Specificity and mutational analysis of the metal-dependent 3-deoxy-d-manno-octulosonate 8-phosphate synthase from Acidithiobacillus ferrooxidans

3-Deoxy-d-manno-octulosonate 8-phosphate synthase (KDO8PS) catalyzes the reaction between phosphoenol pyruvate and d-arabinose 5-phosphate to generate KDO8P. This reaction is part of the biosynthetic pathway to 3-deoxy-d-manno-octulosonate, a component of the lipopolysaccharide of the Gram-negative bacterial cell wall. Two distinct groups of KDO8PSs exist, differing by the absolute requirement of a divalent metal ion. In this study Acidithiobacillus ferrooxidans KDO8PS has been expressed and purified and shown to require a divalent metal ion, with Mn²⁺, Co²⁺ and Cd²⁺ (in decreasing order) being able to restore activity to metal-free enzyme. Cd²⁺ significantly enhanced the stability of the enzyme, raising the T_m by 14 °C. d-Glucose 6-phosphate and d-erythrose 4-phosphate were not substrates for A. ferrooxidans KDO8PS, whereas 2-deoxy-d-ribose 5-phosphate was a poor substrate and there was negligible activity with d-ribose 5-phosphate. The ²⁴³AspGlyPro²⁴⁵ motif is absolutely conserved in the metal-independent group of synthases, but the Gly and Pro sites are variable in the metal-dependent enzymes. Substitution of the putative metal-binding Asp243 to Ala in A. ferrooxidans KDO8PS gave inactive enzyme, whereas substitutions Asp243Glu or Pro245Ala produced active enzymes with altered metal-dependency profiles. Prior studies indicated that exchange of a metal-binding Cys for Asn converts metal-dependent KDO8P synthase into a metal-independent form. Unexpectedly, this mutation in A. ferrooxidans KDO8P synthase (Cys21Asn) gave inactive enzyme. This finding, together with modest activity towards 2-deoxy-d-ribose 5-phosphate suggests similarities between the A. ferrooxidans KDO8PS and the related metal-dependent 3-deoxy-d-arabino-heptulosonate phosphate synthase, and highlights the importance of the AspGlyPro loop in positioning the substrate for effective catalysis in all KDO8P synthases.     

A 96-well microtiter plate assay for high-throughput screening of Mycobacterium tuberculosis dTDP-d-glucose 4,6-dehydratase inhibitors

Mycobacterium tuberculosis dTDP-d-glucose 4,6-dehydratase (RmlB) is the second enzyme for the biosynthesis of dTDP-l-rhamnose, which is a sugar donor to the synthesis of the cell wall linker, d-N-acetylglucosamine-l-rhamnose. RmlB is essential to mycobacterial growth and is not found in humans; therefore, it is a potential target for developing new anti-tuberculosis drugs. So far, there has been no suitable method for high-throughput screening of RmlB inhibitors. Here, the recombinant M. tuberculosis RmlB was purified and an absorbance-based microtiter plate assay was developed for RmlB activity. It could be used for high-throughput screening of RmlB inhibitors. The kinetic properties of M. tuberculosis RmlB, including optimal pH, optimal temperature, the effect of metal ions, and the kinetic parameters, were determined with this assay. The inhibitory effects of dTTP and dTDP on M. tuberculosis RmlB were also studied with the assay.     

Effects of 2-deoxy-d-glucose on the glucose metabolism in Saccharomyces cerevisiae studied by multinuclear-NMR spectroscopy and biochemical methods

