Computational approach for prediction of domain organization and substrate specificity of modular polyketide synthases

Modular polyketide synthases (PKSs) are large multi-enzymatic, multi-domain megasynthases, which are involved in the biosynthesis of a class of pharmaceutically important natural products, namely polyketides. These enzymes harbor a set of repetitive active sites termed modules and the domains present in each module dictate the chemical moiety that would add to a growing polyketide chain. This modular logic of biosynthesis has been exploited with reasonable success to produce several novel compounds by genetic manipulation. However, for harnessing their vast potential of combinatorial biosynthesis, it is essential to develop knowledge based in silico approaches for correlating the sequence and domain organization of PKSs to their polyketide products. In this work, we have carried out extensive sequence analysis of experimentally characterized PKS clusters to develop an automated computational protocol for unambiguous identification of various PKS domains in a polypeptide sequence. A structure based approach has been used to identify the putative active site residues of acyltransferase (AT) domains, which control the specificities for various starter and extender units during polyketide biosynthesis. On the basis of the analysis of the active site residues and molecular modelling of substrates in the active site of representative AT domains, we have identified a crucial residue that is likely to play a major role in discriminating between malonate and methylmalonate during selection of extender groups by this domain. Structural modelling has also explained the experimentally observed chiral preference of AT domain in substrate selection. This computational protocol has been used to predict the domain organization and substrate specificity for PKS clusters from various microbial genomes. The results of our analysis as well as the computational tools for prediction of domain organization and substrate specificity have been organized in the form of a searchable computerized database (PKSDB). PKSDB would serve as a valuable tool for identification of polyketide products biosynthesized by uncharacterized PKS clusters. This database can also provide guidelines for rational design of experiments to engineer novel polyketides.     

Isolation of etiological agent of hydropericardium syndrome in chicken embryo liver cell culture and its serological characterization

The virus causing hydropericardium syndrome was isolated in chicken embryo liver (CEL) cell culture from livers obtained from naturally infected broilers. The cytopathic effects characterized by rounding and degeneration of cells were visible 36 hr post infection in first passage. At 4th passage level, the infectivity titre was 5.24 log10 TCID50/ml. In May-Grunwald and Giemsa stained cells, basophilic intranuclear inclusions ('bird eye' inclusion), typical of aviadenovirus infection, were observed. The specificity of inclusion was confirmed by indirect immunofluorescence. Various serological tests, such as agar gel precipitation test, counter immuno electrophoresis, micro serum neutralization test and enzyme linked immunosorbent assay were also standardized to confirm the isolation of etiological agent of hydropericardium syndrome in CEL cell culture and to diagnose the disease in poultry.     

Sulfonate protecting groups. Regioselective sulfonylation of myo-inositol orthoesters-improved synthesis of precursors of D- and L-myo-inositol 1,3,4,5-tetrakisphosphate,myo-inositol 1,3,4,5,6-pentakisphosphate and related derivatives

The regioselectivity of sulfonylation of myo-inositol orthoesters was controlled by the use of different bases to obtain the desired sulfonate. Monosulfonylation of myo-inositol orthoesters in the presence of one equivalent of sodium hydride or triethylamine resulted in the sulfonylation of the 4-hydroxyl group. The use of pyridine as a base for the same reaction resulted in sulfonylation of the 2-hydroxyl group. Disulfonylation of these orthoesters in the presence of excess sodium hydride yielded the 4,6-di-O-sulfonylated orthoesters. However, the use of triethylamine or pyridine instead of sodium hydride yielded the 2,4-di-O-sulfonylated orthoester. Sulfonylated derivatives of myo-inositol orthoesters were stable to conditions of O-alkylation but were cleaved using magnesium/methanol or sodium methoxide in methanol to regenerate the corresponding myo-inositol orthoester derivative. These new methods of protection-deprotection have been used: (i) for the efficient synthesis of enantiomers of 2,4-di-O-benzyl-myo-inositol, which are precursors for the synthesis of D- and L-myo-inositol 1,3,4,5-tetrakisphosphate; (ii) for the preparation of 2-O-benzyl-myo-inositol which is a precursor for the preparation of myo-inositol 1,3,4,5,6-pentakisphosphate.     

