Acid mediated formation of an N-acyliminium ion from tubulysins: a new methodology for the synthesis of natural tubulysins and their analogs

Tubuylsins are extremely potent cytotoxic agents which inhibit tubulin polymerization and lead to cell cycle arrest and apoptosis. Tubulysins have been isolated from fermentation mixtures and have been chemically synthesized; however, these efforts have been hampered by poor yields and arduous purifications. In contrast, treatment of a mixture of natural tubulysins A, B, C, G, and I, obtained from a fermentation batch with trifluoroacetic acid results in the formation of a single N-acyliminium ion. Subsequent addition of butyric, isopentyl, or acetic acid results in the formation of tubulysin B, A, or I, respectively, as a single species. New tubulysin analogs can be formed upon treatment of the acyliminium ion with other nucleophiles such as alcohols, thiols, and nitriles, resulting in corresponding N-acyl-N,O-acetals, N-acyl-N,S-thioacetals, and N,N'-diacyl-aminals. Carbon-carbon bond formation is also possible with a modification of this protocol. The cytotoxicies of the natural tubulysins and tubulysin analogs synthesized by this method were evaluated on KB cells.     

Immobilization of Candida rugosa lipase on glass beads for enantioselective hydrolysis of racemic Naproxen methyl ester

Candida rugosa lipase (CRL) was immobilized on glutaraldehyde-activated aminopropyl glass beads by using covalent binding method or sol-gel encapsulation procedure and improved considerably by fluoride-catalyzed hydrolysis of mixtures of RSi(OCH₃)₃ and Si(OCH₃)₄. The catalytic properties of the immobilized lipases were evaluated into model reactions, i.e. the hydrolysis of p-nitrophenylpalmitate (p-NPP). It has been observed that the percent activity yield of the encapsulated lipase was 166.9, which is 5.5 times higher than that of the covalently immobilized lipase. The enantioselective hydrolysis of racemic Naproxen methyl ester by immobilized lipase was studied in aqueous buffer solution/isooctane reaction system and it was noticed that particularly, the glass beads based encapsulated lipases had higher conversion and enantioselectivity compared to covalently immobilized lipase. In short, the study confirms an excellent enantioselectivity (E > 400) for the encapsulated lipase with an ee value of 98% for S-Naproxen.     

Improvement of catalytic activity of lipase from Candida rugosa via sol-gel encapsulation in the presence of calix(aza)crown

Lipase from Candida rugosa (CRL) was encapsulated within a chemically inert sol-gel support in the presence of calix(aza)crowns as the new additives. The catalytic activity of the encapsulated lipases was evaluated both in the hydrolysis of p-nitrophenyl palmitate (p-NPP) and the enantioselective hydrolysis of racemic Naproxen methyl ester. It has been observed that the percent activity yields of the calix(aza)crown based encapsulated lipases were higher than that of the free lipase. Improved enantioselectivity was observed with the calix(aza)crown-based encapsulated lipases as compared to encapsulated free lipase. The reaction of Naproxen methyl ester resulted in 48.4% conversion for 24 h and 98% enantiomeric excess for the S-acid, corresponding to an E value of >300 (E = 166 for the encapsulated free enzyme). Moreover, the encapsulated lipases were still retained about 18% of their conversion ratios after the sixth reuse in the enantioselective reaction.     

Structure based design of compounds from natural sources for diabetes and inflammation

Medicinal plants and marine sources are important elements of indigenous medical systems worldwide. The natural drugs frommedicinal plants and marine sources have received considerable interest in treatment of diabetes and inflammation. Based onliterature, alpha glucosidase, aldose reductase and PTP1B enzymes were chosen as anti-diabetes targets and PLA₂ was chosen forthe anti-inflammatory target. In our study, plant and bromophenols (BPs) inhibitors were screened using High Throughput Virtualscreening (HTVS) followed by Induced Fit Docking (IFD) studies were carried out against diabetes and inflammation targets. TheIFD result of natural inhibitors has showed favorable docking score, glide energy and hydrogen bonds interactions with the activesite residues. Some of the natural inhibitors successively satisfied all the in silico parameters among the others and seem to bepotent inhibitors against diabetes and inflammation.     

