist of glutaminase and synthase subunits that catalyze in a concerted reaction the incorporation of nitrogen within various metabolic pathways. An important feature of GATases is the strong stimulation of glutaminase activity by the associated synthase. To understand the mechanism of this tight activity regulation, we probed by site-directed mutagenesis four residues of the glutaminase subunit TrpG from anthranilate synthase that are located between the catalytic Cys-His-Glu triad and the synthase subunit TrpE. In order to minimize structural perturbations induced by the introduced exchanges, the amino acids from TrpG were substituted with the corresponding residues of the closely related glutaminase HisH from imidazole glycerol phosphate synthase. Steady-state kinetic characterization showed that, in contrast to wild-type TrpG, two TrpG variants with single exchanges constitutively hydrolyzed glutamine in the absence of TrpE. A reaction assay performed with hydroxylamine as a stronger nucleophile replacing water and a filter assay with radiolabeled glutamine indicated that the formation of the thioester intermediate is the rate-limiting step of constitutive glutamine hydrolysis. Molecular dynamics simulations with wild-type TrpG and constitutively active TrpG variants suggest that the introduced amino acid exchanges result in a distance reduction between the active site Cys-His pair, which facilitates the deprotonation of the sulfhydryl group of the catalytic cysteine and thus enables its nucleophilic attack onto the carboxamide group of the glutamine side chain. We propose that native TrpG in the anthranilate synthase complex is activated by a similar mechanism. PMID:22432907

In this study, three trehalose gene clusters, treX-Y-Z, tpS1, and treS, of the acarbose-producing strain, Actinoplanes sp. SN223/29, have been identified. In particular, five trehalose synthetic genes were sequenced and characterized in detail. They were cloned and expressed in Escherichia coli BL21(DE3)pLysS using the His-tag vector pET19b. The recombinant proteins were purified by Ni2+-nitrilotriacetic acid agarose affinity chromatography, and their functions were characterized biochemically. Both the maltooligosyltrehalose synthase (TreY?TreZ) pathway and the trehalose synthase (TreS) pathway have maximum activity at 40?C and at pH?7.5 and 7.0, respectively, in 100-mM phosphate buffer. Meanwhile, the trehalose-6-phosphate synthase (TpS1) showed maximum activity at 35?C and at pH?7.5 in ...

Diabetes is one of the major life threatening diseases worldwide. It creates major health problems in urban India. Glycogen Synthase Kinase-3 (GSK-3) protein of human is known for phosphorylating and inactivating glycogen synthase which also acts as a negative regulator in the hormonal control of glucose homeostasis. In traditional medicine, Momordica charantia is used as antidiabetic plant because of its hypoglycemic effect. Hence to block the active site of the GSK-3 protein three anti-diabetic compounds namely, charantin, momordenol & momordicilin were taken from Momordica charantia for docking study and calculation of binding energy. The aim of present investigation is to find the binding energy of three major insulin-like active compounds against glycogen synthase kinase-3 (GSK-3), one of the key proteins involved in carbohydrate metabolism, with the help of molecular docking using ExomeTM Horizon suite. The study recorded minimum binding energy by momordicilin in comparison to the others. PMID:16759816

Starch synthase I (SSI) contributes the majority of the starch synthase activity in developing maize endosperm. In this work, the effects of various plant hormones and sugars on the expression of the starch synthase I gene (ZmSSI) in developing maize endosperms were examined. The accumulation of ZmSSI mRNA was induced using abscisic acid (ABA) but not with glucose, sucrose, or gibberellin treatment. To investigate the molecular mechanism underlying this effect, the ZmSSI promoter region (–1537 to +51) was isolated and analysed. A transient expression assay in maize endosperm tissue showed that the full-length ZmSSI promoter is activated by ABA. The results of deletion and mutation assays demonstrated that a CACCG motif in the ZmSSI promoter is responsible for the ABA inducibi...

Callose ((1-3){beta}-D-glucan), measured as incorporation of {sup 14}C-glucose into ethanol-insoluble product, is produced within 2h in victorin treated mesophyll protoplasts of victorin-sensitive cultivars of oat (Avena sativa). This production is ten-fold higher in the presence of 6 ng victorin/ml than untreated protoplasts after 2h. Microsomes of victorin-treated and untreated protoplasts are assayed for callose synthase activity using radiolabeled substrate, UDP-glucose. Preliminary studies indicate that microsomes of victorin-treated protoplasts have up to four times more callose synthase activity than microsomes of untreated protoplasts. Therefore, stimulation of callose synthase by some means that survives microsome isolation, at least in part, may account for the effect of victorin.

