Synthesis and transformations of a pyrazole containing <Emphasis Type="Italic">α</Emphasis>,<Emphasis Type="Italic">β</Emphasis>-didehydro-<Emphasis Type="Italic">α</Emphasis>-amino acid derivatives

Summary.  2H-Pyran-2-ones 1 were transformed with various hydrazines into (E)- or (Z)-α,β-didehydro-α-amino acid (DDAA) derivatives 4 (and 7) containing a highly substituted pyrazolyl moiety attached at the β-position. With heterocyclic hydrazines, the products 4 were accompanied also by decarboxylated enamines E-6. In order to separate (E/Z)-mixtures of acids, they were transformed to the corresponding methyl esters 9 and 10 by the application of diazomethane. Catalytic hydrogenation under high pressures with Pd/C as a catalyst resulted in the formation of racemic alanine derivatives 11. Received January 29, 2002 Accepted May 27, 2002 Published online December 18, 2002 RID="*" ID="*"  Dedicated with deep respect to Professor Waldemar Adam on the occasion of his 65^th birthday. Acknowledgements We thank the Ministry of Education, Science and Sport of the Republic of Slovenia for the financial support (P0-0503-103). Dr. B. Kralj and Dr. D. Žigon (Center for Mass Spectroscopy, “Jožef Stefan” Institute, Ljubljana, Slovenia) are gratefully acknowledged for the mass measurements. Authors' address: Prof. Marijan Kočevar, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Aškerčeva 5, SI-1000 Ljubljana, Slovenia, E-mail: marijan.kocevar@uni-lj.si     

Characteristics of β-lactamases and their genes (<Emphasis Type="Italic">blaA</Emphasis> and <Emphasis Type="Italic">blaB</Emphasis>) in <Emphasis Type="Italic">Yersinia intermedia</Emphasis> and <Emphasis Type="Italic">Y. frederiksenii</Emphasis>

Background  

The presence of β-lactamases in Y. enterocolitica has been reported to vary with serovars, biovars and geographical origin of the isolates. An understanding of the β-lactamases in other related species is important for an overall perception of antibiotic resistance in yersiniae. The objective of this work was to study the characteristics of β-lactamases and their genes in strains of Y. intermedia and Y. frederiksenii, isolated from clinical and non-clinical sources in India.     

In-fusion expression and characterization of <Emphasis Type="Italic">β</Emphasis>-xylanase and <Emphasis Type="Italic">β</Emphasis>-1,3-1,4-glucanase in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Two chimeric genes, XynA-Bs-Glu-1 and XynA-Bs-Glu-2, encoding Aspergillus sulphureus β-xylanase (XynA, 26 kDa) and Bacillus subtilis β-1,3-1,4-glucanase (Bs-Glu, 30 kDa), were constructed via in-fusion by different linkers and expressed successfully in Pichia pastoris. The fusion protein (50 kDa) exhibited both β-xylanase and β-1,3-1,4-glucanase activities. Compared with parental enzymes, the moiety activities were decreased in fermentation supernatants. Parental XynA and Bs-Glu were superior to corresponding moieties in each fusion enzymes because of lower K_n higher k_cat. Despite some variations, common optima were generally 50°C and pH 3.4 for the XynA moiety and parent, and 40°C and pH 6.4 for the Bs-Glu counterparts. Thus, the fusion enzyme XynA-Bs-Glu-1 and XynA-Bs-Glu-2 were bifunctional.     

Synthesis of disaccharides using β-glucosidases from <Emphasis Type="Italic">Aspergillus niger</Emphasis>, <Emphasis Type="Italic">A. awamori</Emphasis> and <Emphasis Type="Italic">Prunus dulcis</Emphasis>

Objective

Glucose conversion into disaccharides was performed with β-glucosidases from Prunus dulcis (β-Pd), Aspergillus niger (β-An) and A. awamori (β-Aa), in reactions containing initial glucose of 700 and 900 g l^?1.