Slow formation of stable complexes during coincubation of minimal rRNA and ribosomal protein S4

Ribosomal protein S4 binds and stabilizes a five-helix junction or five-way junction (5WJ) in the 5′ domain of 16S ribosomal RNA (rRNA) and is one of two proteins responsible for nucleating 30S ribosome assembly. Upon binding, both protein S4 and 5WJ reorganize their structures. We show that labile S4 complexes rearrange into stable complexes within a few minutes at 42 °C, with longer coincubation leading to an increased population of stable complexes. In contrast, prefolding the rRNA has a smaller effect on stable S4 binding. Experiments with minimal rRNA fragments show that this structural change depends only on 16S residues within the S4 binding site. SHAPE (selective 2′-hydroxyl acylation analyzed by primer extension) chemical probing experiments showed that S4 strongly stabilizes 5WJ and the helix (H) 18 pseudoknot, which become tightly folded within the first minute of S4 binding. However, a kink in H16 that makes specific contacts with the S4 N-terminal extension, as well as a right-angle motif between H3, H4, and H18, requires a minute or more to become fully structured. Surprisingly, S4 structurally reorganizes the 530-loop and increases the flexibility of H3, which is proposed to undergo a conformational switch during 30S assembly. These elements of the S4 binding site may require other 30S proteins to reach a stable conformation.     

ITIS,a bioinformatics tool for accurate identification of transposon insertion sites using next-generation sequencing data

Background

Transposable elements constitute an important part of the genome and are essential in adaptive mechanisms. Transposition events associated with phenotypic changes occur naturally or are induced in insertional mutant populations. Transposon mutagenesis results in multiple random insertions and recovery of most/all the insertions is critical for forward genetics study. Using genome next-generation sequencing data and appropriate bioinformatics tool, it is plausible to accurately identify transposon insertion sites, which could provide candidate causal mutations for desired phenotypes for further functional validation.

Results

We developed a novel bioinformatics tool, ITIS (Identification of Transposon Insertion Sites), for localizing transposon insertion sites within a genome. It takes next-generation genome re-sequencing data (NGS data), transposon sequence, and reference genome sequence as input, and generates a list of highly reliable candidate insertion sites as well as zygosity information of each insertion. Using a simulated dataset and a case study based on an insertional mutant line from Medicago truncatula, we showed that ITIS performed better in terms of sensitivity and specificity than other similar algorithms such as RelocaTE, RetroSeq, TEMP and TIF. With the case study data, we demonstrated the efficiency of ITIS by validating the presence and zygosity of predicted insertion sites of the Tnt1 transposon within a complex plant system, M. truncatula.

Conclusion

This study showed that ITIS is a robust and powerful tool for forward genetic studies in identifying transposable element insertions causing phenotypes. ITIS is suitable in various systems such as cell culture, bacteria, yeast, insect, mammal and plant.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-015-0507-2) contains supplementary material, which is available to authorized users.     

Heparin-benzyl alcohol enhancement of biofilms formation and antifungal susceptibility of vaginal Candida species isolated from pregnant and nonpregnant Saudi women