Prostaglandin-H (PGH) synthase from ram seminal vesicles is a dimeric integral membrane protein of molecular weight 140 kDa. PGH synthase is a key enzyme in the biosynthesis of prostaglandins, has cyclooxygenase and peroxidase activities, and contains heme as a coenzyme. In the peroxidation step of its reaction, PGH synthase can use xenobiotics as co-substrates and can catalyze the metabolic activation of carcinogens such as diethylstilbestrol. To gain a detailed understanding of the inner workings of PGH synthase, the authors are investigating its three-dimensional structure by X-ray crystallography. A purification procedure was established that yields stable homogeneous PGH synthase that is at least 80% holoenzyme. Manipulation of these crystals is very difficult due to the small volume of the growth phase. The crystals dissolved rapidly in all aqueous media into which they were transferred for mounting in X-ray capillaries. Therefore, the authors have not yet been able to demonstrate their true X-ray scattering power. A crystal provisionally dry mounted diffracted to about 8 {angstrom} resolution.

The chain oxidation of glyceraldehyde-3-phosphate dehydrogenase NADH by perhydroxyl radicals and propagated by molecular oxygen was studied by the xanthine-xanthine oxidase system, /sup 60/Co ..gamma..-ray, and pulse radiolysis. The chain length, amount of NADH oxidized per HO/sub 2/ generated, increases with increasing acidity of the medium and reaches a value of 73 at pH 5.0. The rate constant for the oxidation of the glyceraldehyde-3-phosphate dehydrogenase NADH complex by HO/sub 2/ was estimated to be 2 x 10/sup 7/ m/sup -1/s/sup -1/ at ambient temperatures (23-24/sup 0/C). Rate studies as a function of pH indicate that O/sub 2//sup -/ is unreactive toward the glyceraldehyde-3-phosphate dehydrogenase NADH complex. Other dehydrogenases (malate dehydrogenase, glutamate dehydrogenase, and isocitric dehydrogenase) studied showed no catalytic activity in the oxidation of NADH by HO/sub 2//O/sub 2//sup -/.

Soluble proteins were extracted from the vegetative cells of four pentose-fermenting yeasts, Candida shehatae, Pichia stipitis, R-1, and R-2. The R strains were recycled from fermentations of biomass hydrolyzates, and are the best pentose fermenters found to date, although they are of uncertain taxonomy. Isoenzyme patterns, protein patterns, and two-dimensional polypeptide mapping of these four strains were compared by polyacrylamide gel electrophoresis. The two R strains showed great similarity in two-dimensional polypeptide mapping, the pattern of sodium dodecyl sulfate - polyacrylamide gel electrophoresis, isoelectrofocusing, and isoenzymes, and may be one species. Each of the other two yeasts had its own characteristic electrophoretic pattern. The R strains showed the presence of three alcohol dehydrogenase isoenzymes compared with one for the culture collection yeasts, as well as much higher activity of malate dehydrogenase, isocitrate dehydrogenase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase, which further the formation of pyruvate and ethanol. 26 refs., 5 figs.

The synthesis, characterization, anti-hyperglycemic activity, oxidative DNA damage capacity, and acute toxicity of chromium(III) malate complex [Cr2(LMA)3] were described. [Cr2(LMA)3] was synthesized in a single-step reaction by chelating chromium(III) with L-malic acid in aqueous solution. Based on elemental analysis, thermodynamic analysis, and spectroscopy studies, the molecular formula of [Cr2(LMA)3] was inferred as Cr2(C4H4O5)3?5H2O. Daily treatment with 2.85?17.10?mg/kg body mass of [Cr2(LMA)3] in alloxan-induced diabetic rats for 2?weeks indicated that low-molecular-weight organic chromium complex [Cr2(LMA)3] had better bioavailability and more beneficial influences on the improvement of controlling blood glucose, serum lipid, and liver glycogen levels compared with CrCl3?6H2O. [Cr2...

The mitochondrial damage in the lung was assessed by examining the levels of reactive oxygen species (ROS), lipid peroxides, reduced glutathione, and the activities of isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, complexes I to IV, and cytochrome c. The oxidative phosphorylation (levels of adenosine triphosphatase) was evaluated for the assessment of mitochondrial functional capacity. We found significantly elevated levels of ROS, lipid peroxides, and decreased levels of mitochondrial enzymes in the mice administered with benzo[a]pyrene (B[a]p). Measurement of oxidative phosphorylation revealed a marked depletion in all the variables studied. Administration of crocetin prevented the structural and functional impairment of mitochondria upon administration to B[a]p. From the results, we suggest that administration of B[a]p induces damage to the lung mitochondria and crocetin protects the lung from damage by maintaining the structural and functional integrity of the mitochondrial membrane.   

Thyroid diseases are one of the most common metabolic disorders in the human population. In this work, we present data concerning changes in the activity and kinetic parameters of several enzymes associated with both anabolic (glucose-6-phosphate dehydrogenase?G6PDH, EC 1.1.1.49; 6-phosphogluconate dehydrogenase?6PGDH, EC 1.1.1.44; malic enzyme?ME, EC 1.1.1.40; and isocitrate dehydrogenase?IDH, EC 1.1.1.42) and catabolic (NAD-dependent malate dehydrogenase?NAD-MDH, EC 1.1.1.37; and lactate dehydrogenase?LDH, EC 1.1.1.27) processes under conditions of hypothyroidism and T3 treatment. Hypothyroidism was induced in rats by the surgical removal of the thyroid gland. T3-treated rats were injected by T3 (0.5?mg?T3/kg body weight daily during 10?days). We have found that T3 treatment caused an in...

