Functional analyses of a highly thermostable hexokinase from Pyrobaculum calidifontis.

The gene encoding a repressor open reading frame sugar kinase (ROK) family protein from hyperthermophilic crenarchaeon Pyrobaculum calidifontis, Pcal-HK, was cloned and expressed in Escherichia coli. The recombinant protein was produced in soluble and highly active form. Purified Pcal-HK was highly thermostable and existed in a monomeric form in solution. The enzyme was specific to ATP as phosphoryl donor but showed broad specificity to phosphoryl acceptors. It catalyzed the phosphorylation of a number of hexoses, including glucose, glucosamine, N-acetyl glucosamine, fructose and mannose, at nearly the same rate and similar affinity. The enzyme was metal ion dependent exhibiting highest activity at 90-95 °C and pH 8.5. Mg2+ was most effective metal ion, which could be partially replaced by Mn2+, Ni2+ or Zn2+. Kinetic parameters were determined at 90 °C and the enzyme showed almost similar catalytic efficiency (kcat/Km) towards the above mentioned hexoses. To the best of our knowledge, Pcal-HK is the most active thermostable ROK family hexokinase characterized to date which catalyzes the phosphorylation of various hexoses with nearly similar affinity.

ADP-dependent glucose/glucosamine kinase from Thermococcus kodakarensis: cloning and characterization.

The genome sequence of Thermococcus kodakarensis contains an open reading frame, TK1110, annotated as ADP-dependent glucokinase. The encoding gene was expressed in Escherichia coli and the gene product, TK-GLK, was produced in soluble and active form. The recombinant enzyme was extremely thermostable. Thermostability was increased significantly in the presence of ammonium sulfate. ADP was the preferred co-factor for TK-GLK, which could be replaced with CDP but with a 60% activity. TK-GLK was a metal ion-dependent enzyme which exhibited glucokinase, glucosamine kinase and glucose 6-phosphatase activities. It catalyzed the phosphorylation of both glucose and glucosamine with nearly the same rate and affinity. The apparent Km values for glucose and glucosamine were 0.48 ± 0.03 and 0.47 ± 0.09 mM, respectively. The catalytic efficiency (kcat/Km) values against these two substrates were 6.2 × 105 ± 0.25 and 5.8 × 105 ± 0.75 M-1 s-1. The apparent Km value for dephosphorylation of glucose 6-phosphate was ~14-fold higher than that of glucose phosphorylation. Similarly, catalytic efficiency (kcat/Km) for phosphatase reaction was ~19-fold lower than that for the kinase reaction. To the best of our knowledge, this is the first report that describes the reversible nature of a euryarchaeal ADP-dependent glucokinase.

Biochemical characterization of a highly active ADP-dependent phosphofructokinase from Thermococcus kodakarensis.

The genome sequence of Thermococcus kodakarensis contains an open reading frame, TK0376, annotated as ADP-dependent phosphofructokinase belonging to pfkC family. The encoding gene was expressed in Escherichia coli and the gene product was characterized. The recombinant protein was produced in soluble and active form. Phosphofructokinase activity of TK0376 was metal-ion dependent and the highest activity (5090 µmol min-1 mg-1) was found in the presence of Co2+ followed by Mg2+ (3280 µmol min-1 mg-1) at 90°C and pH 7.5. TK0376 preferred ADP as phosphoryl donor, however, it could be replaced by ATP but with a 5-fold lower activity. It catalyzed the phosphorylation of fructose 6-phosphate and dephosphorylation of fructose 1,6-bisphosphate. In addition, it was able to phosphorylate glucose and nucleosides but with a much lower rate compared to that of fructose 6-phosphate. The apparent kcat and Km values against fructose 6-phosphate were 4238 s-1 and 0.74 mM, respectively. The rate of dephosphorylation of fructose 1,6-bisphosphate was 3-times lower at 50°C than the phosphorylation of fructose 6-phosphate. Similarly, the rate of phosphorylation of glucose was 450-fold lower than that of fructose 6-phosphate. Phosphofructokinase activity was not allosterically regulated, but it was slightly enhanced by phosphoenol pyruvate, and inhibited by ATP and AMP in a competitive manner.

A Phosphofructokinase Homolog from Pyrobaculum calidifontis Displays Kinase Activity towards Pyrimidine Nucleosides and Ribose 1-Phosphate.