Results

The reactions’ time courses were followed regarding glucose and product concentrations. In all cases, there was a predominant formation of gentiobiose over cellobiose and also of oligosaccharides with a higher molecular mass. For reactions containing 700 g glucose l^?1, the final substrate conversions were 33, 38, and 23.5% for β-An, β-Aa, and β-Pd, respectively. The use of β-An yielded 103 g gentiobiose l^?1 (15.5% yield), which is the highest reported for a fungal β-glucosidase. The increase in glucose concentration to 900 g l^?1 resulted in a significant increase in disaccharide synthesis by β-Pd, reaching 128 g gentiobiose l^?1 (15% yield), while for β-An and β-Aa, there was a shift toward the synthesis of higher oligosaccharides.

Conclusion

β-Pd and the fungal β-An and β-Aa β-glucosidases present quite dissimilar kinetics and selective properties regarding the synthesis of disaccharides; while β-Pd showed the highest productivity for gentiobiose synthesis, β-An presented the highest specificity.

     

A Synthesis of Heteroaromatic <Emphasis Type="Italic">S</Emphasis>- and <Emphasis Type="Italic">N</Emphasis>-β-Glycosides of <Emphasis Type="Italic">N</Emphasis>-Acetylglucosamine under Phase-Transfer Conditions

The use of crown ethers for a phase transfer-catalyzed synthesis of heteroaromatic glycosides of N-acetylglucosamine was studied. The solid-liquid system and catalysis by 15-crown-5 were found to provide for both the 100% conversion of α-D-glucosaminyl chloride peracetate and a high reaction rate. The interaction of α-D-glucosaminyl chloride peracetate and oxadiazole and triazole mercapto derivatives capable of thiol-thione tautomerism carried out at room temperature in acetonitrile in the presence of anhydrous potassium carbonate and crown ethers was shown to lead to both S- and N-glucosides. The structures of the compounds synthesized were confirmed by X-ray analysis and ¹³C and ¹H NMR spectroscopy.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Codonopsis lanceolata</Emphasis> using the <Emphasis Type="Italic">γ-TMT</Emphasis> gene

Efficient transformation of leaf disc-derived callus of Codonopsis lanceolata was obtained using Agrobacterium tumefaciens strain LBA4404 harboring a binary vector, pYBI121, that carries the neomycin phosphotransferase (npt II) gene as a selectable marker. The green shoots recovered from agroinfected explants on selection medium (containing 0.1 mg/l α-naphthaleneacetic acid (NAA), 1 mg/l 6-benzylaminopurine (BAP), 100 mg/l kanamycin, and 250 mg/l cefotaxime) were rooted on Murashige and Skoog (MS) medium supplemented with 2 mg/l IBA and 10 mg/l kanamycin. To optimize the transformation conditions, several factors were assessed, including the co-cultivation period, the duration of pre- and post-culture in darkness and light, the kanamycin concentration, and the Agrobacterium densities. We produced transgenic Codonopsis lanceolata overexpressing γ-tocopherol methyltransferase (γ-TMT) by this protocol. Moreover, the α-tocopherol content of the plants was enhanced by the overexpression of this gene. Bimal Kumar Ghimire and Eun Soo Seong contributed equally to this work.     

Biosynthesis of ω-alicyclic fatty acids induced by cyclic precursors and change of membrane fluidity in thermophilic bacteria <Emphasis Type="Italic">Geobacillus stearothermophilus</Emphasis> and <Emphasis Type="Italic">Meiothermus</Emphasis><Emphasis Type="Italic">ruber</Emphasis>

Two thermophilic strains belonging to Geobacillus stearothermophilus and Meiothermus ruber, which naturally do not synthesize ω-alicyclic fatty acids (ω-FAs) were cultivated with cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl carboxylic acids. Gas chromatography–mass spectrometry analysis of fatty acid methyl and picolinyl esters showed that both strains are able to synthesize ω-FAs when cultivated with the appropriate precursor. The incorporation of cyclic acids influenced the whole FA composition as well as membrane fluidity. Membrane fluidity of intact cells was studied by measuring the fluorescence polarisation of the probe l,6-diphenyl-1,3,5-hexatriene incorporated into membrane lipid bilayers. Cytoplasmic membrane became more fluid with increasing content of ω-FAs. This is caused by considerable changes in lipid packing within the membrane induced by the presence of ω-FAs not found in the natural environment of Geobacillus and Meiothermus strains.     