Lead (Pb) and zinc (Zn) are two typical metal contaminants with high levels in both seawater and sediment in the intertidal zones of the Bohai Sea. Suaeda salsa is the pioneer halophyte plant in the intertidal zones of the Bohai Sea. In the present work, the short (1?week) and long term (1?month) toxicological effects of environmentally relevant concentrations of Pb and Zn were characterized in S. salsa using NMR-based metabolomics combined with antioxidant enzyme activities. After metal exposure for 1?week, no significant metabolic responses were detected in root tissues of S. salsa. The significant metabolic responses included the increase of isocaproate, glucose and fructose, and decrease of malate, citrate and sucrose in root tissues of S. salsa exposed to Pb for 1?month. The increased...

Oenococcus oeni is recognized as the principal microorganism responsible for malolactic fermentation, and the control of its activity is of primary importance in winemaking. The aim of this study was to quantify the levels of expression of the malate transporter gene (mleP) and of two genes putatively involved in the ATP-binding cassette transport system (oeoe_1651, oeoe_0550) to better understand the physiological response of bacteria during rehydration. These genes coding for transporters were studied in different rehydration media. Initially, three different statistical algorithms were used to identify suitable reference genes to be used for the normalization of expression data in O. oeni during rehydration, and to this purpose, the best genes found were ddl and gyrB. The results showed...

Redesigning of an enzyme for a new catalytic reaction and modified substrate specificity was exploited with 3-isopropylmalate dehydrogenase (IPMDH). Point-mutation on Gly-89, which is not in the catalytic site but near it, was done by changing it to Ala, Ser, Val, and Pro, and all the mutations changed the substrate specificity. The mutant enzymes showed higher catalytic efficiency (kcat/Km) than the native IPMDH when malate was used as a substrate instead of 3-isopropylmalate. More interestingly, an additional insertion of Gly between Gly-89 and Leu-90 significantly altered the substrate-specificity, although the overall catalytic activity was decreased. Particularly, this mutant turned out to efficiently accept D-lactic acid, which was not accepted as a substrate by wild-type IPMDH at all. These results demonstrate the opportunity for creating novel enzymes by modification of amino acid residues that do not directly participate in catalysis, or by insertion of additional residues.   

Compared to the group I chaperonins, such as Escherichia coli GroEL, which facilitate protein folding, many aspects of the functional mechanism of archaeal group II chaperonins are unclear. Sequence homology between the chaperonin from Pyrococcus furiosus (PfCPN) and other group II chaperonins, together with the homo-oligomeric nature of PfCPN, suggest that PfCPN may serve as a model to clarify the role of the homologous position Gly-345 in the chaperonin-mediated protein folding. Here, we show that the purified chaperonin mutant in which the conserved residue Gly-345 is replaced by Asp (G345D) displays only about 25% ATP/ADP hydrolysis activities of the wild-type in the presence of Co2+ and has a reduced capacity to promote folding of denatured malate dehydrogenase in vitro. This may be a...

Acidity levels greatly affect the taste and flavor of fruit, and consequently its market value. In mature apple fruit, malic acid is the predominant organic acid. Several studies have confirmed that the major quantitative trait locus Ma largely controls the variation of fruit acidity levels. The Ma locus has recently been defined in a region of 150?kb that contains 44 predicted genes on chromosome 16 in the Golden Delicious genome. In this study, we identified two aluminum-activated malate transporter-like genes, designated Ma1 and Ma2, as strong candidates of Ma by narrowing down the Ma locus to 65?82?kb containing 12?19 predicted genes depending on the haplotypes. The Ma haplotypes were determined by sequencing two bacterial artificial chromosome clones from G.41 (an apple rootstock of g...

Abstract Root efflux of organic acid anions underlies a major mechanism of plant aluminium (Al) tolerance on acid soils. This efflux is mediated by transporters of the Al-activated malate transporter (ALMT) or the multi-drug and toxin extrusion (MATE) families. ZmALMT2 was previously suggested to be involved in Al tolerance based on joint association-linkage mapping for maize Al tolerance. In the current study, we functionally characterized ZmALMT2 by heterologously expressing it in Xenopus laevis oocytes and transgenic Arabidopsis. In oocytes, ZmALMT2 mediated an Al-independent electrogenic transport product of organic and inorganic anion efflux. Ectopic overexpression of ZmALMT2 in an Al-hypersensitive Arabidopsis KO/KD line lacking the Al tolerance genes, AtALMT1 and AtMATE, resulted in...