Biofilm formation by Candida species is a major contribute to their pathogenic potential.The aim of this study was to determine in vitro effects of EDTA, cycloheximide, and heparin-benzyl alcohol preservative on C. albicans (126) and non-albicans (31)vaginal yeast isolates biofilm formations and their susceptibility against three antifungal Etest strips. Results of the crystal violet-assay, indicated that biofilms formation were most commonly observed [100%] for C. kefyr, C. utilis, C. famata, and Rhodotorula mucilaginosa, followed by C. glabrata [70%], C. tropicalis [50%], C. albicans [29%], Saccharomyces cerevisiae [0.0%]. EDTA (0.3mg/ml) significantly inhibited biofilm formation in both C. albicans and non-albicans isolates (P=0.0001) presumably due to chelation of necessary metal cations for the process-completion. In contrast, heparin (-benzyl alcohol preservative) stimulated biofilm formation in all tested isolates, but not at significant level (P=0.567). Conversely, cycloheximide significantly (P=0.0001) inhibited biofilm formation in all C. albicans strains(126) and its effect was even 3 fold more pronounced than EDTA inhibition, probably due to its attenuation of proteins (enzymes) and/or complex molecules necessary for biofilm formation. Results also showed that all nonalbicans yeasts isolates were susceptible to 5-flucytosine (MIC₅₀, 0.016 µg/ml; MIC₉₀, 0.064 µg/ml), but 14% of C. albicans isolates were resistant (MIC₅₀, 0.064 µg/ml; MIC₉₀ >32 µg/ml). The MIC₅₀ value of amphotricin B for all C. albicans and non-albicans isolates was at a narrow range of 0.023 µg /ml, and the MIC₉₀ values were 0.047 µg/ml and 0.064 µg/ml respectively, thereby confirming its efficacy as a first line empiric- treatment of Candida spp infections.     

Application of supercritical CO2 for extraction of polyisoprenoid alcohols and their esters from plant tissues

In this study, a method of supercritical fluid extraction (SFE) with carbon dioxide of polyisoprenoids from plant photosynthetic tissues is described. SFE was an effective extraction method for short- and medium-chain compounds with even higher yield than that observed for the “classical extraction” method with organic solvents. Moreover, SFE-derived extracts contained lower amounts of impurities (e.g., chlorophylls) than those obtained by extraction of the same tissue with organic solvents. Elevated temperature and extended extraction time of SFE resulted in a higher rate of extraction of long-chain polyisoprenoids. Ethanol cofeeding did not increase the extraction efficiency of polyisoprenoids; instead, it increased the content of impurities in the lipid extract. Optimization of SFE time and temperature gives the opportunity of prefractionation of complex polyisoprenoid mixtures accumulated in plant tissues. Extracts obtained with application of SFE are very stable and free from organic solvents and can further be used directly in experimental diet supplementation or as starting material for preparation of semisynthetic polyisoprenoid derivatives, e.g., polyisoprenoid phosphates.     

Probing electron transfer reactions between two azurins from Alcaligenes xylosoxidans GIFU 1051 with optically active Ru complexes as molecular recognition probes: Importance of the 43rd residue

Electron transfer reactions between optically-active Ru^II/III complexes incorporating (S)-/(R)-amino acids, and the two azurins, azurin-1 (az-1Cu) and azurin-2 (az-2Cu) isolated from Alcaligenes xylosoxidans GIFU 1051, have been studied to probe molecular recognition sites on the two azurins. The Ru^II/III complexes are K[Ru^II(L)(bpy)] and [Ru^III(L)(bpy)], and have a tripodal ligand (L) derived from the (S)-/(R)-amino acids, which are in turn exchanged for other functional substituent groups, such as (S)-/(R)-phenylalanine, -leucine, -valine, -alanine, and -glutamic acid (L = (S)-/(R)-BCMPA, -BCMLE, -BCMVA, -BCMAL, and -BCMGA). In the oxidation reaction of az-1Cu^I promoted by the Ru^III complexes, the kinetic parameters exhibited enantio- and stereo-selectivities, while the same reaction of az-2Cu^I was less enantio- and stereo-selective. These differences suggest that the processes of formation of the activated states are different for the two azurins. On the other hand, such a difference has not been observed for az-1 and az-2 with respect to the reduction reactions promoted by both azurins Cu^II by the Ru^II complexes within the experimental error. This suggests that the neutrality of the Ru complexes is important for precise molecular recognition of azurins. His117 has been proposed as the electron transfer site. The local structures in the vicinity of the His117 side chain in the two azurins, are essentially identical with the exception of the 43rd residue, Val43 and Ala43 for az-1 and az-2, respectively. Electron transfer reactions between Ru^III complexes and a mutant azurin, V43A-az-1, were also carried out. Interestingly, the activation parameters estimated were very similar to those of az-2, indicating that the 43rd residue acts as the electron transfer site in azurins and provides rationalization for the different mechanisms of az-1 and az-2 in redox reactions.     

