Total synthesis of aurachins C,D, and L,and a structurally simplified analog of aurachin C

The quinoline antibiotics aurachins C, D, and L, and a structurally simplified analog of aurachin C were synthesized from 1-(2-nitrophenyl)butane-1,3-dione via reductive cyclizations of δ-nitro ketone intermediates, with zinc or iron as key steps. The results of antimicrobial tests indicate that the N-hydroxyquinolone nucleus mimics the electron carrier in the respiratory chain more strongly than the quinoline N-oxide nucleus.     

Structure of the quinoline N-hydroxylating cytochrome P450 RauA,an essential enzyme that confers antibiotic activity on aurachin alkaloids

The cytochrome P450 RauA from Rhodococcus erythropolis JCM 6824 catalyzes the hydroxylation of a nitrogen atom in the quinolone ring of aurachin, thereby conferring strong antibiotic activity on the aurachin alkaloid. Here, we report the crystal structure of RauA in complex with its substrate, a biosynthetic intermediate of aurachin RE. Clear electron density showed that the quinolone ring is oriented parallel to the porphyrin plane of the heme cofactor, while the farnesyl chain curls into a U-shape topology and is buried inside the solvent-inaccessible hydrophobic interior of RauA. The nearest atom from the heme iron is the quinolone nitrogen (4.3 Å), which is consistent with RauA catalyzing the N-hydroxylation of the quinolone ring to produce mature aurachin RE.     

Aaptolines A and B,Two New Quinoline Alkaloids from the Marine Sponge Aaptos aaptos

Two new quinoline alkaloids, aaptolines A and B, were isolated from the marine sponge Aaptos aaptos. Their structures were determined by HR‐ESI‐MS data, NMR analysis, and X‐ray crystallography. Structurally, aaptoline A is characterized as having a quinoline skeleton fused with a 1,4‐dioxane motif at the C(7)?C(8) position, whereas aaptoline B possessed an intriguing 1H‐pyrrolo[2,3‐g]quinoline moiety. The cytotoxic assay of these compounds showed no cytotoxicity towards HepG2, A549, and PC9 cancer cell lines and had IC₅₀ values greater than 20 μm .     

Completing the puzzle of aurachin biosynthesis in Stigmatella aurantiaca Sg a15

The aurachins are a family of secondary metabolites, with the main members aurachin A, B, C, and D, produced by the myxobacterium Stigmatella aurantiaca Sg a15. These isoprenoid quinoline alkaloids are classified as A-type or C-type aurachins according to the position of the farnesyl residue either at C4 or C3 of the quinoline core, respectively. Previous feeding studies revealed that the C-type aurachins are converted to A-type aurachins by late stage tailoring reactions. While the core gene cluster coding for the functionalities required for the biosynthesis of the basic structure aurachin D is known, neither of the genes encoding for the successively acting tailoring enzymes was known up to date, which was assumed to be due to a split cluster organisation. Here we describe the identification of a total of five genes, located upstream of the aurachin core cluster and at additional two loci elsewhere in the genome, encoding for the aforementioned functionalities. The generation and evaluation of respective inactivation mutants of S. aurantiaca Sg a15 allowed for the first time to propose an exhaustive model for aurachin biosynthesis. One of the deduced biosynthetic transformations corresponds to a pinacol rearrangement, an unprecedented tailoring reaction in secondary metabolite biosynthesis.     

Effect of heat shock on protein synthesis in Locusta migratoria epidermis

Heat shock induced by an increase in temperature from 30°C to 47°C led to changes in protein synthesis in wing pads of the fifth larval instar of Locusta migratoria. Synthesis of heat shock proteins in the molecular weight range of 85,000, 70,000 and 18,000–22,000 was first detected at a threshold temperature of 45°C and was found to be highest at 47°C. A marked decline in the synthesis of many other proteins was also evident at 47°C. Recovery of general protein synthesis was observed when wing pads were shifted back to 30°C after a 2-h heat shock at 47°C. Heat shock protein patterns in Locusta and Drosophila were compared.     