Peritoneal exudate mononuclear cells obtained from BCG-vaccinated rabbits showed higher utilization of succinate, glycerophosphate, beta - hydroxybutyrate, and glycerol than cells from control animals. No differences in utilization of the following substrates were noted: lactate, glucose-6-phosphate, malate, isocitrate, alpha -ketoglutarate, and glutamic acid. A second, later stage of elevated metabolic activity was associated with heightened resistance to infection. When rabbits which had been irradiated with 400 r 2 years previously were vaccinated with BCG, they failed to respond as shown by their lack of resistance to infection and failure of their mononuclear cells to show the biphasic metabolic stimulation. The results demonstrate the close relation between the metabolic capabilities of reticuloendothelial cells and their resistance to tuberculosis. (H.H.D.)

Effects of alphamethrin (0.04ppm, 0.08ppm, 0.16ppm, 0.32ppm) on some metabolic dehydrogenases and proteins for 1, 2, 3, 4, 8 and 16days using three ecologically different earthworm species (Perionyx sansibaricus, Lampito mauritii and Metaphire posthuma) were studied. The first significant effect was on 2nd/3rd day and maximum response was achieved on 16th day of exposure of alphamethrin at all concentrations. Similarly, maximum effect was obtained at 0.32ppm alphamethrin at different exposure periods. It showed a dose- and duration-dependent inhibitory effects of a pyrethroid on enzymes and proteins of earthworms. There were approximately 47%, 43% and 41% declines in specific activities of cytoplasmic malate dehydrogenase (cMDH) of P. sansibaricus, L. mauritii and M. posthuma, respectively...

The succulent, cylindrical leaves of the C4 dicot Portulaca grandiflora possess three distinct green cell types: bundle sheath cells (BSC) in radial arrangement around the vascular bundles; mesophyll cells (MC) in an outer layer adjacent to the BSC; and water storage cells (WSC) in the leaf center. Unlike typical Kranz leaf anatomy, the MC do not surround the bundle sheath tissue but occur only in the area between the bundle sheath and the epidermis. Intercellular localization of photosynthetic enzymes was characterized using protoplasts isolated enzymatically from all three green cell types. Like other C4 plants, P. grandiflora has ribulose 1,5-bisphosphate carboxylase and the decarboxylating enzyme, NADP+-malic enzyme, in the BSC. Unlike other C4 plants, however, phosphoenolpyruvate carboxylase, pyruvate, Pi dikinase, and NADP+-malate dehydrogenase of the C4 pathway were present in all three green cell types, indicating that all are capable of fixing CO2 via phosphoenolpyruvate carboxylase and regenerating phosphoenolpyruvate. Other enzymes were about equally distributed between MC and BSC similar to other C4 plants. The enzyme profile of the WSC was similar to that of the MC but with reduced activity in most enzymes, except mitochondrion-associated enzymes. Intracellular localization of enzymes was studied in organelles partitioned by differential centrifugation using mechanically ruptured mesophyll and bundle sheath protoplasts. Phosphoenolpyruvate carboxylase was a cytosolic enzyme in both cells; whereas, ribulose 1,5-bisphosphate carboxylase and NADP+-malic enzyme were exclusively compartmentalized in the bundle sheath chloroplasts. NADP+-malate dehydrogenase, pyruvate, Pi dikinase, aspartate aminotransferase, 3-phosphoglycerate kinase, and NADP+-triose-P dehydrogenase were predominantly localized in the chloroplasts while alanine aminotransferase and NAD+-malate dehydrogenase were mainly present in the cytosol of both cell types. Based on enzyme localization, a scheme of C4 photosynthesis in P. grandiflora is proposed. Well-watered plants of P. grandiflora exhibit a diurnal fluctuation of total titratable acidity, with an amplitude of 61 and 54 microequivalent per gram fresh weight for the leaves and stems, respectively. These changes were in parallel with changes in malic acid concentration in these tissues. Under severe drought conditions, diurnal changes in both titratable acidity and malic acid concentration in both leaves and stems were much reduced. However, another C4 dicot Amaranthus graecizans (nonsucculent) did not show any diurnal acid fluctuation under the same conditions. These results confirm the suggestion made by Koch and Kennedy (Plant Physiol. 65: 193-197, 1980) that succulent C4 dicots can exhibit an acid metabolism similar to Crassulacean acid metabolism plants in certain environments. Images

