Computational modeling and <Emphasis Type="Italic">in silico</Emphasis> analysis of differential regulation of <Emphasis Type="Italic">myo</Emphasis>-inositol catabolic enzymes in <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis>

Background  

Inositol is a key cellular metabolite for many organisms. Cryptococcus neoformans is an opportunistic pathogen which primarily infects the central nervous system, a region of high inositol concentration, of immunocompromised individuals. Through the use of myo-inositol oxygenase C. neoformans can catabolize inositol as a sole carbon source to support growth and viability.     

Degradation of <Emphasis Type="Italic">myo</Emphasis>-inositol Hexakisphosphate by a Phytate-degrading Enzyme from <Emphasis Type="Italic">Pantoea agglomerans</Emphasis>

High-pressure liquid chromatography (HPLC) analysis established myo-inositol pentakisphosphate as the final product of phytate dephosphorylation by the phytate-degrading enzyme from Pantoea agglomerans. Neither product inhibition by phosphate nor inactivation of the Pantoea enzyme during the incubation period were responsible for the limited phytate hydrolysis as shown by addition of phytate-degrading enzyme and phytate, respectively, after the observed stop of enzymatic phytate degradation. In additon, the Pantoea enzyme did not possess activity toward the purified myo-inositol pentakisphosphate. Using a combination of High-Performance Ion Chromatography (HPIC) analysis and kinetic studies, the nature of the generated myo-inositol pentakisphosphate was established. The data demonstrate that the phytate-degrading enzyme from Pantoea agglomerans dephosphorylates myo-inositol hexakisphosphate in a stereospecific way to finally D-myo-inositol(1,2,4,5,6)pentakisphosphate.     

<Emphasis Type="Italic">In silico</Emphasis> and <Emphasis Type="Italic">in situ</Emphasis> characterization of the zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) <Emphasis Type="Italic">gnrh3</Emphasis> (sGnRH) gene

Background

Gonadotropin releasing hormone (GnRH) is responsible for stimulation of gonadotropic hormone (GtH) in the hypothalamus-pituitary-gonadal axis (HPG). The regulatory mechanisms responsible for brain specificity make the promoter attractive for in silico analysis and reporter gene studies in zebrafish (Danio rerio).

Results

We have characterized a zebrafish [Trp⁷, Leu⁸] or salmon (s) GnRH variant, gnrh 3. The gene includes a 1.6 Kb upstream regulatory region and displays the conserved structure of 4 exons and 3 introns, as seen in other species. An in silico defined enhancer at -976 in the zebrafish promoter, containing adjacent binding sites for Oct-1, CREB and Sp1, was predicted in 2 mammalian and 5 teleost GnRH promoters. Reporter gene studies confirmed the importance of this enhancer for cell specific expression in zebrafish. Interestingly the promoter of human GnRH-I, known as mammalian GnRH (mGnRH), was shown capable of driving cell specific reporter gene expression in transgenic zebrafish.

Conclusions

The characterized zebrafish Gnrh3 decapeptide exhibits complete homology to the Atlantic salmon (Salmo salar) GnRH-III variant. In silico analysis of mammalian and teleost GnRH promoters revealed a conserved enhancer possessing binding sites for Oct-1, CREB and Sp1. Transgenic and transient reporter gene expression in zebrafish larvae, confirmed the importance of the in silico defined zebrafish enhancer at -976. The capability of the human GnRH-I promoter of directing cell specific reporter gene expression in zebrafish supports orthology between GnRH-I and GnRH-III.

     

Complete hydrolysis of <Emphasis Type="Italic">myo</Emphasis>-inositol hexakisphosphate by a novel phytase from <Emphasis Type="Italic">Debaryomyces castellii</Emphasis> CBS 2923

Debaryomyces castellii phytase was purified to homogeneity in a single step by hydrophobic interaction chromatography. Its molecular mass is 74 kDa with 28.8% glycosylation. Its activity was optimal at 60°C and pH 4.0. The K _m value for sodium phytate was 0.532 mM. The enzyme exhibited a low specificity and hydrolyzed many phosphate esters. The phytase fully hydrolyzed myo-inositol hexakisphosphate (or phytic acid, Ins P₆) to inositol and inorganic phosphate. The sequence of Ins P₆ hydrolysis was determined by combining results from high-performance ionic chromatography and nuclear magnetic resonance. D. castellii phytase is a 3-phytase that sequentially releases phosphate groups through Ins (1,2,4,5,6) P₅, Ins (1,2,5,6) P₄, Ins (1,2,6) P₃, Ins (1,2) P₂, Ins (1 or 2) P₁, and inositol (notation 3/4/5/6/1 or 2).     