H NMR spectroscopy as a tool to determine accurate photoisomerization quantum yields of stilbene-like ligands coordinated to rhenium(I) polypyridyl complexes

In this work, the use of proton nuclear magnetic resonance, ¹H NMR, was fully described as a powerful tool to follow a photoreaction and to determine accurate quantum yields, so called true quantum yields (Φ_true), when a reactant and photoproduct absorption overlap. For this, Φ_true for the trans-cis photoisomerization process were determined for rhenium(I) polypyridyl complexes, fac-[Re(CO)₃(NN)(trans-L)]⁺ (NN = 1,10-phenanthroline, phen, or 4,7-diphenyl-1,10-phenanthroline, ph₂phen, and L = 1,2-bis(4-pyridyl)ethylene, bpe, or 4-styrylpyridine, stpy). The true values determined at 365 nm irradiation (e.g. Φ_NMR = 0.80 for fac-[Re(CO)₃(phen)(trans-bpe)]⁺) were much higher than those determined by absorption spectral changes (Φ_UV-Vis = 0.39 for fac-[Re(CO)₃(phen)(trans-bpe)]⁺). Φ_NMR are more accurate in these cases due to the distinct proton signals of trans and cis-isomers, which allow the actual determination of each component concentration under given irradiation time. Nevertheless when the photoproduct or reactant contribution at the probe wavelength is negligible, one can determine Φ_true by regular absorption spectral changes. For instance, Φ_313 nm for free ligand photoisomerization determined both by absorption and ¹H NMR variation are equal within the experimental error (bpe: Φ_UV-Vis = 0.27, Φ_NMR = 0.26; stpy: Φ_UV-Vis = 0.49, Φ_NMR = 0.49). Moreover, ¹H NMR data combined with electronic spectra allowed molar absorptivity determination of difficult to isolate cis-complexes.     

High-light induced singlet oxygen formation in cytochrome b6f complex from Bryopsis corticulans as detected by EPR spectroscopy

Electron paramagnetic resonance (EPR) spectroscopy was used to detect the light-induced formation of singlet oxygen (¹O₂*) in the intact and the Rieske-depleted cytochrome b₆f complexes (Cyt b₆f) from Bryopsis corticulans, as well as in the isolated Rieske Fe–S protein. It is shown that, under white-light illumination and aerobic conditions, chlorophyll a (Chl a) bound in the intact Cyt b₆f can be bleached by light-induced ¹O₂*, and that the ¹O₂* production can be promoted by D₂O or scavenged by extraneous antioxidants such as l-histidine, ascorbate, β-carotene and glutathione. Under similar experimental conditions, ¹O₂* was also detected in the Rieske-depleted Cyt b₆f complex, but not in the isolated Rieske Fe–S protein. The results prove that Chl a cofactor, rather than Rieske Fe–S protein, is the specific site of ¹O₂* formation, a conclusion which draws further support from the generation of ¹O₂* with selective excitation of Chl a using monocolor red light.     