Because accumulation of altered proteins is the most common biochemical symptom of aging, it is at least possible that such proteotoxicity may cause aging and influence life span. The life span of the nematode worm Caenorhabditis elegans is strongly influenced by changes in the intracellular concentration of methylglyoxal (MG), a putative source of much age-related proteotoxicity and organelle, cellular, and molecular dysfunction. Glycerol has recently been shown to shorten, whereas oxaloacetate has been found to extend, life span in C. elegans. It is suggested here that glycerol and oxaloacetate exert opposing effects on MG formation in C. elegans. It is proposed that, if not secreted by aquaporin, glycerol is converted to glycerol phosphate and then to dihydroxyacetone phosphate (DHAP) via a reaction requiring nicotinamide adenine dinucleotide (NAD(+)). This inhibits operation of the glycerol phosphate cycle in which DHAP is converted into glycerol phosphate, which concomitantly regenerates NAD(+) from NADH, thereby ensuring glycolytic oxidation of glyceraldehyde-3-phosphate (G3P). Because DHAP and G3P spontaneously decompose into MG, and NAD(+) is required for conversion of G3P into phosphoglycerate, the glycerol-induced increased DHAP formation and decreased NAD(+) availability will increase the potential for MG generation. In contrast, oxaloacetate may decrease MG generation by stimulating the operation of the malate-oxaloacetate shuttle, in which oxaloacetate is converted to malate, which regenerates NAD(+) from NADH. By the ensuing G3P oxidation, increased NAD(+) availability will decrease the potential for MG formation. It should be noted that mitochondria are involved in the operation of the above cycle/shuttles and that increased NAD(+) availability also stimulates those sirtuin activities that increase mitogenesis and mitochondrial activity via effects on signal transduction and gene expression, which frequently accompany dietary restriction-induced life span extension. PMID:20645869

We investigated whether the reductive pentose phosphate path in guard cells of Pisum sativum had the capacity to contribute significantly to the production of osmotica during stomatal opening in the light. Amounts of ribulose 1,5-bisphophate carboxylase/oxygenase (Rubisco) were determined by the ({sup 14}C) carboxyarabinitol bisphosphate assay. A guard cell contained about 1.2 and a mesophyll cell about 324 picograms of the enzyme; the ratio was 1:270. The specific activities of Rubisco in guard cells and in mesophyll cells were equal; there was no indication of a specific inhibitor of Rubisco in guard cells. Rubisco activity was 115 femtomol per guard-cell protoplast and hour. This value was different from zero with a probability of 0.99. After exposure of guard-cell protoplasts to {sup 14}CO{sub 2} for 2 seconds in the light, about one-half of the radioactivity was in phosphorylated compounds and <10% in malate. Guard cells in epidermal strips produced a different labelling pattern; in the light, <10% of the label was in phosphorylated compounds and about 60% in malate. The rate of solute accumulation in intact guard cells was estimated to have been 900 femto-osmol per cell and hour. If Rubisco operated at full capacity in guard cells, and hexoses were produced as osmotica, solutes could be supplied at a rate of 19femto-osmol per cell and hour, which would constitute 2% of the estimated requirement. The capacity of guard-cell Rubisco to meet the solute requirement for stomatal opening in leaves of Pisum sativum is insignificant.

During hibernation, animals cycle between periods of torpor, during which body temperature (T(b)) and metabolic rate (MR) are suppressed for days, and interbout euthermia (IBE), during which T(b) and MR return to resting levels for several hours. In this study, we measured respiration rates, membrane potentials, and reactive oxygen species (ROS) production of liver and skeletal muscle mitochondria isolated from ground squirrels (Ictidomys tridecemlineatus) during torpor and IBE to determine how mitochondrial metabolism is suppressed during torpor and how this suppression affects oxidative stress. In liver and skeletal muscle, state 3 respiration measured at 37°C with succinate was 70% and 30% lower, respectively, during torpor. In liver, this suppression was achieved largely via inhibition of substrate oxidation, likely at succinate dehydrogenase. In both tissues, respiration by torpid mitochondria further declined up to 88% when mitochondria were cooled to 10°C, close to torpid T(b). In liver, this passive thermal effect on respiration rate reflected reduced activity of all components of oxidative phosphorylation (substrate oxidation, phosphorylation, and proton leak). With glutamate + malate and succinate, mitochondrial free radical leak (FRL; proportion of electrons leading to ROS production) was higher in torpor than IBE, but only in liver. With succinate, higher FRL likely resulted from increased reduction state of complex III during torpor. With glutamate + malate, higher FRL resulted from active suppression of complex I ROS production during IBE, which may limit ROS production during arousal. In both tissues, ROS production and FRL declined with temperature, suggesting ROS production is also reduced during torpor by passive thermal effects. PMID:21993528