<Emphasis Type="Italic">In silico</Emphasis> physical mapping software for the <Emphasis Type="Italic">Triticum aestivum</Emphasis> genome

The large size of the Triticum aestivum genome makes it unlikely that a complete genome sequence for wheat will be available in the near future. Exploiting the conserved genome organization between wheat and rice and existing genomic resources, we have constructed in silico physical mapping software for wheat, assigning a gross physical location(s) into chromosome bins to 22,626 representative wheat gene sequences. To validate the predictions from the software we compared the predicted locations of ten ESTs to their positions experimentally determined by SNP marker analysis. Six of the sequences were correctly positioned on the map including four that demonstrated a high level of colinearity with their orthologous rice genomic region. This tool will facilitate the development of molecular markers for regions of interest and the creation of map-based cloning strategies in areas demonstrating high levels of sequence conservation and organization between wheat and rice.     

<Emphasis Type="Italic">In silico</Emphasis> prediction of horizontal gene transfer in <Emphasis Type="Italic">Streptococcus thermophilus</Emphasis>

A combination of gene loss and acquisition through horizontal gene transfer (HGT) is thought to drive Streptococcus thermophilus adaptation to its niche, i.e. milk. In this study, we describe an in silico analysis combining a stochastic data mining method, analysis of homologous gene distribution and the identification of features frequently associated with horizontally transferred genes to assess the proportion of the S. thermophilus genome that could originate from HGT. Our mining approach pointed out that about 17.7% of S. thermophilus genes (362 CDSs of 1,915) showed a composition bias; these genes were called ‘atypical’. For 22% of them, their functional annotation strongly support their acquisition through HGT and consisted mainly in genes encoding mobile genetic recombinases, exopolysaccharide (EPS) biosynthesis enzymes or resistance mechanisms to bacteriophages. The distribution of the atypical genes in the Firmicutes phylum as well as in S. thermophilus species was sporadic and supported the HGT prediction for more than a half (52%, 189). Among them, 46 were found specific to S. thermophilus. Finally, by combining our method, gene annotation and sequence specific features, new genome islands were suggested in the S. thermophilus genome.     

<Emphasis Type="Italic">In silico</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis> analysis reveal a novel gene in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> trehalose metabolism

Background

The ability to respond rapidly to fluctuations in environmental changes is decisive for cell survival. Under these conditions trehalose has an essential protective function and its concentration increases in response to enhanced expression of trehalose synthase genes, TPS1, TPS2, TPS3 and TSL1. Intriguingly, the NTH1 gene, which encodes neutral trehalase, is highly expressed at the same time. We have previously shown that trehalase remains in its inactive non-phosphorylated form by the action of an endogenous inhibitor. Recently, a comprehensive two-hybrid analysis revealed a 41-kDa protein encoded by the YLR270w ORF, which interacts with NTH1p.

Results

In this work we investigate the correlation of this Trehalase Associated Protein, in trehalase activity regulation. The neutral trehalase activity in the ylr270w mutant strain was about 4-fold higher than in the control strain. After in vitro activation by PKA the ylr270w mutant total trehalase activity increased 3-fold when compared to a control strain. The expression of the NTH1 gene promoter fused to the heterologous reporter lacZ gene was evaluated. The mutant strain lacking YLR270w exhibited a 2-fold increase in the NTH1-lacZ basal expression when compared to the wild type strain.

Conclusions

These results strongly indicate a central role for Ylr270p in inhibiting trehalase activity, as well as in the regulation of its expression preventing a wasteful futile cycle of synthesis-degradation of trehalose.

