Anthocyanins from Ocimum sanctum L., a promising biomolecule for development of cost-effective and widely applicable pH indicator.

A novel cost-effective and widely applicable pH indicator was developed using anthocyanins extracted from the purple subtype of Ocimum sanctum L. and common lab filter paper. This pH indicator was successfully tested to monitor the pH of a wide range of buffers, solutions, irrigation water, and soil solution. Upon testing, the indicator displayed specific colors at corresponding pH ranges. Sucrose showed a stabilizing effect for the color of the extracted anthocyanins. Further, molecular analysis indicated that the leaves from the purple subtypes showed higher transcripts abundance for chalcone synthase, chalcone isomerase, anthocyanidin synthase, and dihydroflavonol 4-reductase than that of the green subtype. Similarly, transcription factors HY5 and a bHLH putatively involved in the biosynthesis of anthocyanins showed up-regulation in the purple subtype of O. sanctum.

NADP⁺ binding effects tryptophan accessibility, folding and stability of recombinant B. malayi G6PD.

Brugia malayi Glucose 6-phosphate dehydrogenase apoenzyme (BmG6PD) was expressed and purified by affinity chromatography to study the differences in kinetic properties of enzyme and the effect of the cofactor NADP(+) binding on enzyme stability. The presence of cofactor NADP(+) influenced the tertiary structure of enzyme due to significant differences in the tryptophan microenvironment. However, NADP(+) binding have no effect on secondary structure of the enzyme. Quenching with acrylamide indicated that two or more tryptophan residues became accessible upon cofactor binding. Unfolding and cross linking study of BmG6PD showed that NADP(+) stabilized the protein in presence of high concentration of urea/GdmCl. A homology model of BmG6PD constructed using human G6PD (PDB id: 2BH9) as a template indicated 34% α-helix, 19% ß-sheet and 47% random coil conformations in the predicted model of the enzyme. In the predicted model binding of NADP(+) to BmG6PD was less tight with the structural sites (-10.96 kJ/mol binding score) as compared with the coenzyme site (-15.47 kJ/mol binding score).

Identification and in silico analysis of cattle DExH/D box RNA helicases.

The helicases are motor proteins participating in a range of nucleic acid metabolisms. RNA helicase families are characterized by the presence of conserved motifs. This article reports a comprehensive in silico analysis of Bos taurus DExH/D helicase members. Bovine helicases were identified using the helicase domain sequences including 38 DDX (DEAD box) and 16 DHX (DEAH box) members. Signature motifs were used for the validation of these proteins. Putative sub cellular localization and phylogenetic relationship for these RNA helicases were established. Comparative analysis of these proteins with human DDX and DHX members was carried out. These bovine helicase have been assigned putative physiological functions. Present study of cattle DExH/D helicase will provides an invaluable source for the detailed biochemical and physiological research on these members.

Molecular cloning and characterization of Brugia malayi thymidylate kinase.

Thymidylate kinase (TMK) is a potential chemotherapeutic target because it is directly involved in the synthesis of deoxythymidine triphosphate, which is an essential component for DNA synthesis. The gene encoding thymidylate kinase of Brugia malayi was amplified by PCR and expressed in Escherichia coli. The native molecular weight of recombinant B. malayi thymidylate kinase (rBmTMK) was estimated to be ∼52kDa by gel filtration chromatography, suggesting a homodimeric structure. rBmTMK activity required divalent cation and Mg(2+) was found to be the most effective cation. The enzyme was sensitive to pH and temperature, it showed maximum activity at pH 7.4 and 37°C. The Km values for dTMP and ATP were 17 and 66µM, respectively. The turnover number kcat was found to be 38.09s(-1), a value indicating the higher catalytic efficiency of the filarial enzyme. The nucleoside analogues 5-bromo-2'-deoxyuridine (5-BrdU), 5-chloro-2'-deoxyuridine (5-CldU) and 3'-azido-3'-deoxythymidine (AZT) showed specific inhibitory effect on the enzyme activity and these effects were in good association with binding interactions and the scoring functions as compared to human TMK. Differences in kinetic properties and structural differences in the substrate binding site of BmTMK model with respect to human TMK can serve as basis for designing specific inhibitors against parasitic enzyme.

Behavior of Plasmodium falciparum purine nucleoside phosphorylase in macromolecular crowded environment.

Biochemical and biophysical properties of enzymes have been studied in dilute buffer system, which are far from the crowded physiological condition of cell. We report the enzyme kinetics and refolding of Plasmodium falciparum purine nucleoside phosphorylase under crowded conditions. Enzyme catalytic efficiency was inversely affected in the presence of polyethylene glycols and Dextran whereas it was increased in the presence of osmolytes. We detected a non-linear relationship between Km and increasing macromolecular crowding agents. At low concentrations of PEGs and Dextran, we observed decreased substrate binding whereas higher concentrations of PEGs and Dextran favored substrate binding. The presence of sucrose decreased the Km values. We detected decrease in Kcat value in the presence of PEGs and Dextran, whereas osmolytes increased the Kcat values. Thermal resistance of enzyme was increased in the presence of crowding agents. Intrinsic and extrinsic fluorescence analysis indicated change around active site loop region having single tryptophan residue. Preferential exclusions of polyols favor the catalytic mechanism of the enzyme. Urea denatured enzyme showed fast refolding when diluted and rate of refolding was not affected by the presence of crowding agents. It is important to draw together significant knowledge about modulation of inherent properties of this enzyme in crowded environment which will be helpful in better understanding of this drug-target enzyme and in further inhibitor design.

Single tryptophan of disordered loop from Plasmodium falciparum purine nucleoside phosphorylase: involvement in catalysis and microenvironment.

Among various tropical diseases, malaria is a major life-threatening disease caused by Plasmodium parasite. Plasmodium falciparum is responsible for the deadliest form of malaria, so-called cerebral malaria. Purine nucleoside phosphorylase from P. falciparum is a homohexamer containing single tryptophan residue per subunit that accepts inosine and guanosine but not adenosine for its activity. This enzyme has been exploited as drug target against malaria disease. It is important to draw together significant knowledge about inherent properties of this enzyme which will be helpful in better understanding of this drug target. The enzyme shows disorder to order transition during catalysis. The single tryptophan residue residing in conserved region of transition loop is present in purine nucleoside phosphorylases throughout the Plasmodium genus. This active site loop motif is conserved among nucleoside phosphorylases from apicomplexan parasites. Modification of tryptophan residue by N-bromosuccinamide resulted in complete loss of activity showing its importance in catalysis. Inosine was not able to protect enzyme against N-bromosuccinamide modification. Extrinsic fluorescence studies revealed that tryptophan might not be involved in substrate binding. The tryptophan residue localised in electronegative environment showed collisional and static quenching in the presence of quenchers of different polarities.

Genome wide computational analysis of Brugia malayi helicases: a comparison with human host.

The availability of Brugia malayi genome sequence has paved ways for the search of homologues for a variety of genes. Helicases are ubiquitous enzymes involved in all the nucleic acid metabolic pathways and are essential for the development and growth. The genome wide analysis of B. malayi for different helicases showed the presence of a number of DEAD box helicases, 7 DEAH box helicases, RecQ helicases, repair helicases, super killer helicases, MCM2-7 complex, Rad54 and two subunits of Ku helicase. The comparison of protein sequence of each helicase with its human counterpart indicated characteristic differences in filarial helicases. There are noticeable differences in some of the filarial helicases such as DHX35, RecQL1 and Ku. Further characterization of these helicases will help in understanding physiological significance of these helicases in filarial parasites, which in future can be utilized for chemotherapy of parasitic infection.