Sphingolipids are ubiquitous and essential components of eukaryotic membranes, particularly the plasma membrane. The biosynthetic pathway for the formation of these lipid species is conserved up to the formation of sphinganine. However, a divergence is apparent in the synthesis of complex sphingolipids. In animal cells, ceramide is a substrate for sphingomyelin (SM) production via the enzyme SM synthase. In contrast, fungi utilize phytoceramide in the synthesis of inositol phosphorylceramide (IPC) catalyzed by IPC synthase. Because of the absence of a mammalian equivalent, this essential enzyme represents an attractive target for anti-fungal compounds. In common with the fungi, the kinetoplastid protozoa (and higher plants) synthesize IPC rather than SM. However, orthologues of the gene believed to encode the fungal IPC synthase (AUR1) are not readily identified in the complete genome data bases of these species. By utilizing bioinformatic and functional genetic approaches, we have isolated a functional orthologue of AUR1 in the kinetoplastids, causative agents of a range of important human diseases. Expression of this gene in a mammalian cell line led to the synthesis of an IPC-like species, strongly indicating that IPC synthase activity is reconstituted. Furthermore, the gene product can be specifically inhibited by an anti-fungal-targeting IPC synthase. We propose that the kinetoplastid AUR1 functional orthologue encodes an enzyme that defines a new class of protozoan sphingolipid synthase. The identification and characterization of the protozoan IPC synthase, an enzyme with no mammalian equivalent, will raise the possibility of developing anti-protozoal drugs with minimal toxic side affects. PMID:16861742

The glyoxylate cycle, a metabolic pathway required for generating C(4) units from C(2) compounds, is an important factor in virulence, in both animal and plant pathogens. Here, we report the localization of the key enzymes of this cycle, isocitrate lyase (Icl1; EC 4.1.3.1) and malate synthase (Mls1; EC 2.3.3.9), in the human fungal pathogen Candida albicans. Immunocytochemistry in combination with subcellular fractionation showed that both Icl1 and Mls1 are localized to peroxisomes, independent of the carbon source used. Although Icl1 and Mls1 lack a consensus type I peroxisomal targeting signal (PTS1), their import into peroxisomes was dependent on the PTS1 receptor Pex5p, suggesting the presence of non-canonical targeting signals in both proteins. Peroxisomal compartmentalization of the glyoxylate cycle is not essential for proper functioning of this metabolic pathway because a pex5Delta/Delta strain, in which Icl1 and Mls1 were localized to the cytosol, grew equally as well as the wild-type strain on acetate and ethanol. Previously, we reported that a fox2Delta/Delta strain that is completely deficient in fatty acid beta-oxidation, but has no peroxisomal protein import defect, displayed strongly reduced growth on non-fermentable carbon sources such as acetate and ethanol. Here, we show that growth of the fox2Delta/Delta strain on these carbon compounds can be restored when Icl1 and Mls1 are relocated to the cytosol by deleting the PEX5 gene. We hypothesize that the fox2Delta/Delta strain is disturbed in the transport of glyoxylate cycle products and/or acetyl-CoA across the peroxisomal membrane and discuss the possible relationship between such a transport defect and the presence of giant peroxisomes in the fox2Delta/Delta mutant. PMID:18832312

Phosphatidylinositol (PtdIns) synthase in microsomal fractions derived from Tetrahymena vorax was studied to determine its activity requirements. The suitability of inositol isomers as substrates for the synthase and in headgroup exchange reactions also was investigated. Tetrahymena PtdIn synthase activity was optimum in the presence of 2 mM MgCl2 plus 2 mM MnCl2, a pH of 7.8, and a temperature of 30 degrees C. The enzyme retained approximately 80% of its activity after incubation at 70 degrees C for 10 min. PtdIns headgroup exchange activity was maximal in the presence of cytidine monophosphate. By following either the accumulation of radiolabeled reaction products or the loss of radiolabel from precursors, each of the inositol isomers tested appeared to serve as substrates for both the PtdIns synthase and PtdIns:inositol phosphatidyl transferase activities. In each case, myo-inositol and scyllo-inositol were the preferred substrates. The data suggest two routes for the formation of phosphatidyl-non-myo-inositols in Tetrahymena and the potential for the production of novel, non-myo-inositol-containing second messengers. PMID:17403152

The objective of this study was to elucidate the roles of sugar in the formation of root systems. Several parts of the seminal root were investigated to determine their sucrose, glucose and fructose contents, and the activity and the in situ localization of the activities of two kinds of metabolic enzymes, invertase and sucrose synthase, which hydrolyze sucrose. The sucrose, glucose and fructose concentrations in the 0-1 cm section from the root apex were three to five times those in the other sections. The invertase and sucrose synthase activities were also higher in the apical section. The in situ localization of invertase activity was detected in the cell elongation zone of the seminal root using histochemical method. The sucrose synthase activity was detected in the cell elongation zone of the seminal root and the root apices of lateral roots. These results suggested that sucrose is transported to the root elongation zone and the surrounding tissue of the lateral root primordia, and is cleaved into glucose, fructose, and UDP-glucose by invertase or sucrose synthase. This suggested that sucrose contributes to root formation by serving as the energy source, the carbon source for cell wall synthesis, and as a compatible solute for cell elongation.   