Integration of sequence tagged microsatellite sites to the chickpea genetic map

Fifty sequence-tagged microsatellite site (STMS) markers and a resistant gene-analog (RGA) locus were integrated into a chickpea ( Cicer arietinum L., 2n = 2 x = 16 chromosomes) genetic map that was previously constructed using 142 F(6)-derived recombinant inbred lines (RILs) from a cross of C. arietinum x Cicer reticulatum Lad. The map covers 1,174.5 cM with an average distance of 7.0 cM between markers in nine linkage groups (LGs). Nine markers including the RGA showed distorted segregation ( P < 0.05). The majority of the newly integrated markers were mapped to marker-dense regions of the LGs. Six co-dominant STMS markers were integrated into two previously reported major quantitative trait loci (QTLs) conferring resistance to Ascochyta blight caused by Ascochyta rabiei (Pass.) Labr. Using common STMS markers as anchors, three maps developed from different mapping populations were joined, and genes for resistance to Ascochyta blight, Fusarium wilt (caused by Fusarium oxysporum Schlechtend.: Fr. f. sp. ciceris), and for agronomically important traits were located on the combined linkage map. The integration of co-dominant STMS markers improves the map of chickpea and makes it possible to consider additional fine mapping of the genome and also map-based cloning of important disease resistance genes.     

Human genome search in celiac disease: mutated gliadin T-cell-like epitope in two human proteins promotes T-cell activation

Discovery of a number of novel and known human genes whose protein products bear striking similarity to two or more wheat gliadin domains raised the possibility that human intestinal non-HLA peptides homologous to celiac T-cell epitopes could play a role in non-HLA gene specification in celiac disease. Database searching of the entire human genome identified only 11 gut-expressed proteins with high T-cell epitope homology, particularly to the DQ2-gamma-I-gliadin epitope (i.e. TFIIA, FOXJ2 and IgD; mean BestFit quality score=40 versus random value of 24). Others were similar to DQ2-alpha-I-gliadin (i.e. PAX9; BestFit quality 46 versus 20 for random), or DQ2-alpha-II-gliadin (PHLDA1, known in mice as the T-cell death-associated gene; BestFit quality 43 versus 30 for random) epitopes. Among proteins previously screened for gliadin homology, noteworthy was achaete scute homologous protein (DQ2-alpha-I-gliadin; BestFit quality 41 versus 22 for random). With the exception of IgD, all are nuclear factors. Paying particular attention to the position of potential major histocompatibility complex (MHC) anchor residues, several were selected for testing in a DQ2-gamma-I-gliadin-restricted T-cell system. All native 10-mer peptides were inactive, even when deamidated, but V96F substitution of deamidated TFIIA amino acid residues 91-100 stimulated IL-2 release at levels exceeding the wheat gliadin positive control. Also active, but only slightly, was L1009F substitution of AIB3 amino acid residues 1004-1013. PlotSimilarity alignment of TFIIAs from eight species revealed subthreshold similarity score in the peptide region, in contrast to the highly conserved amino and carboxy termini. Molecular modeling of TFIIA[V96F] peptide points to an important juxtaposition of an upwardly projecting phenylalanine residue at peptide position 6 that likely contacts a receptor complementarity-determining region, and a downwardly projecting glutamic acid residue that fits into the shallow MHC P7 pocket. These observations tentatively point to a new multi-gene hypothesis for the initiation of celiac disease in which deamidated free human peptides with T-cell epitope homology (particularly those made more homologous by mutation) escape negative selection, as per deamidation of the HEL(48-62) peptide in the hen egg lysozyme model of autoimmunity. Deamidation following peptide release due to injury triggers inflammation, thereafter repeatedly provoked by dietary gliadin immunodominant peptides concentrated in the proximal small intestine.     

Differential action of iodine on mitochondria from human tumoral- and extra-tumoral tissue in inducing the release of apoptogenic proteins

Iodide is actively concentrated in the thyroid gland for thyroid hormone biosynthesis. Excess iodine has been observed to induce apoptosis in thyrocytes and mammary cells. The mechanism of iodine induced apoptosis is poorly understood. Among various cell organelles, mitochondria is known to provide conducive environment for the organification of iodine, i.e. iodination of different proteins. Mitochondria also play a central role in execution of apoptosis. To study the role of mitochondria in iodine induced apoptosis, we investigated the direct interaction of iodine and human breast mitochondria vis-a-vis its role in the initiation of apoptosis in vitro. We observed that mitochondria isolated from the tumor (TT) and extra-tumoral tissue (ET) of human breast display significant uptake of iodine. Mitochondrial proteins were observed to be predominantly iodinated in ET but not in TT mitochondria. Treatment with iodine showed an increase in mitochondrial permeability transition of TT and decrease in ET. Iodine induced released factor(s) other than cytochrome c from tumor mitochondria initiate(s) apoptosis in vitro, while those from ET mitochondria were non-apoptogenic in nature. To our knowledge, this is first report demonstrating that iodine acts differentially on mitochondria of tumor and extratumoral origin to release apoptogenic proteins from TT and has a protective effect on ET.     