The effects of various concentrations of deoxyglucose (DG) on the aerobic metabolism of glucose in glucose-grown repressed Saccharomyces cerevisiae cells were studied at 30°C in a standard pyrophosphate medium containing 4.5 10⁷ cells/ml. ³¹P-nuclear magnetic resonance (NMR) spectroscopy was used to monitor DG phosphorylation and the formation of polyphosphates. The production of soluble metabolites of glucose was evaluated by ¹³C- and ¹H-NMR and biochemical techniques. The cells were aerobically incubated with 25 mM of glucose and various concentrations of DG (0, 5 and 10 mM) in order to determine the DG concentration leading to optimum of 2-deoxy-d-glucose 6-phosphate (DG6P) formation without over-inhibiting the synthesis of other metabolites. The production of DG6P increased by about 25% when the external DG concentration was doubled (from 5 to 10 mM). The formation of polyphosphates (polyP), on the other hand, was found to be mainly conditioned by the DG concentration. The amount of polyP decreased by a factor of four upon addition of 5 mM DG and became undetectable in the presence of 10 mM DG. The glucose consumption and the production of soluble metabolites of [1-¹³C]glucose were then evaluated as a function of time in both the absence and presence of 5 mM DG. The effect of DG is to decrease the glucose consumption and the formation of polyphosphates, ethanol, glycerol, trehalose, glutamate, aspartate and succinate while stimulating the formation of arginine and citrate. Upon co-addition of 25 mM glucose and 5 mM DG, the ratio between the initial rates of glucose consumption (0.16 mM/min) and DG6P production (0.027 mM/min) is about (5.9 ± 1.2), not very different from the ratio of the initial concentration of glucose and DG (= 5.0). Therefore, hexokinase can phosphorylate deoxyglucose as well as glucose. However, after 100 min of incubation, the glucose concentration in the external medium decreased by about 64% while only 10% of DG was phosphorylated. DG6P was formed and quickly reached the limiting value about 30 min after co-addition of glucose and DG. Nevertheless, when the maximum quantity of DG6P was obtained, the DG consumption became negligible. By contrast, the glucose consumption and the production of ethanol and glycerol, although substantially reduced by about 42%, varied linearly with time up to 80 min of incubation. Thus even in the presence of an excess of DG, glycolysis is only slowed but not gradually or completely inhibited by DG. The reasons why DG6P cannot accumulate indefinitely in cells are discussed, together with the reasons why the consumption of DG, but not glucose, becomes negligible after 30 min of incubation. In the absence of DG, the amount of polyphosphates (polyP) increased regularly with time as long as glucose was sufficiently present (≥ 5 mM) in the suspension. When glucose was exhausted, long chain polyphosphates disappeared to give rise, at first, to polyP with shorter chains and finally to inorganic phosphate. In the presence of 5 mM DG, the reduction in quantity of polyP can be explained by the fact that ATP, normally used for the polyP synthesis, is now diverted to phosphorylation of DG to DG6P. The presence of 5 mM DG also had significant effects on the glutamate C2, C3 and C4 signal intensity and the production of all aminoacids. The results seem to indicate that the enzymes involved in the Krebs cycle are also affected by the presence of DG.     

Isolation of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> (Bals.) Vuill. (Deuteromycotina: Hyphomycetes) from the Chagas disease vector, <Emphasis Type="Italic">Triatoma infestans</Emphasis> (Hemiptera: Reduviidae) in Argentina

A survey for natural entomopathogenic fungi of the Chagas disease vector Triatoma infestans was conducted in five provinces of Argentina since 2001. Nymphs (1.5%) and adults (3.3%) infected with a strain of the fungus Beauveria bassiana were found at Dean Funes, Córdoba province, Argentina. Field collected insects that died in the laboratory were maintained in moist chambers and incubated at 22 °C. Beauveria bassiana from infected insects was cultured on SDAY media. Pathogenicity tests were conducted with a conidial suspension (1 × 10⁷ conidia/ml) of this isolate on T. infestans adults. A mortality rate of 100% was obtained at 15 days post-infection. This is the first record of natural infection of T. infestans by B. bassiana.     