Background: Coronary artery disease (CAD) is the leading cause of death worldwide, and the major cause of hospital admissions in the Western countries. The pathogenesis of CAD is closely related to nitric oxide release and formation. The purpose of this study was to investigate the status of platelets nitric oxide in patients with coronary artery disease.Methods: We measured platelets aggregation, cGMP, NO (nitrite/nitrate level), NO synthase activity, plasma NO, and ionized Ca2+ in 40 healthy volunteers and 120 patients with myocardial infarction, unstable and stable angina, with 40 subjects in each group. The subjects’ age mean range was from 40–51 years.Results: Platelets aggregation, NO, cGMP, NO synthase activity, plasma NO and ionized Ca2+ have increased significantly (P <0.001) across the patients groups compared to controls. Platelets NO synthase activity (mean ± SD / U / 109 platelets) in healthy controls, MI, unstable angina and stable angina patients were 1.19 ± 0.56, 1.21 ± 0.64, 1.64 ± 0.98 and 1.57 ± 0.81 respectively. The cGMP (mean ± SD / pmole / 109 platelets) levels were 0.95 ± 0.41, 1.53 ± 0.64, 3.18 ± 0.77, and 5.12 ± 1.5 respectively.Conclusions: The present study demonstrated that platelets aggregation, NO, cGMP, NO synthase activity, plasma NO, and ionized Ca2+ profoundly increased in CAD. The increases in NO-cGMP components may have resulted as a compensatory response to ameliorate platelet activity and Ca2+ levels in CAD patients.   

Dimeric Salmonella typhimurium orotate phosphoribosyltransferase (OMP synthase, EC 2.4.2.10), a key enzyme in de novo pyrimidine nucleotide synthesis, has been cocrystallized in a complete substrate E·MgPRPP·orotate complex and the structure determined to 2.2 Å resolution. This structure resembles that of Saccharomyces cerevisiae OMP synthase in showing a dramatic and asymmetric reorganization around the active site-bound ligands but shares the same basic topology previously observed in complexes of OMP synthase from S. typhimurium and Escherichia coli. The catalytic loop (residues 99-109) contributed by subunit A is reorganized to close the active site situated in subunit B and to sequester it from solvent. Furthermore, the overall structure of subunit B is more compact, because of movements of the amino-terminal hood and elements of the core domain. The catalytic loop of subunit B remains open and disordered, and subunit A retains the more relaxed conformation observed in loop-open S. typhimurium OMP synthase structures. A non-proline cis-peptide formed between Ala71 and Tyr72 is seen in both subunits. The loop-closed catalytic site of subunit B reveals that both the loop and the hood interact directly with the bound pyrophosphate group of PRPP. In contrast to dimagnesium hypoxanthine-guanine phosphoribosyltransferases, OMP synthase contains a single catalytic Mg(2+) in the closed active site. The remaining pyrophosphate charges of PRPP are neutralized by interactions with Arg99A, Lys100B, Lys103A, and His105A. The new structure confirms the importance of loop movement in catalysis by OMP synthase and identifies several additional movements that must be accomplished in each catalytic cycle. A catalytic mechanism based on enzymic and substrate-assisted stabilization of the previously documented oxocarbenium transition state structure is proposed. PMID:22531064

Acetate salts are emerging as potentially attractive bulk chemicals for a variety of environmental applications, for example, as catalysts to facilitate combustion of high-sulfur coal by electrical utilities and as the biodegradable noncorrosive highway deicing salt calcium magnesium acetate. The structural gene coding for citrate synthase from the gram-positive soil isolate Bacillus sp. strain C4 (ATCC 55182) capable of secreting acetic acid at pH 5.0 to 7.0 in the presence of dolime has been cloned from a genomic library by complementation of an Escherichia coli auxotrophic mutant lacking citrate synthase. The nucleotide sequence of the entire 3.1-kb HindIII fragment has been determined, and one major open reading frame was found coding for citrate synthase (ctsA). Citrate synthase from Bacillus sp. strain C4 was found to be a dimer (M{sub r}, 84,500) with a sub unit with an M{sub r} of 42,000. The N-terminal sequence was found to be identical with that predicted from the gene sequence. The kinetics were best fit to a bisubstrate enzyme with an ordered mechanism. Bacillus sp. strain C4 citrate synthase was not activated by potassium chloride and was not inhibited by NADH, ATP, ADP, or AMP at levels up to 1 mM. The predicted amino acid sequence was compared with that of the E. coli, Acinetobacter anitratum, Pseudomonas aeruginosa, Rickettsia prowazekii, porcine heart, and Saccharomyces cerevisiae cytoplasmic and mitochondrial enzymes.

Platelets metabolize arachidonic acid to thromboxane A{sub 2}, a potent platelet aggregator and vasoconstrictor compound. The first step of this transformation is catalyzed by prostaglandin (PG) G/H synthase, a target site for nonsteroidal antiinflammatory drugs. We have isolated the cDNA for both human platelet and human erythroleukemia cell PGG/H synthase using the polymerase chain reaction and conventional screening procedures. The cDNA encoding the full-length protein was expressed in COS-M6 cells. Microsomal fractions from transfected cells produced prostaglandin endoperoxide derived products which were inhibited by indomethacin and aspirin. Mutagenesis of the serine residue at position 529, the putative aspirin acetylation site, to an asparagine reduced cyclooxygenase activity to barely detectable levels, an effect observed previously with the expressed sheep vesicular gland enzyme. Platelet-derived growth factor and phorbol ester differentially regulated the expression of PGG/H synthase mRNA levels in the megakaryocytic/platelet-like HEL cell line. The PGG/H synthase gene was assigned to chromosome 9 by analysis of a human-hamster somatic hybrid DNA panel. The availability of platelet PGG/H synthase cDNA should enhance our understanding of the important structure/function domains of this protein and it gene regulation.