Differential chromosomal and mitochondrial DNA synthesis in temperature-sensitive mutants ofUstilago maydis

Summary The amount and type of residual DNA synthesis was determined in eight temperature-sensitive mutants of the smut fungusUstilago maydis after incubation at the restrictive temperature (32° C) for eight hours. Mutantsts-220,ts-207,ts-432 andts-346 were found to have an overall reduction in the synthesis of both nuclear and mitochondrial DNA in comparison to the wild-type. In mutantsts-20,tsd 1-1,ts-84 andpol 1-1 nuclear DNA synthesis was depressed relative to mitochondrial synthesis. The DNA-polymerase mutantpol 1-1 had persistent nuclear synthesis at about 50% of the rate of synthesis of mitochondrial DNA and similar behavior was observed in a diploid homozygous strain. Mutantts-84 had an initial burst of DNA synthesis which was reduced for nuclear but not mitochondrial synthesis after three hours preincubation at 32°C.tsd 1-1 andts-20 had nuclear residual synthesis amounting to about 25% of the relative rate of mitochondrial synthesis with correlates to increasing UV sensitivity of these strains on incubation at 32° C. Apol 1-1ts-84 double mutant had an additive loss of nuclear DNA synthesis which indicates that the steps of replication involved may be sequential.     

DNA supercoiling and thermal regulation of unsaturated fatty acid synthesis in Bacillus subtilis

Bacillus subtilis growing at 37° C synthesizes, almost exclusively, saturated fatty acids. However, when a culture growing at 37°C is transferred to 20°C, the synthesis of unsaturated fatty acids is induced. The addition of the DNA gyrase inhibitor novobiocin specifically prevented the induction of unsaturated fatty acid synthesis at 20° C. Furthermore, it was determined that plasmid DNA isolated from cells growing at 20°C was significantly more negatively supercoiled than the equivalent DNA isolated from cells growing at 37°C. The overall results agree with the hypothesis that an increase in DNA supercoiling associated with a temperature downshift could regulate the unsaturated fatty acids synthesis in B. subtilis.     

The quinone-binding sites of the cytochrome bo3 ubiquinol oxidase from Escherichia coli

Cytochrome bo₃ is the major respiratory oxidase located in the cytoplasmic membrane of Escherichia coli when grown under high oxygen tension. The enzyme catalyzes the 2-electron oxidation of ubiquinol-8 and the 4-electron reduction of dioxygen to water. When solubilized and isolated using dodecylmaltoside, the enzyme contains one equivalent of ubiquinone-8, bound at a high affinity site (Q_H). The quinone bound at the Q_H site can form a stable semiquinone, and the amino acid residues which hydrogen bond to the semiquinone have been identified. In the current work, it is shown that the tightly bound ubiquinone-8 at the Q_H site is not displaced by ubiquinol-1 even during enzyme turnover. Furthermore, the presence of high affinity inhibitors, HQNO and aurachin C1–10, does not displace ubiquinone-8 from the Q_H site. The data clearly support the existence of a second binding site for ubiquinone, the Q_L site, which can rapidly exchange with the substrate pool. HQNO is shown to bind to a single site on the enzyme and to prevent formation of the stable ubisemiquinone, though without displacing the bound quinone. Inhibition of the steady state kinetics of the enzyme indicates that aurachin C1–10 may compete for binding with quinol at the Q_L site while, at the same time, preventing formation of the ubisemiquinone at the Q_H site. It is suggested that the two quinone binding sites may be adjacent to each other or partially overlap.     

EVIDENCE FOR THE BIOSYNTHESIS OF MANNOSYLPHOSPHORYLDOLICHOL AND N-ACETYLGLUCOSAMINYLPYROPHOSPHORYLDOLICHOL BY AN AXOLEMMA-ENRICHED MEMBRANE PREPARATION FROM BOVINE WHITE MATTER

An axolemma-enriched membrane fraction prepared by an improved procedure from bovine white matter catalyzes the enzymatic transfer of [¹⁴C]mannose and N-acetyl[¹⁴C]glucosamine from their nucleotide derivatives into a mannolipid and an N-acetylglucosaminyl lipid in the presence of exogenous dolichyl monophosphate. The labeled glycolipid products have the chemical and chromatographic characteristics of mannosylphosphoryldolichol and N-acetylglucosaminylpyrophosphoryldolichol. The initial rates of synthesis of the glycolipids by the axolemma-enriched membrane fraction have been compared with the initial rates of glycolipid formation catalyzed by a microsomal preparation and myelin in the presence or absence of dolichyl monophosphate. Essentially no glycolipid synthesis was observed when either GDP-[¹⁴C]mannose or UDP-N-acetyl[¹⁴C]glucosamine were incubated with myelin in the presence or absence of exogenous dolichyl monophosphate. A comparison of the initial rates of synthesis of the glycolipids using endogenous acceptor lipid revealed that the rate of formation of mannolipid was 7 times faster for the microsomal membranes than the axolemma-enriched membranes. In the presence of an amount of dolichyl monophosphate approaching saturation the initial rate of glycolipid synthesis was markedly enhanced for both membrane preparations. However, due to a more dramatic enhancement in the axolemma-enriched membranes the initial rate of mannolipid synthesis was only approx. 2.5 times greater in the microsomal membranes. A similar observation was made when the initial rates of N-acetylglucosaminyl lipid synthesis were compared for axolemma-enriched and microsomal preparations in the presence and absence of exogenous dolichyl monophosphate. These studies indicate that the axolemma-enriched membranes have a relatively lower content of dolichyl monophosphate than the microsomal membranes although the difference in the amount of mannosyltransferase is only two to three-fold lower. The presence of a sugar nucleotide pyrophosphatase activity capable of degrading GDP-mannose and UDP-N-acetylglucosamine has also been demonstrated in the axolemma-enriched membrane fraction.     