Expression and purification of <Emphasis Type="Italic">Zantedeschia aethiopica</Emphasis> agglutinin in <Emphasis Type="Italic">Escherichia </Emphasis><Emphasis Type="Italic">coli</Emphasis>

Recombinant Zantedeschia aethiopica agglutinin (ZAA) was expressed in Escherichia coli as N-terminal His-tagged fusion. After induction with isopropylthio-β-d-galactoside (IPTG), the recombinant ZAA was purified by metal-affinity chromatography. The purified ZAA protein was applied in anti-fungal assay and the result showed that recombinant ZAA had anti-fungal activity towards leaf mold (Fulvia fulva), one of the most serious phytopathogenic fungi causing significant yield loss of crops. This study suggests that ZAA could be an effective candidate in genetic engineering of plants for the control of leaf mold.     

Pistil-function breakdown in a new <Emphasis Type="Italic">S</Emphasis>-allele of European pear, <Emphasis Type="Italic">S</Emphasis><Subscript><Emphasis Type="Italic">21</Emphasis></Subscript>°, confers self-compatibility

European pear exhibits RNase-based gametophytic self-incompatibility controlled by the polymorphic S-locus. S-allele diversity of cultivars has been extensively investigated; however, no mutant alleles conferring self-compatibility have been reported. In this study, two European pear cultivars, ‘Abugo’ and ‘Ceremeño’, were classified as self-compatible after fruit/seed setting and pollen tube growth examination. S-genotyping through S-PCR and sequencing identified a new S-RNase allele in the two cultivars, with identical deduced amino acid sequence as S ₂₁ , but differing at the nucleotide level. Test-pollinations and analysis of descendants suggested that the new allele is a self-compatible pistil-mutated variant of S ₂₁ , so it was named S ₂₁ °. S-genotypes assigned to ‘Abugo’ and ‘Ceremeño’ were S ₁₀ S ₂₁ ° and S ₂₁ °S ₂₅ respectively, of which S ₂₅ is a new functional S-allele of European pear. Reciprocal crosses between cultivars bearing S ₂₁ and S ₂₁ ° indicated that both alleles exhibit the same pollen function; however, cultivars bearing S ₂₁ ° had impaired pistil-S function as they failed to reject either S ₂₁ or S ₂₁ ° pollen. RT-PCR analysis showed absence of S ₂₁ °-RNase gene expression in styles of ‘Abugo’ and ‘Ceremeño’, suggesting a possible origin for S ₂₁ ° pistil dysfunction. Two polymorphisms found within the S-RNase genomic region (a retrotransposon insertion within the intron of S ₂₁ ° and indels at the 3′UTR) might explain the different pattern of expression between S ₂₁ and S ₂₁ °. Evaluation of cultivars with unknown S-genotype identified another cultivar ‘Azucar Verde’ bearing S ₂₁ °, and pollen tube growth examination confirmed self-compatibility for this cultivar as well. This is the first report of a mutated S-allele conferring self-compatibility in European pear.     

<Emphasis Type="Italic">Carex</Emphasis> × <Emphasis Type="Italic">involuta</Emphasis> and <Emphasis Type="Italic">Carex juncella</Emphasis> in the flora of Slovakia

The paper brings information on an isolated occurrence and morphological characters of Carex × involuta and C. juncella populations in the Vel’ká Fatra Mts. Their presence has been known neither from the territory of Slovakia nor from the whole Western Carpathians till now.     