The hyperthermophilic archaeon Pyrobaculum islandicum uses the citric acid cycle in the oxidative and reductive directions for heterotrophic and autotrophic growth, respectively, but the control of carbon flow is poorly understood. P. islandicum was grown at 95°C autotrophically, heterotrophically, and mixotrophically with acetate, H2, and small amounts of yeast extract and with thiosulfate as the terminal electron acceptor. The autotrophic growth rates and maximum concentrations of cells were significantly lower than those in other media. The growth rates on H2 and 0.001% yeast extract with and without 0.05% acetate were the same, but the maximum concentration of cells was fourfold higher with acetate. There was no growth with acetate if 0.001% yeast extract was not present, and addition of H2 to acetate-containing medium greatly increased the growth rates and maximum concentrations of cells. P. islandicum cultures assimilated 14C-labeled acetate in the presence of H2 and yeast extract with an efficiency of 55%. The activities of 11 of 19 enzymes involved in the central metabolism of P. islandicum were regulated under the three different growth conditions. Pyruvate synthase and acetate:coenzyme A (CoA) ligase (ADP-forming) activities were detected only in heterotrophically grown cultures. Citrate synthase activity decreased in autotrophic and acetate-containing cultures compared to the activity in heterotrophic cultures. Acetylated citrate lyase, acetate:CoA ligase (AMP forming), and phosphoenolpyruvate carboxylase activities increased in autotrophic and acetate-containing cultures. Citrate lyase activity was higher than ATP citrate synthase activity in autotrophic cultures. These data suggest that citrate lyase and AMP-forming acetate:CoA ligase, but not ATP citrate synthase, work opposite citrate synthase to control the direction of carbon flow in the citric acid cycle.

SummaryMonoterpene indole alkaloids from Catharanthus roseus (Madagascar periwinkle), such as the anticancer agents vinblastine and vincristine, have important pharmacological activities. Metabolic engineering of alkaloid biosynthesis can provide an efficient and environmentally friendly route to analogs of these synthetically challenging and pharmaceutically valuable natural products. However, the narrow substrate scope of strictosidine synthase, the enzyme at the entry point of the pathway, limits a pathway engineering approach. We demonstrate that with a different expression system and screening method it is possible to rapidly identify strictosidine synthase variants that accept tryptamine analogs not turned over by the wild-type enzyme. The variants are used in stereoselective synthes...

Diagnosis of porphyria is a clinical and biochemical procedure. Acute hepatic porphyrias are molecular regulation diseases which are characterized by a relative enzyme deficiency of the ferro-chelatase chain and an induction of hepatic delta-aminoacid synthase. There are indistinct clinical and pathobiochemical transitions between the three acute hepatic types of porphyria: acute intermittent porphyria, hereditary coproporphyria and porphyria variegata. They develop a similar acute clinical syndrome. The differential diagnosis is made possible by a differentiation of porphyrins and porphyrin precursers in the urine and the porphyrines in the stool and by the determination of uroporphyrinogen synthase activity in the erythrocytes. PMID:117341

A small library of boron-cluster- and metallacarborane-cluster-based ligands was designed, prepared, and tested for isoform-selective activation or inhibition of the three nitric oxide synthase isoforms. On the basis of the concept of creating a hydrophobic analogue of a natural substrate, a stable and nontoxic basic boron cluster system, previously used for boron neutron capture therapy, was modified by the addition of positively charged moieties to its periphery, providing hydrophobic and nonclassical hydrogen bonding interactions with the protein. Several of these compounds show efficacy for inhibition of NO synthesis with differential effects on the various nitric oxide synthase isoforms. PMID:23075390

Inorganic nitrogen metabolism in the obligate anaerobic thermophiles Chlostridium thermosaccharolyticum and Clostridium thermoautotrophicum differs in several respects. C. thermosaccharolyticum contains a nitrogenase as inferred from NH4+ repressible C2H2 reduction, a glutamine synthetase which is partially repressed by ammonium, very labile glutamate synthase activities with both NADH and NADPH, NADPH-dependent glutamate dehydrogenase, and NH4+-dependent asparagine synthetase. C. thermoautotrophicum contains no nitrogenase, but glutamine synthetase, no glutamate synthase, no glutamate dehydrogenase, but a NADH-dependent alanine dehydrogenase and a NH4+-dependent asparagine synthetase. PMID:2870691