In situ and in vitro colonization of Cathaya argyrophylla (Pinaceae) by ectomycorrhizal fungi

Cathaya argyrophylla, a critically endangered conifer, is found to grow at four isolated areas located in subtropical mountains of China. To examine the involvement and usefulness of mycorrhizas for sustaining the population of this tree, we compared the root system, morphology, and structure of mycorrhizal roots of C. argyrophylla, which were collected from a natural stand and an artificial stand, each grown at a different location. More mycorrhizal roots were found for trees from an artificial stand. The presence of extramatrical mycelium, mantle, and Hartig net revealed that C. argyrophylla formed an ectomycorrhizal association in both sampling sites. Starch granules were found in mycorrhizal roots collected only from a natural stand. The aseptic synthesis of C. argyrophylla and Cenococcum geophilum was established for the first time in vitro. Typical ectomycorrhizas formed on seedlings on RM medium containing 0.1 g/l glucose, 5 weeks after inoculation. By light microscopy, the synthesized mycorrhizas showed a thin mantle from which emanated extramatrical hyphae and highly branched Hartig net. A simple, rapid, and convenient mycorrhiza synthesis system was developed, which facilitates further studies on ectomycorrhizal development of C. argyrophylla.     

In VivoC NMR Study of the Bidirectional Reactions of the Wood–Werkman Cycle and around the Pyruvate Node in Propionibacterium freudenreichii subsp. shermanii and Propionibacterium acidipropionici

This study used in vivo¹³C NMR spectroscopy to directly examine bidirectional reactions of the Wood–Werkman cycle involved in central carbon metabolic pathways of dairy propionibacteria during pyruvate catabolism. The flow of [2-¹³C]pyruvate label was monitored on living cell suspensions of Propionibacterium freudenreichii subsp. shermanii and Propionibacterium acidipropionici under acidic conditions. P. shermanii and P. acidipropionici cells consumed pyruvate at apparent initial rates of 161 and 39 μmol min⁻¹ g⁻¹ (cell dry weight), respectively. The bidirectionality of reactions in the first part of the Wood–Werkman cycle was evident from the formation of intermediates such as [3-¹³C]pyruvate and [3-¹³C]malate and of products like [2-¹³C]acetate from [2-¹³C]pyruvate. For the first time alanine labeled on C2 and C3 and aspartate labeled on C2 and C3 were observed during [2-¹³C]pyruvate metabolism by propionibacteria. The kinetics of aspartate isotopic enrichment was evidence for its production from oxaloacetate via aspartate aminotransferase. Activities of a partial tricarboxylic acid pathway, acetate synthesis, succinate synthesis, gluconeogenesis, aspartate synthesis, and alanine synthesis pathways were evident from the experimental results.     

Arginine metabolism in the sheep abomasal nematode parasites Haemonchus contortus and Teladorsagia circumcincta

The ornithine urea cycle, polyamine synthesis, nitric oxide synthesis and metabolism of arginine to putrescine have been investigated in L3 and adult Haemonchus contortus and Teladorsagia circumcincta. Neither parasite had a detectable arginine deiminase/dihydrolase pathway nor a functional ornithine urea cycle. Nitric oxide synthase was present in central and peripheral nerves, but was not detected in whole parasite homogenates. Both arginase (E.C. 3.5.3.1) and agmatinase (E.C. 3.5.3.11) activities were present in both species. Arginase did not require added Mn²⁺ and had an optimal pH of 8.5. Polyamine metabolism differed in the two species and from that in mammals. Ornithine decarboxylase (E.C. 4.1.1.17) was present in both parasites, but no arginine decarboxylase (E.C. 4.1.1.19) activity was detected in T. circumcincta. The flexibility of synthesis of putrescine in H. contortus may make this pathway less useful as a target for parasite control than in T. circumcincta, in which only the ornithine decarboxylase pathway was detected.     

