Purification and N-terminal amino acid sequence of solanapyrone synthase, a natural Diels-Alderase from Alternaria solani

The first natural Diels-Alderase, solanapyrone synthase, was purified 1,630-fold from a crude extract. The 41-kDa protein on SDS-polyacrylamide gel electrophoresis was identified as truncated solanapyrone synthase, and its N-terminal amino acid sequence was found to be QETQNLNNFLESNAINP.     

Isolation of Solanapyrones A, B and C from Culture Filture and Spore Germination Fluids of Ascochyta rabiei and Aspects of Phytotoxin Action

Nine isolates of the fungus Ascochyta rabiei have been assayed for their ability to produce solanapyrone toxins. All isolates formed solanapyrone A, B and C which were secreted into the culture medium. Pronounced production of the toxins only occurred after onset of sporulation. The identification of the fungal products was achieved by cochromatography (TLC, HPLC), ¹H-NMR (solanapyrone A and B) and mass spectrometry (solanapyrone B). Work with A. rabiei isolate X showed that cultivation in chickpea seed extract medium in a surface culture provided best conditions for maximal toxin production. The accumulation of solanapyrones over the growth cycle was monitored. Germinating spores produced solanapyrones C and B whereas solanapyrone A was formed from the 6th day of the culture period on. Application of a mixture of solanapyrones A, B and C to leaflets of intact plants from an A. rabiei resistant cultivar (ILC 3279) and a susceptible cultivar (ILC 1929) led to characteristic changes in leaf morphology which had earlier been obsevad in susceptible plants following infection with spores of A. rabiei. Attempts to demonstrate the occurrence of toxins in the infected leaf were unsuccessful. Application of solanapyrones to solanapyrones to chickpea cell suspension cultures (derived from both cultivars) led to pronounced losses in viability and to plasmolysis of cells.     

The effect of essential oil of Ammoides pusilla (brot.) breistr on the growth and the production of solanapyrone a by Ascochyta rabiei

Biological control such as the use of plant extracts has emerged as promising option to the phenomena of fungi resistance to chemical. Several constituent of essential oil have been studied for their biological activity including antibacterial and antifungal activity. In this study the effect of Ammoides pusilla essential oil with different concentrations was test against the growth of Ascochyta rabiei and the production of solanapyrone A by the fungus. After 14 days the mycelium was collected and the dry weight measured. A. rabiei did not grow at a final concentration of 6 and 3 mg/ml, at 1.5 mg/ml and 0.625ml there was little growth of the fungus with a dry weight of 55 mg and 99 mg respectively compared to the control with 519 mg dry weight, but there was no solanapyrone A produced. However a new compound appeared at the HPLC at 10 min. 30 sec. compared with the solanapyrone A which elutes at nearly 14 minutes.     

The effect of solanapyrone a produced by Ascochyta rabiei on seed germination and the elongation of radicles and hypocotyls of chickpea (Cicer areitinum L.)

Three Algerian isolates of A. rabiei (72, Mat 1.2 and 9216) were grown on Czapek Dox medium supplemented with cations and incubated for 14 days. After incubation, the mycelium of the fungus was removed by filtration through four layers of muslin cloth and spores were removed from the filtrate by centrifugation at 10,000 g for 20 min. Solanapyrone A was partially purified by liquid phase extraction into ethyl acetate and, after removal of the ethyl acetate, the toxin samples were dissolved in methanol and quantified by analytical High Performance Liquid Chromatography (HPLC). Solanapyrone A was identified by superimposition of its UV spectrum, obtained from the diode array detector of the HPLC, on the spectrum of an authentic sample. The action of solanapyrone A solution on seed germination and elongation of radicles and hypocotyls was tested using a concentration of 18.2 microg/ml and a two-fold dilution series of this solution in distilled water. The three Isolates, 72, Mat1.2 and 9216 produced solanapyrone A at concentrations of 37.2, 14.2 and 11.09 microg/ml, respectively. When probit % inhibition of seed germination was plotted against log2 of solanapyrone A concentration, there was a linear relationship and the EC50 concentration was determined as 7.2 microg/ml. Similarly, when radicle and hypocotyl elongation was plotted against log2 of solanapyrone A concentration, both gave linear relationships and the EC50 concentrations were determined as 5.37 and 6.02 microg/ml, respectively. It was concluded that solanapyrone A has a considerable inhibition of chickpea. However radicles and hypocotyls were susceptible than seed germination.     