Rapid induction of <Emphasis Type="Italic">Agrobacterium</Emphasis> <Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transgenic roots directly from adventitious roots in <Emphasis Type="Italic">Panax</Emphasis> <Emphasis Type="Italic">ginseng</Emphasis>

Root segments from seedlings of Panax ginseng produced adventitious roots directly when cultured on 1/2 MS solid medium lacking NH₄NO₃ and containing 3.0 mg l⁻¹ IBA. Using this adventitious root formation, we developed rapid and efficient transgenic root formation directly from adventitious root segments in P. ginseng. Root segments were co-cultivated with Agrobacterium tumefaciens (GV3101) caring β-glucuronidase (GUS) gene. Putative transgenic adventitious roots were formed directly from root segments on medium with 400 mg l⁻¹ cefotaxime and 50 mg l⁻¹ kanamycin. Kanamycin resistant adventitious roots were selected and proliferated as individual lines by subculturing on medium with 300 mg l⁻¹ cefotaxime and 50 mg l⁻¹ kanamycin at two weeks subculture interval. Frequency of transient and stable expression of GUS gene was enhanced by acetosyringon (50 mg l⁻¹) treatment. Integration of transgene into the plants was confirmed by the X-gluc reaction, PCR and Southern analysis. Production of transgenic plants was achieved via somatic embryogenesis from the embryogenic callus derived from independent lines of adventitious roots. The protocol for rapid induction of transgenic adventitious roots directly from adventitious roots can be applied for a new Agrobacterium tumefaciens-mediated genetic transformation protocol in P. ginseng.     

In vitro growth response of <Emphasis Type="Italic">Phytophthora cactorum</Emphasis>, <Emphasis Type="Italic">P. nicotianae</Emphasis> and <Emphasis Type="Italic">P.</Emphasis> × <Emphasis Type="Italic">pelgrandis</Emphasis> to antibiotics and fungicides

The reactions of isolates of Phytophthora cactorum, P. nicotianae and P. × pelgrandis to metalaxyl, mancozeb, dimethomorph, streptomycin and chloramphenicol were tested to obtain information about the variability of resistance in these pathogens. Distinct genetic groups showed significant differences in resistance to all tested substances except streptomycin. In response to streptomycin, the growth inhibition rates of distinct groups did not differ significantly. The most remarkable differences were detected in the reactions to chloramphenicol and metalaxyl. Discriminant analysis evaluating the effect of all substances confirmed the differences among the groups, which are in agreement with the differences revealed by earlier DNA analyses.     

Innate immunity to <Emphasis Type="Italic">Legionella</Emphasis> and toll-like receptors — <Emphasis Type="Italic">review</Emphasis>

Legionella is a parasite of eukaryotic cells, able to survive and replicate in a wide range of protozoan hosts. It can also infect humans as an opportunistic pathogen, primarily by interaction with alveolar macrophages. These bacteria can cause life-threatening pneumonia, especially in immunocompromised individuals. However, most infections triggered by Legionella are cleared by an efficient host immune system. The protective immune responses against Legionella are complex and multifaceted, involving many components of the immune system. Recognition of such components as LPS, flagellum, and peptidoglycan of L. pneumophila by the TLRs, which orchestrates the innate immune responses to Legionella, lays an important role in activation of monocytes and alveolar macrophages and, thus, in inhibition of intracellular proliferation of bacteria. MyD88-dependent signaling pathways are important for host protection against Legionella.     

Carbohydrate and Ethane Release with <Emphasis Type="Italic">Erwinia carotovora</Emphasis> Subspecies <Emphasis Type="Italic">betavasculorum</Emphasis><Emphasis Type="Italic">–</Emphasis>Induced Necrosis

Erwinia carotovora subspecies betavasculorum, also known as E. betavasculorum and Pectobacterium betavasculorum, is a soil bacterium that has the capacity to cause root rot necrosis of sugarbeets. The qualitatively different pathogenicity exhibited by the virulent E. carotovora strain and two avirulent strains, a Citrobacter sp. and an Enterobacter cloacae, was examined using digital analysis of photographic evidence of necrosis as well as for carbohydrate, ethane, and ethylene release compared with uninoculated potato tuber slices. Visual scoring of necrosis was superior to digital analysis of photographs. The release of carbohydrates and ethane from potato tuber slices inoculated with the soft rot necrosis-causing Erwinia was significantly greater than that of potato tuber slices that had not been inoculated or that had been inoculated with the nonpathogenic E. cloacae and Citrobacter sp. strains. Interestingly, ethylene production from potato slices left uninoculated or inoculated with the nonpathogenic Citrobacter strain was 5- to 10-fold higher than with potato slices inoculated with the pathogenic Erwinia strain. These findings suggest that (1) carbohydrate release might be a useful measure of the degree of pathogenesis, or relative virulence; and that (2) bacterial suppression of ethylene formation may be a critical step in root rot disease formation.     