Accumulation of glycinebetaine and its synthesis from radioactive precursors under salt-stress in the cyanobacterium Aphanothece halophytica

Growth in salt-stressed (2.0 M NaCl) Aphanothece halophytica was initially delayed during the first two days of cultivation and eventually attained the same growth rate as the control (0.5 M NaCl) cells. Glycinebetaine accumulation increased slightly in control cells but a dramatic increase of glycinebetaine occurred in salt-stressed cells during a growth period of six days. There was no apparent increase in the synthesis of [¹⁴C] glycinebetaine in the control cells, in contrast to the marked increase in its synthesis in the salt-stressed cells. Increasing NaCl concentration in the growth medium induced both the accumulation and the synthesis of glycinebetaine. Time course experiments provided evidence that [¹⁴C] choline was first oxidized to [¹⁴C] betaine aldehyde which was further oxidized to [¹⁴C] glycinebetaine in A. halophytica. The supporting data for such a pathway were obtained from the presence of choline and betaine aldehyde dehydrogenase activities found in the membrane and cytoplasmic fractions, respectively. The activities of these two enzymes were also enhanced upon increasing NaCl concentration in the growth medium from 0.5 M to 2.0 M. Under this condition an increaseof approximately 1.5-fold was observed for choline dehydrogenase activity as compared to 2.5-fold for betaine aldehyde dehydrogenase activity, suggesting a preferable induction of the latter enzyme by salt stress. A. halophytica was able to utilize [¹⁴C] ethanolamine and [¹⁴C] glycine for the synthesis of [¹⁴C] glycinebetaine. This revised version was published online in August 2006 with corrections to the Cover Date.     

A carotenoid synthesis gene cluster from Algoriphagus sp. KK10202C with a novel fusion-type lycopene β-cyclase gene

A carotenoid synthesis gene cluster was isolated from a marine bacterium Algoriphagus sp. strain KK10202C that synthesized flexixanthin. Seven genes were transcribed in the same direction, among which five of them were involved in carotenoid synthesis. This cluster had a unique gene organization, with an isoprenoid gene, ispH (previously named lytB), being present among the carotenoid synthesis genes. The lycopene β-cyclase encoded by the crtY _cd gene appeared to be a fusion of bacterial heterodimeric lycopene cyclase CrtY_c and CrtY_d. This was the first time that a fusion-type of lycopene β-cyclase was reported in eubacteria. Heterologous expression of the Algoriphagus crtY _cd gene in lycopene-accumulating Escherichia coli produced bicyclic β-carotene. A biosynthesis pathway for monocyclic flexixanthin was proposed in Algoriphagus sp. strain KK10202C, though several of the carotenoid synthesis genes not linked with the cluster have not yet been cloned.     

Fatty acid synthesis by slices from developing leaves

Fatty acid synthesis was studied in successive leaf sections from the base to the tip of developing barley (Hordeum vulgare L.), maize (Zea mays L.), rye grass (Lolium perenne L.) and wheat (Triticum aestivium L.) leaves. The basal regions of the leaves had the lowest rates of fatty acid synthesis and accumulated small amounts of very long chain fatty acids. Fatty acid synthesis was highest in the middle leaf sections in all four plants. Linolenic acid synthesis from [1-¹⁴C]acetate was highest in the distal leaf sections of rye grass. The labelling of the fatty acids of individual lipids of rye grass was examined and it was found that [¹⁴C]linolenic acid was highest in the galactolipids. Synthesis of this acid in the galactolipids was most active in leaf segment C. Only traces of [¹⁴C]linolenic acid were ever found in phosphatidylcholine and it is concluded that this phospholipid cannot serve as a substrate for linoleic acid desaturation in rye grass. The synthesis of fatty acids was sensitive to arsenite, fluoride and the herbicide EPTC. The latter was only inhibitory towards those leaf segments which made very long chain fatty acids. Formation of fatty acids from [1-¹⁴C]acetate was also studied in chloroplasts prepared from successive leaf sections of rye grass. Chloroplasts isolated from the middle leaf sections had the highest activity. Palmitic and oleic acids were the main fatty acid products in all chloroplast preparations. Linolenic acid synthesis was highest in chlorplasts isolated from the distal leaf sections of rye grass.     