Prodigiosin is not a determinant factor in lysis of Leishmania (Viannia) braziliensis after interaction with Serratia marcescensd-mannose sensitive fimbriae

In this paper, the lytic activity of two variants of Serratia marcescens against promastigotes of Leishmania braziliensis was studied. In vitro assays showed that S. marcescens variant SM365 lyses L. braziliensis promastigotes, while the variant DB11 did not. Scanning electron microscopy (SEM) revealed that S. marcescens SM365 adheres to all cellular body and flagellum of the parasite. Several filamentous structures were formed and identified as biofilms. After 120 min incubation, they connect the protozoan to the developing bacterial clusters. SEM also demonstrated that bacteria, adhered onto L. braziliensis promastigote surface, formed small filamentous structures which apparently penetrates into the parasite membrane. d-mannose protects L. braziliensis against the S. marcescens SM365 lytic effect in a dose dependent manner. SM365 variant pre cultivated at 37 °C did not synthesize prodigiosin although the adherence and lysis of L. braziliensis were similar to the effect observed with bacteria cultivated at 28 °C, which produce high concentrations of prodigiosin. Thus, we suggest that prodigiosin is not involved in the lysis of promastigotes and that adherence promoted by bacterial mannose-sensitive (MS) fimbriae is a determinant factor in the lysis of L. braziliensis by S. marcescens SM365.     

Novel and facile synthesis of furanodictines A and B based on transformation of 2-acetamido-2-deoxy-d-glucose into 3,6-anhydro hexofuranoses

A novel synthesis of furanodictines A [2-acetamido-3,6-anhydro-2-deoxy-5-O-isovaleryl-d-glucofuranose (1)] and B [2-acetamido-3,6-anhydro-2-deoxy-5-O-isovaleryl-d-mannofuranose (2)] is described starting from 2-acetamido-2-deoxy-d-glucose (GlcNAc). The synthetic protocol is based on deriving the epimeric bicyclic 3,6-anhydro sugars [2-acetamido-3,6-anhydro-2-deoxy-d-glucofuranose (4) and 2-acetamido-3,6-anhydro-2-deoxy-d-mannofuranose (5)] from GlcNAc. Reaction with borate upon heating led to a facile transformation of GlcNAc into the desired epimeric 3,6-anhydro sugars. The C5 hydroxyl group of the 3,6-anhydro compounds 4 and 5 was regioselectively esterified with the isovaleryl chloride to complete the synthesis of furanodictines A and B, respectively. The targets 1 and 2 were synthesized in only two steps requiring no protection/deprotection.     

Synthesis of new N-substituted 3,4,5-trihydroxypiperidin-2-ones from d-ribono-1,4-lactone

d-Ribono-1,4-lactone was treated with ethylamine in DMF to afford N-ethyl-d-ribonamide 8a in quantitative yield. Using this reaction procedure, N-butyl, N-hexyl, N-dodecyl, N-benzyl, N-(3-methyl-pyridinyl)-, N-(2-hydroxy-ethyl)-, and N-(2-cyano-ethyl)-d-ribonamides 8b-h were obtained in quantitative yield. Bromination of the amides 8a-e with acetyl bromide in dioxane followed by acetylation gave 2,3,4-tri-O-acetyl-5-bromo-5-deoxy-N-ethyl, N-butyl, N-hexyl, N-dodecyl, and N-benzyl-d-ribonamides 9a-e in 40-54% yields. To obtain 2,3,4-tri-O-acetyl-5-bromo-5-deoxy-N-(3-methyl-pyridinyl)-, N-(2-hydroxy-ethyl)-, and N-(2-cyano-ethyl)-9f-h, the bromination is necessary before the amidation reaction. Treatment of the bromoamides 9a-h with NaH in DMF followed by methanolysis affords N-alkyl-d-ribono-1,5-lactams 12a-h in quantitative yield.     