Isolation of Paenibacillus sp. and Variovorax sp. strains from decaying woods and characterization of their potential for cellulose deconstruction

Prospection of cellulose-degrading bacteria in natural environments allows the identification of novel cellulases and hemicellulases that could be useful in second-generation bioethanol production. In this work, cellulolytic bacteria were isolated from decaying native forest soils by enrichment on cellulose as sole carbon source. There was a predominance of Gram positive isolates that belonged to the phyla Proteobacteria and Firmicutes. Many primary isolates with cellulolytic activity were not pure cultures. From these consortia, isolation of pure constituents was attempted in order to test the hypothesis whether microbial consortia are needed for full degradation of complex substrates. Two isolates, CB1-2-A-5 and VG-4-A-2, were obtained as the pure constituents of CB1-2 and VG-4 consortia, respectively. Based on 16S RNA sequence, they could be classified as Variovorax paradoxus and Paenibacillus alvei. Noteworthy, only VG-4 consortium showed measurable xylan degrading capacity and signs of filter paper degradation. However, no xylan or filter paper degrading capacities were observed for the pure cultures isolated from it, suggesting that other members of this consortium were necessary for these hydrolyzing activities. Our results indicated that Paenibacillus sp. and Variovorax sp. as well as VG-4 consortium, might be a useful source of hydrolytic enzymes. Moreover, although Variovorax sp. had been previously identified in metagenomic studies of cellulolytic communities, this is the first report on the isolation and characterization of this microorganism as a cellulolytic genus.     

Genetic Diversity of Taenia asiatica from Thailand and Other Geographical Locations as Revealed by Cytochrome c Oxidase Subunit 1 Sequences

Twelve 924 bp cytochrome c oxidase subunit 1 (cox1) mitochondrial DNA sequences from Taenia asiatica isolates from Thailand were aligned and compared with multiple sequence isolates from Thailand and 6 other countries from the GenBank database. The genetic divergence of T. asiatica was also compared with Taenia saginata database sequences from 6 different countries in Asia, including Thailand, and 3 countries from other continents. The results showed that there were minor genetic variations within T. asiatica species, while high intraspecies variation was found in T. saginata. There were only 2 haplotypes and 1 polymorphic site found in T. asiatica, but 8 haplotypes and 9 polymorphic sites in T. saginata. Haplotype diversity was very low, 0.067, in T. asiatica and high, 0.700, in T. saginata. The very low genetic diversity suggested that T. asiatica may be at a risk due to the loss of potential adaptive alleles, resulting in reduced viability and decreased responses to environmental changes, which may endanger the species.     

Feruloyl esterases as a tool for the release of phenolic compounds from agro-industrial by-products

Agro-industrial by-products are a potential source of added-value phenolic acids with promising applications in the food and pharmaceutical industries. Here two purified feruloyl esterases from Aspergillus niger, FAEA and FAEB were tested for their ability to release phenolic acids such as caffeic acid, p-coumaric acid and ferulic acid from coffee pulp, apple marc and wheat straw. Their hydrolysis activity was evaluated and compared with their action on maize bran and sugar beet pulp. The specificity of both enzymes against natural and synthetic substrates was evaluated; particular attention was paid to quinic esters and lignin monomers. The efficiency of both enzymes on model substrates was studied. We show the ability of these enzymes to hydrolyze quinic esters and ester linkages between phenolic acids and lignin monomer.     

Functional characterization of recombinant prefoldin complexes from a hyperthermophilic archaeon, Thermococcus sp. strain KS-1

Prefoldin is a heterohexameric molecular chaperone complex that is found in the eukaryotic cytosol and also in archaea. It captures a nonnative protein and subsequently delivers it to a group II chaperonin for proper folding. Archaeal prefoldin is a heterocomplex containing two α subunits and four β subunits with the structure of a double β-barrel assembly, with six long coiled coils protruding from it like a jellyfish with six tentacles. We have studied the protein folding mechanism of group II chaperonin using those of Thermococcus sp. strain KS-1 (T. KS-1) because they exhibit high protein folding activity in vitro. We have also demonstrated functional cooperation between T. KS-1 chaperonins and prefoldin from Pyrococcus horikoshii OT3. Recent genome analysis has shown that Thermococcus kodakaraensis KOD1 contains two pairs of prefoldin subunit genes, correlating with the existence of two different chaperonin subunits. In this study, we characterized four different recombinant prefoldin complexes composed of two pairs of prefoldin subunits (α1, α2, β1, and β2) from T. KS-1. All of them (α1-β1, α2-β1, α1-β2, and α2-β2) exist as α₂β₄ heterohexamers and can protect several proteins from forming aggregates with different activities. We have also compared the collaborative activity between the prefoldin complexes and the cognate chaperonins. Prefoldin complexes containing the β1 subunit interacted with the chaperonins more strongly than those with the β2 subunit. The results suggest that Thermococcus spp. express different prefoldins for different substrates or conditions as chaperonins.     