     

<Emphasis Type="Italic">In silico</Emphasis> analysis of paraoxon binding by human and bovine serum albumin

Albumin is known to be able to cleave ether bonds in organophosphates (OPs). Amino acids responsible for esterase and pseudo-esterase albumin activity towards OPs are not yet finally identified. Presumably, Sudlow’s site I with the Tyr150 residue shows a “true” esterase activity, while Sudlow’s II site with the Tyr411 residue—a pseudo-esterase one. Both human (HSA) and bovine (BSA) serum albumins were used in in vitro studies of albumin (pseudo)esterase activity towards OPs. There is a body of evidence that the efficiency of interaction of different xenobiotics differs for these two proteins. Using paraoxon as an example, the aim of this study was to conduct an in silico study of the OP interaction with the previously identified potential sites of HSA and BSA (pseudo)esterase activity, to estimate the possibility of enzymatic reactions at these sites, to comparatively analyze these proteins from the evolutionary viewpoint, and to assess the possibility of extrapolating the experimental data obtained on BSA to a human organism. Molecular docking of paraoxon into the sites of HSA and BSA potential (pseudo)esterase activity has been performed. Conformational changes occurring in the resultant complexes with time have been studied by molecular dynamics simulation. It has been shown that Sudlow’s site II is less liable to evolutionary changes. Binding of modulators at other sites is not required for productive sorption of OPs and the phosphorylation reaction at Sudlow’s site II. It has been concluded that simi lar results for HSA and BSA could be expected for the irreversible binding of OPs at Sudlow’s site II. Since Sudlow’s site I is less conservative, diff erent binding efficiency could be expected for rigid molecules or optically active compounds. Both for HSA and BSA, productive binding of OPs at Sudlow’s site I is possible only after changes in the albumin molecule structure induced by binding of modulators at other sites.     

Effects of exogenous <Emphasis Type="Italic">myo</Emphasis>-inositol on leaf water status and oxidative stress of <Emphasis Type="Italic">Capsicum annuum</Emphasis> under drought stress

Drought-stressed plants accumulate cyclitols such as myo-inositol, pinitol, quercitol in the cytosol. These solutes (compatible solutes) protect plants from stress effects. Synthetic myo-inositol was used in the investigation of drought stress tolerance in pepper plants. Hydrogen peroxide (H₂O₂), membrane damage, ascorbate peroxidase (AP), catalase (CAT), proline and calcium increased in plants under drought conditions. Water status, calcium level, glutathione reductase activities increased in myo-inositol treated Capsicum annuum L. (pepper) under drought stress. Exogenous myo-inositol significantly decreased H₂O₂, membrane damage and proline levels and AP (except for 5 µM) and CAT activity, compared with untreated plants. Myo-inositol can play a role as effective as proline in signal transduction and in regulating concentrations of reactive oxygen species within tolerable ranges and in maintaining cell turgor by binding water molecules. Myo-inositol may become a useful instrument to eliminate the negative effects of drought environments.     

<Emphasis Type="Italic">In silico</Emphasis> analysis and experimental improvement of taxadiene heterologous biosynthesis in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The biosynthesis of terpenoids in heterologous hosts has become increasingly popular. Isopentenyl diphosphate (IPP) is the central precursor of all isoprenoids, and the synthesis can proceed via two separate pathways in different organisms: The 1-deoxylulose 5-phosphate (DXP) pathway and the mevalonate (MVA) pathway. In this study, an in silico comparison was made between the maximum theoretical IPP yields and the thermodynamic properties of the DXP and MVA pathways using different hosts and carbon sources. We found that Escherichia coli and its DXP pathway have the most potential for IPP production. Consequently, codon usage redesign, and combinations of chromosomal engineering and various strains were considered for optimizing taxadiene biosynthesis through the endogenic DXP pathway. A high production strain yielding 876 ± 60 mg/L taxadiene, with an overall volumetric productivity of 8.9 mg/(L × h), was successfully obtained by combining the chromosomal engineered upstream DXP pathway and the downstream taxadiene biosynthesis pathway. This is the highest yield thus far reported for taxadiene production in a heterologous host. These results indicate that genetic manipulation of the DXP pathway has great potential to be used for production of terpenoids, and that chromosomal engineering is a powerful tool for heterologous biosynthesis of natural products.     