Biological activity of l-2-azetidinecarboxylic acid,isolated from Polygonatum odoratum var. pluriflorum, against several algae

The biological activities of an aqueous fraction extracted from Polygonatum odoratum var. pluriflorum Owhi and of l-2-azetidinecarboxylic acid (AZC), purified from the extract, on the growth of several types of algae were tested. The aqueous fraction was prepared by methanol extraction of P. odoratum var. pluriflorum rhizomes followed by reverse partitioning with butanol. The aqueous extraction inhibited growth of the green alga Chlorella vulgaris by less than 10% at a concentration of 1000 mg L⁻¹. However, growth of the blue-green alga Microcystis aeruginosa was inhibited by 22.0%, 67.9%, and 87.1%, respectively, at 3.1, 6.2, and 12.5 mg extract L⁻¹. AZC was isolated from the aqueous extract and was shown to be the major active substance inhibiting algal growth. AZC concentrations higher than 25 μM inhibited growth, while at 400 μM, growth of the green algae C. vulgaris and Scenedesmus spp. was inhibited by 71.2% and 70.4%, respectively. In contrast, growth of the blue-green algae Anabaena affinis and M. aeruginosa was inhibited at concentrations greater than 1.6 and 0.2 μM, respectively, whereas 92% control required concentrations of 6.3 and 1.6 μM, respectively. AZC also suppressed the growth of the red-tide microalga Cochlodinium polykrikoides by 86.9% and 100% at concentrations of 6.3 and 12.5 μM, respectively. Azetidine and 2-azetidinone showed little activity on the tested algae. The results demonstrate that AZC selectively inhibits algal growth at low concentrations. The green algae C. vulgaris and Scenedesmus spp. were tolerant, whereas M. aeruginosa, A. affinis, and C. polykrikoides were relatively sensitive. Thus, extract and AZC, prepared from P. odoratum rhizomes, showed a potential as natural selective algicide for the control of harmful algae in laboratory assay.     

The Hypocrea jecorina (syn. Trichoderma reesei) lxr1 gene encodes a d-mannitol dehydrogenase and is not involved in l-arabinose catabolism

The Hypocrea jecorina LXR1 was described as the first fungal l-xylulose reductase responsible for NADPH dependent reduction of l-xylulose to xylitol in l-arabinose catabolism. Phylogenetic analysis now reveals that LXR1 forms a clade with fungal d-mannitol 2-dehydrogenases. Lxr1 and the orthologous Aspergillus nigermtdA are not induced by l-arabinose but expressed at low levels during growth on different carbon sources. Deletion of lxr1 does not affect growth on l-arabinose and l-xylulose reductase activity remains unaltered whereas d-mannitol 2-dehydrogenase activities are reduced. We conclude that LXR1 is a d-mannitol 2-dehydrogenase and that a true LXR1 is still awaiting discovery.     

A novel l-isoleucine hydroxylating enzyme, l-isoleucine dioxygenase from Bacillus thuringiensis, produces (2S,3R,4S)-4-hydroxyisoleucine

The unique function of 4-hydroxyisoleucine (4-HIL) is to stimulate glucose-induced insulin secretion in a glucose-dependent manner. 4-HIL is distributed only in certain kinds of plants and mushrooms, but the biosynthetic mechanism of 4-HIL has not been elucidated. Moreover, 4-HIL-producing microorganisms have not been reported. l-isoleucine (l-Ile) hydroxylating activity producing 4-HIL was detected in a cell lysate of Bacillus thuringiensis strain 2e2 AKU 0251 obtained from the mid-late exponential phase of growth. Properties of the purified hydroxylase demonstrated that it is a α-ketoglutaric acid (α-KG) dependent l-Ile dioxygenase (IDO) and requires α-KG, ferric ion, and ascorbic acid for its maximum activity. IDO showed high stereoselectivity in l-Ile hydroxylation producing only (2S,3R,4S)-4-HIL. The N-terminal 22 amino acids sequence revealed high homology to a hypothetical protein (GenBank ID: RBTH_06809) in B. thuringiensis serovar israelensis ATCC 35646. The histidine motif, which is conserved in α-KG dependent dioxygenases, is found in RBTH_06809.     