Structural insights into the membrane-anchoring mechanism of a cholesterol-dependent cytolysin

Perfringolysin O (PFO), a cytolytic toxin secreted by pathogenic Clostridium perfringens, forms large pores in cholesterol-containing membranes. Domain 4 (D4) of the protein interacts first with the membrane and is responsible for cholesterol recognition. By using several independent fluorescence techniques, we have determined the topography of D4 in the membrane-inserted oligomeric form of the toxin. Only the short hydrophobic loops at the tip of the D4 beta-sandwich are exposed to the bilayer interior, whereas the remainder of D4 projects from the membrane surface and is surrounded by water, making little or no contact with adjacent protein monomers in the oligomer. Thus, a limited interaction of D4 with the bilayer core seems to be sufficient to accomplish cholesterol recognition and initial binding of PFO to the membrane. Furthermore, D4 serves as the fulcrum around which extensive structural changes occur during the formation and insertion of the large transmembrane beta-barrel into the bilayer.     

Reductive unfolding and oxidative refolding of a Bowman-Birk inhibitor from horsegram seeds (Dolichos biflorus): evidence for "hyperreactive" disulfide bonds and rate-limiting nature of disulfide isomerization in folding

Horsegram protease inhibitor belongs to the Bowman-Birk class (BBIs) of low molecular weight (8-10 kDa), disulfide-rich, "dual" inhibitors, which can bind and inhibit trypsin and chymotrypsin either independently or simultaneously. They have seven conserved disulfide bonds. Horsegram BBI exhibits remarkable stability against denaturants like urea, guanidine hydrochloride (GdmCl) and heat, which can be attributed to these conserved disulfide bonds. On reductive denaturation, horsegram BBI follows the "two-state" mode of unfolding where all the disulfide bonds are reduced simultaneously resulting in the fully reduced protein without any accumulation of partially reduced intermediates. Reduction with dithiothreitol (DTT) followed apparent first-order kinetics and the rate constants (k(r)) indicated that the disulfide bonds were "hyperreactive" in nature. Oxidative refolding of the fully reduced and denatured inhibitor was possible at very low protein concentration in the presence of "redox" combination of reduced and oxidized glutathiones. Simultaneous recovery of trypsin and chymotryptic inhibitory activities indicated the concomitant folding of both the inhibitory subdomains. Folding efficiency decreased in the absence of the glutathiones and in the presence of denaturants (6 M urea and 4 M GdmCl), indicating the importance of disulfide shuffling and the formation of noncovalent interactions and secondary structural elements, respectively, for folding efficiency. Folding rate was significantly improved in the presence of protein disulfide isomerase (PDI). A 3-fold enhancement of rate was observed in the presence of PDI at molar ratio of 1:20 (PDI/inhibitor), indicating that disulfide bond formation and isomerization to be rate limiting in folding. Peptide prolyl cis-trans isomerase (PPI) did not affect rate at low concentrations, but at molar ratios of 1:1.5 (PPI/inhibitor), there was 1.4-fold enhancement of the folding rate, indicating that the prolyl imidic bond isomerizations may be slowing down the folding reaction but were not rate limiting.     

Plant regeneration from embryo-derived callus of oil palm – the effect of exogenous polyamines

Regeneration in oil palm was achieved through somatic embryogenesis/organogenesis from embryo-derived callus. Callus was induced from mature embryos of the cross 281 (D)×18 (P) on modified MS medium supplemented with 2,4-D (113.12 M) and 2-iP (14.76 M). The embryogenic calluses obtained were transferred to Blaydes medium supplemented with 2,4-D (0.045 M) and one of the following growth regulators: TDZ (4.54 M), zeatin riboside (2.85 M), putrescine (1 mM) and spermine (100 M). Secondary somatic embryogenesis was found to occur in media supplemented with polyamines. The efficiency of formation of somatic embryos, secondary somatic embryos and shoot meristemoids were significantly higher in putrescine containing medium. Histological studies were also undertaken.