Apparent endogenous circadian rhythm inent-kaurene biosynthesis

Net synthesis of [¹⁴C]ent-kaurene from [¹⁴C]2-mevalonic acid was assayed in cell-free enzyme extracts prepared from Alaska pea (Pisum sativum L.) seedlings throughout 44 h of a regimen consisting of a 16-h day and an 8-h night. Activities generally followed an upward trend during the dark period and a downward trend during the photoperiod. Activity was also assayed in enzyme extracts prepared at intervals during a 12-h photoperiod and a following, continuous 36-h dark period after entrainment of plants to a regimen of 12-h days and 12-h nights.Ent-kaurene synthesis activity again followed an upward trend in enzyme extracts prepared during what would have been the entrainment dark period, and a downward trend during the entrainment photoperiod. The apparent endogenous rhythm ofent-kaurene biosynthesis may have implications for the regulation of gibberellin biosynthesis.     

Microbial Metabolism of Quinoline by Comamonas sp.

An aerobic bacterial strain which can use quinoline as the sole carbon and energy source has been isolated from activated sludge and identified as Comamonas sp. The microbial metabolism of quinoline by this strain has been investigated. A pH 8 and a temperature of 30 °C were the optimum degradation conditions of quinoline. Five intermediates including 2-oxo-1,2-dihydroquinoline, 5-hydroxy-6-(2-carboxyethenyl)-1H-2-pyridone, 6-hydroxy-2-oxo-1,2-dihydroquinoline, 5,6-dihydroxy-2-oxo-1,2-dihydroquinoline, and 8-hydroxy-2-oxo-1,2-dihydroquinoline were found during quinoline biodegradation. The presence of these intermediates suggested that at least two pathways were involved for quinoline degradation by Comamonas sp. and a reasonable degradation route was proposed to account for the intermediates observed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Reversibility of DnaA protein activity in the'irreversible'dnaA204 mutant of Escherichia coli

The dnaA204 mutant, one of the so-called irreversible dnaA mutants which cannot reinitiate chromosome replication upon a shift from non-permissive to permissive growth temperature in the absence of protein synthesis, was reinvestigated using flow cytometry and marker frequency analysis. In a temperature downshift experiment and in the presence of protein synthesis the dnaA204 mutant reinitiates chromosome replication very fast. Using a lac promoter-controlled wild type or a dnaA204 mutant gene carried on a plasmid, we have observed instantaneous initiation of replication when synthesis of DnaA protein is induced in the dnaA204 mutant at 42δC. The data indicate that the dnaA204 mutant after a shift to 42δC still contains functional DnaA protein, but that the activity level is below the initiation threshold. Thus, after synthesis of very small amounts of additional DnaA protein, initiation occurs very fast both after a shift to 30δC, and after induction of DnaA protein synthesis at 42 C. A model describing the processing of DnaA protein in mutants and in the wild type Is presented.     

Trigonelline biosynthesis and the pyridine nucleotide cycle in Coffea arabica fruits: Metabolic fate of [carboxyl-C]nicotinic acid riboside

Trigonelline is a major component in coffee seeds and may contribute to the bitter taste of the resultant beverage. To determine the trigonelline biosynthetic pathway in coffee fruits, we investigated the metabolic fate of [carboxyl-¹⁴C]nicotinic acid riboside and in situ activity of related enzymes. Exogenously supplied [carboxyl-¹⁴C]nicotinic acid riboside was rapidly converted to nicotinic acid mononucleotide and was utilized for NAD synthesis. Nicotinic acid riboside was also used for trigonelline synthesis, but this process took longer than NAD synthesis. These results indicate that an efficient nicotinic acid riboside salvage system functions in coffee fruits, and that trigonelline is synthesized mainly from nicotinic acid produced by the degradation of NAD.     

Control of stable RNA synthesis in a temperature-sensitive mutant of elongation factor G of Bacillus subtilis

Summary A temperature-sensitive EFG mutant of Bacillus subtilis was isolated and characterized. This mutant, ts32, synthesizes stable RNA at 48° C with or at 50° C without accompanied protein synthesis. The initial rate of the RNA synthesis at 48° C or 50° C was 1.5 to 2.0 times as much as that at 30° C.This mutant as well as its parent (both leu ^-) showed stringent response for the RNA synthesis upon deprivation of amino acids with an accumulation of the MS nucleotides (pp Gpp and pppGpp). On raising temperature to 48° C or 50° C, the ts-cells immediately began to synthesize the stable RNA with an initial increase of the MS nucleotides. No drastic decrease in amount of the MS was observed during the active RNA synthesis.These results suggest that EFG is somehow involved in repressing the stable RNA synthesis, and have broken the close relationship between the stable RNA synthesis and the MS nucleotides hitherto reported.