Structure of the O-polysaccharide from the lipopolysaccharide of Providencia alcalifaciens O31 containing an ether of d-mannose with (2R,4R)-2,4-dihydroxypentanoic acid

Recently, ether-linked diastereomeric 2,4-dihydroxypentanoic acids have been reported as new components of bacterial glycans [Shashkov, A. S. et al.Nat. Prod. Commun.2008, 3, 1625-1630]. In this work, an ether of (2R,4R)-2,4-dihydroxypentanoic acid (Dhpa) with d-mannose was identified in the O-polysaccharide of Providencia alcalifaciens O31, and the polysaccharide structure was elucidated. Studies by NMR spectroscopy confirmed the ether linkage between O-2 of Dhpa and O-4 of Man, and the absolute configuration of Man was determined after ether cleavage with boron trichloride. In the polysaccharide, Dhpa was found to exist partially in the form of 1,4-lactone. Using sugar and methylation analyses along with ¹H and ¹³C NMR spectroscopy, including 2D ¹H,¹H COSY, TOCSY, ROESY, H-detected ¹H,¹³C HSQC, and gHMBC experiments, the following structure of the tetrasaccharide repeating unit of the polysaccharide was established:     

Mutational analysis of N-glycosylation recognition sites on the biochemical properties of Aspergillus kawachii α-l-arabinofuranosidase 54

A role for N-linked oligosaccharides on the biochemical properties of recombinant α-l-arabinofuranosidase 54 (AkAbf54) defined in glycoside hydrolase family 54 from Aspergillus kawachii expressed in Pichia pastoris was analyzed by site-directed mutagenesis. Two N-linked glycosylation motifs (Asn⁸³–Thr–Thr and Asn²⁰²–Ser–Thr) were found in the AkAbf54 sequence. AkAbf54 comprises two domains, a catalytic domain and an arabinose-binding domain classified as carbohydrate-binding module 42. Two N-linked glycosylation sites are located in the catalytic domain. Asn⁸³, Asn²⁰², and the two residues together were replaced with glutamine by site-directed mutagenesis. The biochemical properties and kinetic parameters of the wild-type and mutant enzymes expressed in P. pastoris were examined. The N83Q mutant enzyme had the same catalytic activity and thermostability as the wild-type enzyme. On the other hand, the N202Q and N83Q/N202Q mutant enzymes exhibited a considerable decrease in thermostability compared to the glycosylated wild-type enzyme. The N202Q and N83Q/N202Q mutant enzymes also had slightly less specific activity towards arabinan and debranched arabinan. However, no significant effect on the affinity of the mutant enzymes for the ligands arabinan, debranched arabinan, and wheat and rye arabinoxylans was detected by affinity gel electrophoresis. These observations suggest that the glycosylation at Asn²⁰² may contribute to thermostability and catalysis.     

The Henry reaction of (1R)-(1,4:3,6-dianhydro-d-mannitol-2-yl)-1,4:3,6-dianhydro-d-fructose 5,5′-dinitrate. Different reactive features of nitromethane to nitroethane

Henry reactions of a novel higher sugar derivative, (1R)-(1,4:3,6-dianhydro-d-mannitol-2-yl)-1,4:3,6-dianhydro-d-fructose 5,5′-dinitrate (Alternate nomenclature: (1R)-(isomannid-2-yl)-1,4:3,6-dianhydro-d-fructose 5,5′-dinitrate), with nitromethane and nitroethane were studied. The kinetic and thermodynamic reactions with nitromethane under different conditions were carried out to afford (2S)- and (2R)-β-nitroalcohols, respectively. But when using nitroethane the reaction gave a (2S)-β-nitroalcohol with an inverted configuration at vicinal carbon C-1. Two stereogenic centers were generated, and one was altered in the reaction.     

Regioselective [3+2] cycloaddition of chalcones with a sugar azide: easy access to 1-(5-deoxy-d-xylofuranos-5-yl)-4,5-disubstituted-1H-1,2,3-triazoles

[3+2] Cycloaddition of 5-azido-5-deoxy-1,2-O-isopropylidene-α-d-xylofuranose with 1,3-diphenyl-prop-3-enones, followed by oxidation of the intermediate triazolines in a tandem manner, led to the regioselective formation of 4-benzoyl-1-(5-deoxy-1,2-O-isopropylidene-α-d-xylofuranos-5-yl)-5-phenyl-1H-1,2,3-triazoles in moderate to good yields.     