Molecular characterization of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> Δ<Emphasis Type="Italic">relA</Emphasis> Δ<Emphasis Type="Italic">spoT</Emphasis> double mutants

In Escherichia coli cellular levels of pppGpp and ppGpp, collectively called (p)ppGpp, are maintained by the products of two genes, relA and spoT. Like E. coli, Vibrio cholerae also possesses relA and spoT genes. Here we show that similar to E. coli, V. cholerae ΔrelA cells can accumulate (p)ppGpp upon carbon starvation but not under amino acid starved condition. Although like in E. coli, the spoT gene function was found to be essential in V. cholerae relA ⁺ background, but unlike E. coli, several V. cholerae ΔrelA ΔspoT mutants constructed in this study accumulated (p)ppGpp under glucose starvation. The results suggest a cryptic source of (p)ppGpp synthesis in V. cholerae, which is induced upon glucose starvation. Again, unlike E. coli ΔrelA ΔspoT mutant (ppGpp⁰ strain), the V. cholerae ΔrelA ΔspoT mutants showed certain unusual phenotypes, which are (a) resistance towards 3-amino-1,2,4-triazole (AT); (b) growth in nutrient poor M9 minimal medium; (c) ability to stringently regulate cellular rRNA accumulation under glucose starvation and (d) initial growth defect in nutrient rich medium. Since these phenotypes of ΔrelA ΔspoT mutants could be reverted back to ΔrelA phenotypes by providing SpoT in trans, it appears that the spoT gene function is crucial in V. cholerae. Part of this work was presented at the International Symposium on Chemical Biology, Kolkata, India, 7–9 March 2007.     

Whole-cell bioreduction of aromatic α-keto esters using <Emphasis Type="Italic">Candida tenuis</Emphasis> xylose reductase and <Emphasis Type="Italic">Candida boidinii</Emphasis> formate dehydrogenase co-expressed in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Whole cell-catalyzed biotransformation is a clear process option for the production of chiral alcohols via enantioselective reduction of precursor ketones. A wide variety of synthetically useful reductases are expressed heterologously in Escherichia coli to a high level of activity. Therefore, this microbe has become a prime system for carrying out whole-cell bioreductions at different scales. The limited capacity of central metabolic pathways in E. coli usually requires that reductase coenzyme in the form of NADPH or NADH be regenerated through a suitable oxidation reaction catalyzed by a second NADP⁺ or NAD⁺ dependent dehydrogenase that is co-expressed. Candida tenuis xylose reductase (CtXR) was previously shown to promote NADH dependent reduction of aromatic α-keto esters with high Prelog-type stereoselectivity. We describe here the development of a new whole-cell biocatalyst that is based on an E. coli strain co-expressing CtXR and formate dehydrogenase from Candida boidinii (CbFDH). The bacterial system was evaluated for the synthesis of ethyl R-4-cyanomandelate under different process conditions and benchmarked against a previously described catalyst derived from Saccharomyces cerevisiae expressing CtXR.     