Reduction in solanapyrone phytotoxin production by Ascochyta rabiei transformed with Agrobacterium tumefaciens

Agrobacterium tumefaciens was used to transform Ascochyta rabiei, the causal agent of chickpea blight. Employing a T-DNA containing a hygromycin resistance gene (hph), 908 transformants were obtained from germinated pycnidiospores on a selective medium containing hygromycin. Transformants were confirmed using PCR and Southern analyses and of four of these that were tested, two had integrated multicopies of the hph gene, one had two copies and one had a single insertion. Transformants were tested for the production of solanapyrone A toxin using a microtitre plate assay. Loss of toxin production by transformants was confirmed by reversed phase high-performance liquid chromatography. Sixteen transformants out of 668 tested produced significantly less solanapyrone A than the wild-type strain.     

Reconstitution of biosynthetic machinery of fungal polyketides: unexpected oxidations of biosynthetic intermediates by expression host

Reconstitution of whole biosynthetic genes in Aspergillus oryzae has successfully applied for total biosynthesis of various fungal natural products. Heterologous production of fungal metabolites sometimes suffers unexpected side reactions by host enzymes. In the studies on fungal polyketides solanapyrone and cytochalasin, unexpected oxidations of terminal olefin of biosynthetic intermediates were found to give one and four by-products by host enzymes of the transformants harboring biosynthetic genes. In this paper, we reported structure determination of by-products and described a simple solution to avoid the undesired reaction by introducing the downstream gene in the heterologous production of solanapyrone C.     

A plant phytotoxin, solanapyrone A, is an inhibitor of DNA polymerase beta and lambda.

Solanapyrone A, a phytotoxin and enzyme inhibitor isolated from a fungus (SUT 01B1-2) selectively inhibits the activities of mammalian DNA polymerase beta and lambda (pol beta and lambda) in vitro. The IC50 values of the compound were 30 microm for pol beta and 37 microm for pol lambda. Because pol beta and lambda are in a family and their three-dimensional structures are thought to be highly similar to each other, we used pol beta to analyze the biochemical relationship with solanapyrone A. On pol beta, solanapyrone A antagonistically competed with both the DNA template and the nucleotide substrate. BIAcore analysis demonstrated that solanapyrone A bound selectively to the N-terminal 8-kDa domain of pol beta. This domain is known to bind single-stranded DNA, provide 5'-phosphate recognition of gapped DNA, and cleave the sugar-phosphate bond 3' to an intact apurinic/apyrimidinic (AP) site (i.e. AP lyase activity) including 5'-deoxyribose phosphate lyase activity. Solanapyrone A inhibited the single-stranded DNA-binding activity but did not influence the activities of the 5'-phosphate recognition in gapped DNA structures and the AP lyase. Based on these results, the inhibitory mechanism of solanapyrone A is discussed.     

Production of Ascochyta rabiei lacking solanapyrone A toxin production

Ascochyta rabiei, agent of Ascochyta blight of chickpea produces three toxins, Solanapyrones A, B and C of which solanapyrone A is the most toxic. All isolates of the fungus so far examined produce at least one of the Solanapyrone toxins, usually Solanapyrone A. The universality of solanapyrone production argues strongly for the importance of the toxins in virulence or pathogenicity. However, further evidence for this awaits the development of mutants lacking toxin production. Generation and isolation of fungal mutants defective in pathogenicity has been very useful for understanding the genetic and enzymatic processes responsible for infectivity in a number of pathosystems. Numerous tools have been used to transform plants and micro-organisms but the most widely micro-organism employed is Agrobacterium tumefaciens. In the present experiments, two strains of A. tumefaciens, AGL1 and LBA1126, harbouring two different plasmids, both encoding a gene for hygromycin resistance in the T-DNA region were used to transform isolate Tk21 of A. rabiei. The transformation of Ascochyta rabiei, gave rise to 498 colonies which grew on media supplemented with the selective agent; hygromycin B. The 30 sporulated transformants produced solanapyrone A on the specific medium at different rates. Solanapyrone A production, as demonstrated by the absorption of light at 327 nm, varied from 2.11 microg/ml to 4.32 microg/ml, representing a reduction of 74.11% to 46.99% in comparison with the wild type (8.15 microg/ml).     

Effect of growth phase on phospholipid biosynthesis in Saccharomyces cerevisiae.