Non-lipolytic and lipolytic sequence-related carboxylesterases: A comparative study of the structure–function relationships of rabbit liver esterase 1 and bovine pancreatic bile-salt-activated lipase

To differentiate esterases from lipases at the structure–function level, we have compared the kinetic properties and structural features of sequence-related esterase 1 from rabbit liver (rLE) and bile-salt-activated lipase from bovine pancreas (bBAL). In contrast to rLE, bBAL hydrolyses water-insoluble medium and long chain esters as vinyl laurate, trioctanoin and olive oil. Conversely, rLE and bBAL are both active on water-soluble short chain esters as vinyl acetate, vinyl propionate, vinyl butyrate, tripropionin, tributyrin and p-nitrophenyl butyrate. However, the enzymes show distinctive kinetic behaviours. rLE displays maximal activity at low substrate concentration, below the critical micelle concentration, whereas bBAL acts preferencially on emulsified esters, at concentration exceeding the solubility limit. Comparison of the 3D structures of rLE and bBAL shows, in particular, that the peptide loop at positions 116–123 in bBAL is deleted in rLE. This peptide segment interacts with a bile salt molecule thus inducing a conformational transition which gives access to the active site. Inhibition studies and manual docking of a bulky ester molecule as vinyl laurate in the catalytic pocket of rLE and bBAL show that the inability of the esterase to hydrolyse large water-insoluble esters is not due to steric hindrance. It is hypothesized that esterases lack specific hydrophobic structures involved both in the stabilization of the lipase–lipid adsorption complex at interfaces and in the spontaneous transfer of a single substrate molecule from interface to the catalytic site.     

The last step in cephalosporin C formation revealed: crystal structures of deacetylcephalosporin C acetyltransferase from Acremonium chrysogenum in complexes with reaction intermediates

Deacetylcephalosporin C acetyltransferase (DAC-AT) catalyses the last step in the biosynthesis of cephalosporin C, a broad-spectrum β-lactam antibiotic of large clinical importance. The acetyl transfer step has been suggested to be limiting for cephalosporin C biosynthesis, but has so far escaped detailed structural analysis. We present here the crystal structures of DAC-AT in complexes with reaction intermediates, providing crystallographic snapshots of the reaction mechanism. The enzyme is found to belong to the α/β hydrolase class of acetyltransferases, and the structures support previous observations of a double displacement mechanism for the acetyl transfer reaction in other members of this class of enzymes. The structures of DAC-AT reported here provide evidence of a stable acyl-enzyme complex, thus underpinning a mechanism involving acetylation of a catalytic serine residue by acetyl coenzyme A, followed by transfer of the acetyl group to deacetylcephalosporin C through a suggested tetrahedral transition state.     

Hypochlorite digestion method for efficient recovery of PHB from <Emphasis Type="Italic">Alcaligenes faecalis</Emphasis>

We reported the optimum amount of PHB accumulated by Alcaligenes faecalis during its 24 h growth under nitrogen deficient conditions. After 24 h incubation decrease in the amount of PHB was recorded. Hypochlorite digestion of biomass of organism followed by extraction with a solvent system consisting of 1:1 mixture of ethanol and acetone resulted in efficient recovery of PHB vis-à-vis earlier methods. This solvent system gave a high recovery yield, i.e. 5.6 gL⁻¹ vis-à-vis earlier reported yield, 1.34 gL⁻¹ (by same method), 0.63 gL⁻¹ (by chloroform extraction method) and 1.1 gL⁻¹ (by dispersion method).