<Emphasis Type="Italic">In silico</Emphasis> identification of essential proteins in <Emphasis Type="Italic">Corynebacterium pseudotuberculosis</Emphasis> based on protein-protein interaction networks

Background

Corynebacterium pseudotuberculosis (Cp) is a gram-positive bacterium that is classified into equi and ovis serovars. The serovar ovis is the etiological agent of caseous lymphadenitis, a chronic infection affecting sheep and goats, causing economic losses due to carcass condemnation and decreased production of meat, wool, and milk. Current diagnosis or treatment protocols are not fully effective and, thus, require further research of Cp pathogenesis.

Results

Here, we mapped known protein-protein interactions (PPI) from various species to nine Cp strains to reconstruct parts of the potential Cp interactome and to identify potentially essential proteins serving as putative drug targets. On average, we predict 16,669 interactions for each of the nine strains (with 15,495 interactions shared among all strains). An in silico sanity check suggests that the potential networks were not formed by spurious interactions but have a strong biological bias. With the inferred Cp networks we identify 181 essential proteins, among which 41 are non-host homologous.

Conclusions

The list of candidate interactions of the Cp strains lay the basis for developing novel hypotheses and designing according wet-lab studies. The non-host homologous essential proteins are attractive targets for therapeutic and diagnostic proposes. They allow for searching of small molecule inhibitors of binding interactions enabling modern drug discovery. Overall, the predicted Cp PPI networks form a valuable and versatile tool for researchers interested in Corynebacterium pseudotuberculosis.

     

<Emphasis Type="Italic">In vitro</Emphasis> and <Emphasis Type="Italic">in silico</Emphasis> studies on fibrinolytic activity of nattokinase: A clot buster from <Emphasis Type="Italic">Bacillus</Emphasis> sp.

Background

Nattokinase (NK) is a serine protease enzyme of the subtilisin family. It exhibits a strong fibrinolytic activity. The fibrinolytic enzymes from Bacillus sp. have attracted interest as thrombolytic agents because of their efficiency in the fibrinolytic process including plasmin activation.

Methods

In the present study, VIT garden soil was collected and subjected to isolation process in order to screen for the NK production. Screening for NK enzyme was performed by radial caseinolytic assay. The production of NK enzyme was done in two different production medium for comparative studies. The NK enzyme was purified by gel permeation chromatography. The activity of the purified NK was checked by clot lysis and casein digestion assay. To investigate the structural basis of NK and fibrinogen interaction and also to identify the best binding mode, molecular dynamics and docking studies were performed.

Results

Based on the morphological and biochemical characterization, the isolate was identified as Bacillus sp. The overall purification fold of NK was about 3 with the specific activity of 664U/mg and 9.9% yield. Homogeneity of the purified enzyme was analyzed and confirmed by the single band obtained in SDS-PAGE. Molecular weight of the purified protease was estimated as 25 kDa. Purified NK enzyme exhibited 97% of effective clot lysis activity. The NK was docked in to the knob region of the fibrinogen at its binding site using Dock server. A total of 26 residues of fibrinogen and 29 residues of NK constitute the interface region. However, 9 residues of fibrinogen (THR238, MET264, LYS266, ARG275, THR277, ALA279, ASN308, MET310, and LYS321) and 8 residues of NK (GLY61, SER63, THR99, PHE189, LEU209, TYR217, ASN218, and MET222) are involved in intact binding.

Conclusions

A significant amount of NK enzyme was obtained from Bacillus sp. The docking analysis revealed that the NK and fibrinogen adopt an extended binding pattern and interacts with the crucial residues to exhibit their activity.

     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Comparative <Emphasis Type="Italic">in silico</Emphasis> analysis of chemotaxis system of <Emphasis Type="Italic">Campylobacter fetus</Emphasis>

Chemoreceptor and chemotaxis signal transduction cascade genes of C. fetus subsp. fetus 82-40 show high level of similarity to that in C. jejuni and appears to include sixteen diverse transducer-like protein (tlp) genes that appear similar to nine of the twelve tlp genes in the C. jejuni NCTC 11168 with a percent identity ranging from 15 to 50%. Sixteen putative C. fetus 82-40 tlp genes belong to three classes: A, B, and C, as well as an aerotaxis gene, based on their predicted structure. C. fetus subsp. fetus 82-40 chemoreceptor and chemotaxis signal transduction pathway genes have close phylogenetic relationship of chemotaxis genes between Campylobacteraceae and Helicobacteraceae.