The effect of growth phase on the membrane-associated phospholipid biosynthetic enzymes CDP-diacylglycerol synthase, phosphatidylserine synthase, phosphatidylinositol synthase, and the phospholipid N-methyltransferases in wild-type Saccharomyces cerevisiae was examined. Maximum activities were found in the exponential phase of cells grown in complete synthetic medium. As cells entered the stationary phase of growth, the activities of the CDP-diacylglycerol synthase, phosphatidylserine synthase, and the phospholipid N-methyltransferases decreased 2.5- to 5-fold. The subunit levels of phosphatidylserine synthase and the cytoplasmic-associated enzyme inositol-1-phosphate synthase were not significantly affected by the growth phase. When grown in medium supplemented with inositol-choline, cells in the exponential phase of growth had reduced CDP-diacylglycerol synthase, phosphatidylserine synthase, and phospholipid N-methyltransferase activities, with repressed subunit levels of phosphatidylserine synthase and inositol-1-phosphate synthase compared with cells grown without inositol-choline. Enzyme activity levels remained reduced in the stationary phase of growth of cells supplemented with inositol-choline. The phosphatidylserine synthase and inositol-1-phosphate synthase subunit levels, however, were depressed. Phosphatidylinositol synthase (activity and subunit) was not affected by growth in medium supplemented with or without inositol-choline or the growth phase of the culture. The phospholipid composition of cells in the exponential and stationary phase of growth was also examined. The phosphatidylinositol to phosphatidylserine ratio doubled in stationary-phase cells. The phosphatidylcholine to phosphatidylethanolamine ratio was not significantly affected by the growth phase of cells.     

Purification and characterization of an unusually large fatty acid synthase from Mycobacterium tuberculosis var. bovis BCG.

Fatty acid synthase was purified from Mycobacterium tuberculosis var. bovis BCG. The method developed gave a 23% yield of the synthase and also yielded purified mycocerosic acid synthase. The fatty acid synthase is of unusually large size and composed of two 500-kDa monomers. The amino acid composition of the two synthases was not identical; the N-terminus of the fatty acid synthase was blocked, whereas that of the mycocerosic acid synthase was not. Western blot analysis of crude mycobacterial extracts with polyclonal antibodies prepared against each synthase showed a single band in each case with no cross-reactivity with the other synthase. Fatty acid synthase required both NADH (Km, 11 microM) and NADPH (Km, 14 microM). The Km for acetyl-CoA and malonyl-CoA were 5 and 6 microM, respectively. Fatty acids were released from the synthase as CoA esters. A bimodal distribution of fatty acids was obtained at around C16 and C26. The primer utilization also reflects the de novo synthesis and elongation capabilities of the enzyme; acetyl-CoA was the preferred primer but CoA esters up to C8 but not C12 and C14 could serve as primers, whereas C16 was readily used as a primer for elongation. Addition of CoA and CoA ester-binding oligosaccharides caused enhanced release of C16. Since this mycobacterial fatty acid synthase is twice as large as other multifunctional fatty acid synthases, it is tempting to suggest that this synthase represents a head to tail fusion of two fatty acid synthase genes coding for a double size protein with one-half producing C16 acid and the other elongating the C16 acid to a C26 acid. The monomer of fatty acid synthase from M. smegmatis was immunologically similar and equal in size to the synthase from M. tuberculosis.     

Regulation of renal glycogen synthase interconversion of two forms in vitro

Glycogen synthase (UDP glucose: glycogen α-4-glucosyltransferase, EC 2.4. 1.11) from rat kidney was stimulated 4- to 5-fold by glucose 6-phosphate. The for glucose 6-phosphate stimulation was about 0.45 mM. Glycogen synthase was not evenly distributed throughout the kidney. Total synthase activity was greatest in the outer cortex and cortico-medullary junction and least in the inner medulla. Glucose 6-phosphate stimulation was greatest in the outer cortex and least in the inner medulla. Glycogen synthase in crude homogenates was not complexed with glycogen and eluted from Sepharose 6-B with an apparent molecular weight of about 390 000.Renal glycogen synthase appeared to exist in two interconvertible forms, synthase I (activity in the absence of glucose 6-phosphate) and synthase D (requires glucose 6-phosphate for activity). The conversion of synthase D to I (synthase D phosphatase) was inhibited by F⁻, glycogen, ATP, Mn²⁺, and Co²⁺. The conversion was not altered by mercaptoethanol, AMP, Mg²⁺, or Ca²⁺. The conversion of synthase I to D (synthase I kinase) required ATP-Mg and was stimulated by cyclic AMP.It was suggested that the interconversion of renal glycogen synthase involved a phosphorylation-dephosphorylation. The significance of glycogen synthase interconversion to the regulation of renal glycogen synthesis is discussed.     