Effects of temperature on two Mediterranean population of Dinophilus gyrociliatus (Polychaeta: Dinophilidae): II. Effects on demographic parameters

The effects of temperature on demographic characteristics of two populations from Ravenna and Genoa of the polychaete Dinophilus gyrociliatus were investigated. Temperature affects age-specific survival and fecundity and all the demographic parameters often to a different degree in the two populations. Individuals from Ravenna survive longer than those from Genoa. The most evident differences in the age-specific fecundity curves of the experimental groups are related to age at maturity and the duration of the reproductive period that are in inverse proportion to temperature. In both populations of D. gyrociliatus, the maximum daily fecundity is observed at intermediate temperatures. In all cases, the Genoa females mature earlier, attain their maximum fecundity more quickly and have a shorter reproductive period than their Ravenna counterparts.Age at maturity, fecundity during the first reproductive events and juvenile survival are by far the most important characteristics in determining the fitness of the two populations at the tested temperatures. Even though the greatest net growth rates and highest expectation of life were recorded at 12 °C in the Ravenna population, the delay in the attainment of sexual maturity means that, at this temperature, the population growth rate is lowest. The higher juvenile survivorship and the greater fecundity observed at 24 °C is counter-balanced by the early attainment of sexual maturity induced at 30 °C. The comparison of the population growth rate calculated in laboratory with field data suggests that temperature is one of the main environmental parameters determining the fitness of D. gyrociliatus.     

Beiträge zur Menschenaffenhaltung im Zoo Berlin nach 1945.: 2. Mitteilung: Bonobos oder Zwergschimpansen (Pan paniscus) Keeping and breeding of Great apes in Berlin Zoo after 1945. 2. Bonobos or Pigmy chimpanzees (Pan paniscus)

The first Bonobo arrived 1987 in Berlin Zoo, the first birth occurred 1995. Up to now four Bonobos were born in Berlin Zoo.     

Effects of temperature on two Mediterranean populations of Dinophilus gyrociliatus (Polychaeta: Dinophilidae): I. Effects on life history and sex ratio

The effects of temperature on the life history characteristics of two populations of the polychaete Dinophilus gyrociliatus, one from Ravenna (northern Adriatic Sea) and the other from Genoa (Ligurian Sea), were investigated. The temperatures tested (6, 12, 18, 24 and 30 °C) cover a wider range than those prevailing in the natural environment. In the populations studied there are broad differences in timing of development and reproduction. At 6 °C, the adults of both populations survive for a long time but they are unable to reproduce. At 12 °C, only the animals from Ravenna manage to reproduce. At the higher temperatures (18, 24 and 30 °C), the development of the animals belonging to the Genoa strain is faster than that of the Ravenna strain. The duration of the various phases of the biological cycle is very similar in both populations, but that from Ravenna exhibits greater tolerance of low temperatures, slower development rate and lower development threshold temperature than does the Genoa population. Temperature and geographical origin also have strong effects on reproductive characteristics. The highest fecundity values were observed at 12 °C in the Ravenna strain, the lowest at 30 °C in both groups. At 18 °C, the Genoa population is more fecund than the Ravenna one, while the situation is reversed at 12 °C. The smallest ovigerous capsules are produced at 30 °C, the biggest at 12 °C, and the Genoa females produce larger capsules than do the females from Ravenna, except at 12 °C. The size of both male and female eggs varies in relation to temperature, the smallest female eggs generally being laid at the higher temperatures. At all the temperatures tested, the sex ratio of the Ravenna population is higher than that of the Genoa population. In the Ravenna strain, temperature has no effect on the sex ratio, while in the Genoa strain the sex ratio at 24 °C is lower than at 18 and 30 °C. Comparison of the two populations at the same temperature reveals considerable differences in the characteristics of their respective life histories and sex ratios. It is very likely that the extreme selectivity of the harbor environments has favored the fragmentation of the species into differentiated populations that have adapted to the conditions prevailing in the different localities.