Expression and characterization of <Emphasis Type="Italic">Trichoderma virens</Emphasis> UKM-1 endochitinase in <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis>

A gene encoding endochitinase from Trichoderma virens UKM-1 was cloned and expressed in E. coli BL21 (DE3). Both the endochitinase gene and its cDNA sequences were obtained. The endochitinase gene encodes 430 amino acids from an open reading frame comprising of 1,690 bp nucleotide sequence with three introns. The endochitinase was expressed as soluble and active enzyme at 20°C when induced with 1 mM IPTG. Maximum activity was observed at 4 h of post-induction time. SDS-PAGE showed that the purified endochitinase exhibited a single band with molecular weight of 42 kDa. Biochemical characterization of the enzyme displayed a near neutral pH characteristic with an optimum pH at 6.0 and optimum temperature at 50°C. The enzyme is stable between pH 3.0–7.0 and is able to retain its activity from 30 to 60°C. The presence of Mg²⁺ and Ca²⁺ ions increased the enzyme activity up to 20%. The purified enzyme has a strong affinity towards colloidal chitin and low effect on ethyl cellulose and D-cellubiose which are non-chitin related substrates. HPLC analysis from the chitin hydrolysis showed the release of (GlcNAc)₃, (GlcNAc)₂ and GlcNAc, in which (GlcNAc)₂ was the main product.     

Conjugal transfer using the bacteriophage ϕC31 <Emphasis Type="Italic">att</Emphasis>/<Emphasis Type="Italic">int</Emphasis> system and properties of the <Emphasis Type="Italic">attB</Emphasis> site in <Emphasis Type="Italic">Streptomyces ambofaciens</Emphasis>

To facilitate molecular genetic studies of Streptomyces ambofaciens that produces spiramycin, a commercially important macrolide antibiotic used in human medicine against Gram-positive pathogenic bacteria, the conditions for the conjugal transfer of DNA from E. coli to S. ambofaciens were established using a bacteriophage ϕC31 att/int system. The transconjugation efficiency of S. ambofaciens varied with the medium used; the highest frequency was obtained on AS-1 medium containing 10 mM MgCl₂ without heat treatment of the spores. In addition, by cloning and sequencing the attB site, we identified that S. ambofaciens contains a single attB site within an ORF coding for a pirin homolog, and its attB site sequence shows 100% nt identity to the sequence of S. coelicolor and S. lividans, which have the highest efficiency in transconjugation using the ϕC31 att/int system.     

Isolation and functional characterization of <Emphasis Type="Italic">Lycopene β-cyclase</Emphasis> (<Emphasis Type="Italic">CYC-B</Emphasis>) promoter from <Emphasis Type="Italic">Solanum habrochaites</Emphasis>

Background  

Carotenoids are a group of C40 isoprenoid molecules that play diverse biological and ecological roles in plants. Tomato is an important vegetable in human diet and provides the vitamin A precursor β-carotene. Genes encoding enzymes involved in carotenoid biosynthetic pathway have been cloned. However, regulation of genes involved in carotenoid biosynthetic pathway and accumulation of specific carotenoid in chromoplasts are not well understood. One of the approaches to understand regulation of carotenoid metabolism is to characterize the promoters of genes encoding proteins involved in carotenoid metabolism. Lycopene β-cyclase is one of the crucial enzymes in carotenoid biosynthesis pathway in plants. Its activity is required for synthesis of both α-and β-carotenes that are further converted into other carotenoids such as lutein, zeaxanthin, etc. This study describes the isolation and characterization of chromoplast-specific Lycopene β-cyclase (CYC-B) promoter from a green fruited S. habrochaites genotype EC520061.     

Functional expression of the lipase gene Lip2 of <Emphasis Type="Italic">Pleurotus</Emphasis> <Emphasis Type="Italic">sapidus</Emphasis> in <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis>

The lipase Lip2 of the edible basidiomycete, Pleurotus sapidus, is an extracellular enzyme capable of hydrolysing xanthophyll esters with high efficiency. The gene encoding Lip2 was expressed in Escherichia coli TOP10 using the gene III signal sequence to accumulate proteins in the periplasmatic space. The heterologous expression under control of the araBAD promoter led to the high level production of recombinant protein, mainly as inclusion bodies, but partially in a soluble and active form. A fusion with a C-terminal His tag was used for purification and immunochemical detection of the target protein. This is the first example of a heterologous expression and periplasmatic accumulation of a catalytically active lipase from a basidiomycete fungus.