Regulation of glycogen synthase activity in muscle by a C-terminal part sequence of human growth hormone

The synthetic peptide hGH 177–191, corresponding to the last 15 residues at the carboxyl terminus of human pituitary growth hormone, promotes the conversion of glycogen synthase α to glycogen synthase b in muscle. When injected, the peptide was found to produce inactivation of glycogen synthase phosphatase activity in rat skeletal muscle. The time course of phosphatase inactivation was closely correlated with that for glycogen synthase. The peptide had no effect either on muscle 3′,5′-cyclic AMP levels or on synthase kinase activity. These results can be explained in terms of a dynamic cycle of interconversion of synthase between active and inactive forms, by the simultaneous action of synthase kinases and synthase phosphatases. A decrease in the ratio of phosphatase to kinase activity would result in a decrease in the steady-state level of synthase α activity.     

Phosphorylation of rat liver glycogen synthase bound to the glycogen particle

Rat liver glycogen synthase bound to the glycogen particle was partially purified by repeated high-speed centrifugation. This synthase preparation was labeled with ³²P by incubations with cAMP-dependent protein kinase and cAMP-independent synthase (casein) kinase-1 in the presence of [γ-³²P]ATP. The phosphorylated synthase was separated from other proteins in the glycogen pellet by immunoprecipitation with rabbit anti-rat liver glycogen synthase serum. Analysis of the immunoprecipitates by sodium dodecyl sulfate-gel electrophoresis showed that synthase subunits of M_r 85,000 and 80,000 were present in varying proportions. The ³²P-labeled synthase in the immunoprecipitate was digested with trypsin, and the resulting peptides were analyzed by isoelectric focusing. Synthase bound to the glycogen particle was phosphorylated by cAMP-dependent protein kinase at more sites and by cAMP-independent synthase (casein) kinase-1 at less sites than when the homogeneous synthase was incubated with these kinases. Phosphorylation of synthase in the glycogen pellet by either cAMP-dependent protein kinase or cAMP-independent synthase (casein) kinase-1 did not cause a significant inactivation as has been observed when the homogeneous synthase was incubated with these kinases. Inactivation of synthase in the glycogen pellet, however, can be achieved by the combination of both kinases. This inactivation appears to result from the phosphorylation of a new site by cAMP-independent synthase (casein) kinase-1 neighboring a site previously phosphorylated by cAMP-dependent protein kinase.     

Glycogen synthase phosphatase of rat liver. Its separation from phosphorylase phosphatase on DE-52 columns.

Glycogen synthase D was prepared from rat liver by chromatographing the glycogen pellet on DE-52 columns. It was free of glycogen and phosphorylase and converted readily into synthase I upon incubation with glycogen synthase phosphatase. With this synthase D as substrate, the identity of rat liver glycogen synthase phosphatase was studied by means of DE-52 column chromatography. Under the conditions developed, synthase phosphatase emerged from the columns as a sharp, single peak, and phosphorylase phosphatase came off later. The two phosphatases were also different from each other in stability, synthase phosphatase being less stable than phosphorylase phosphatase.     

Higher plants express 3-deoxy-D-manno-octulosonate 8-phosphate synthase

Abstract. The enzymatic activity of 3-deoxy- D-manno -octulosonate 8-phosphate (KDOP) synthase was detected in eight diverse plant species, thus providing enzymological data consistent with recent reports of the presence of 3-deoxy- D-manno -octulosonate in plant cell walls. KDOP synthase from spinach was partially purified and characterized. It possessed weak activity as 3-deoxy- D-arabino -heptulosonate 7-phosphate (DAHP) synthase. In the presence of phosphoenolpyruvate, which conferred dramatic thermostability, KDOP synthase had a catalytic temperature optimum of about 53°C. The pH optimum was 6.2, and divalent cations were neither stimulatory nor required for activity. The Km values for arabinose 5-P and phosphoenolpyruvate were 0.27 mol m⁻³ and about 35 mmol m⁻³, respectively. The kinetics of periodate oxidation of KDOP formed by spinach KDOP synthase indicate that the same stereochemical configuration exists as with bacterial KDOP. The possibility that an unregulated species of DAHP synthase found in some bacteria might in fact be a KDOP synthase exhibiting substrate ambiguity of the type seen in higher plants was examined. However, the DAHP synthase isozyme, DS-O, from Acinetobacter calcoaceticus was found to be specific for erythrose 4-P. The KDOP synthase of Acinetobacter calcoaceticus was also found to be specific for arabinose 5-P.     

Phosphatidylglycerophosphate synthease and phosphatidylserine synthase activites in Clostridium perfringens

Cytidine 5'-diphospho (CDP)-1,2-diacyl-sn-glycerol (CDPdiacylglycerol):sn-glycerol-3-phosphate phosphatidyltransferase (EC 2.7.8.5, phosphatidylglycero-P synthase) and CDPdiacylglycerol:L-serine O-phosphatidyltransferase (EC 2.7.8.8, phosphatidylserine synthase) activities were identified in the cell envelope fraction of the gram-positive anaerobe Clostridium perfringens. The association of phosphatidylglycero-P synthase and phosphatidylserine synthase with the cell envelope fraction of cell-free extracts was demonstrated by sucrose density gradient centrifugation, by both activities sedimenting with the 100,000 x g pellet and solubilization of both activities from the 100,000 x g pellet with Triton X-100. The pH optimum for both enzyme activities was 8.0 with tris(hydroxy-methyl)aminomethane-hydrochloride buffer. Phosphatidylglycero-P synthase activity was dependent on magnesium ions (100 mM). Phosphatidylserine synthase activity was dependent on manganese (0.1 mM) or magnesium ions (50 mM). Both enzyme activities were dependent on the addition of the nonionic detergent Triton X-100. Maximum phosphatidylglycero-P synthase and phosphatidylserine synthase activities were obtained when the molar ratio of Triton X-100 to CDP-diacylglycerol was 50:1 and 12:1, respectively. The Km for sn-glycero-3-P in the phosphatidylglycero-P synthase reaction was 0.1 mM. The Km for L-serine in the phosphatidylserine synthase reaction was 0.15 mM. Both enzyme activities were 100% stable for at least 20 min at 60 degrees C.     

Prostaglandin endoperoxide synthase substituted with manganese protoporphyrin IX. Formation of a higher oxidation state and its relation to cyclooxygenase reaction.

The heme in prostaglandin endoperoxide synthase (PGH synthase) was substituted with Mn(III)-protoporphyrin IX. The resulting enzyme, Mn-PGH synthase, showed full cyclooxygenase activity but only 0.9% of the peroxidase activity of the native iron enzyme. During the reaction with exogenous or endogenously produced hydroperoxides, a spectral intermediate of Mn-PGH synthase was observed. The electronic absorption bands of the resting enzyme at 376, 472, and 561 nm decreased, and the intermediate's bands at 417, around 513, and 625 nm appeared. The rate constant of the formation of the intermediate was about 10(4) M-1.s-1 at 22 degrees C, three orders of magnitude lower than with the iron enzyme. Spectral properties, conditions of formation, and the suppressed formation in the presence of electron donors provide evidence for a higher oxidation state of Mn-PGH synthase, tentatively a Mn(IV) species. This species was assigned to an intermediate in the peroxidase reaction of Mn-PGH synthase, the low activity of which was explained by the rate-limiting slow reaction of Mn-PGH synthase with hydroperoxides. The findings and interpretation are consistent with the published properties of other manganese-substituted peroxidases. Although the cyclooxygenase activity was similar to that of Fe-PGH synthase, the cyclooxygenase reaction of Mn-PGH synthase showed distinct differences in comparison with Fe-PGH synthase. A longer activation phase was observed which resembled the time course of the formation of the higher oxidation state. Glutathione peroxidase with glutathione, a hydroperoxide-scavenging system, inhibited the cyclooxygenase of Mn-PGH synthase at concentrations where the activity of Fe-PGH synthase was not affected. It is demonstrated that Mn-PGH synthase requires higher concentrations of hydroperoxides for the activation of the cyclooxygenase. These findings suggest that the substitution of iron with manganese in PGH synthase does not change the mechanism of the enzyme. The main difference is the much lower rate of the reaction with hydroperoxides which affects both the peroxidase activity and the hydroperoxide-dependent activation of the cyclooxygenase. A reaction scheme for Mn-PGH synthase is proposed analogous to that suggested for Fe-PGH synthase (Karthein, R., Dietz, R., Nastainczyk, W., and Ruf, H. H. (1988) Eur. J. Biochem. 171, 313-320).