The genome of the hyperthermophilic archaeon Pyrobaculum calidifontis contains an open reading frame, Pcal_0041, annotated as encoding a PfkB family ribokinase, consisting of phosphofructokinase and pyrimidine kinase domains. Among the biochemically characterized enzymes, the Pcal_0041 protein was 37% identical to the phosphofructokinase (Ape_0012) from Aeropyrum pernix, which displayed kinase activity toward a broad spectrum of substrates, including sugars, sugar phosphates, and nucleosides, and 36% identical to a phosphofructokinase from Desulfurococcus amylolyticus To examine the biochemical function of the Pcal_0041 protein, we cloned and expressed the gene and purified the recombinant protein. Although the Pcal_0041 protein contained a putative phosphofructokinase domain, it exhibited only low levels of phosphofructokinase activity. The recombinant enzyme catalyzed the phosphorylation of nucleosides and, to a lower extent, sugars and sugar phosphates. Surprisingly, among the substrates tested, the highest activity was detected with ribose 1-phosphate (R1P), followed by cytidine and uridine. The catalytic efficiency (kcat/Km ) toward R1P was 11.5 mM-1 · s-1 ATP was the most preferred phosphate donor, followed by GTP. Activity measurements with cell extracts of P. calidifontis indicated the presence of nucleoside phosphorylase activity, which would provide the means to generate R1P from nucleosides. The study suggests that, in addition to the recently identified ADP-dependent ribose 1-phosphate kinase (R1P kinase) in Thermococcus kodakarensis that functions in the pentose bisphosphate pathway, R1P kinase is also present in members of the Crenarchaeota.IMPORTANCE The discovery of the pentose bisphosphate pathway in Thermococcus kodakarensis has clarified how this archaeon can degrade nucleosides. Homologs of the enzymes of this pathway are present in many members of the Thermococcales, suggesting that this metabolism occurs in these organisms. However, this is not the case in other archaea, and degradation mechanisms for nucleosides or ribose 1-phosphate are still unknown. This study reveals an important first step in understanding nucleoside metabolism in Crenarchaeota and identifies an ATP-dependent ribose 1-phosphate kinase in Pyrobaculum calidifontis The enzyme is structurally distinct from previously characterized archaeal members of the ribokinase family and represents a group of proteins found in many crenarchaea.

Optimised synthesis of ZnO-nano-fertiliser through green chemistry: boosted growth dynamics of economically important L. esculentum.

Mounting-up economic losses to annual crops yield due to micronutrient deficiency, fertiliser inefficiency and increasing microbial invasions (e.g. Xanthomonas cempestri attack on tomatoes) are needed to be solved via nano-biotechnology. So keeping this in view, the authors' current study presents the new horizon in the field of nano-fertiliser with highly nutritive and preservative effect of green fabricated zinc oxide-nanostructures (ZnO-NSs) during Lycopersicum esculentum (tomato) growth dynamics. ZnO-NS prepared via green chemistry possesses highly homogenous crystalline structures well-characterised through ultraviolet and visible spectroscopy, Fourier transform infrared spectroscopy, X-ray diffraction and scanning electron microscope. The ZnO-NS average size was found as small as 18 nm having a crystallite size of 5 nm. L. esculentum were grown in different concentrations of ZnO-NS to examine the different morphological parameters includes time of seed germination, germination percentage, the number of plant leaves, the height of the plant, average number of branches, days count for flowering and fruiting time period along with fruit quantity. Promising results clearly predict that bio-fabricated ZnO-NS at optimum concentration resulted as growth booster and dramatically triggered the plant yield.

Enhancement of gene expression in Escherichia coli and characterization of highly stable ATP-dependent glucokinase from Pyrobaculum calidifontis.

The genome of the hyperthermophilic archaeon Pyrobaculum calidifontis contains an open reading frame, Pcal_1032, annotated as glucokinase. Amino acid sequence analysis showed that Pcal_1032 belonged to ROK (repressor, open reading frame, and kinase) family of sugar kinases. To examine the properties of Pcal_1032, the coding gene was cloned and expressed in Escherichia coli. However, expression of the gene was low resulting in a poor yield of the recombinant protein. A single site directed mutation in Pcal_1032 gene, without altering the amino acid sequence, resulted in approximately tenfold higher expression. Purified recombinant Pcal_1032 efficiently phosphorylated various hexoses with a marked preference for glucose. ATP was the most preferred phosphoryl group donor. Optimum temperature and pH for the glucokinase activity of Pcal_1032 were 95 °C and 8.5, respectively. Catalytic efficiency (k cat/K m) towards glucose was 437 mM-1 s-1. The recombinant enzyme was highly stable against temperature with a half-life of 25 min at 100 °C. In addition, Pcal_1032 was highly stable in the presence of denaturants. There was no significant change in the CD spectra and enzyme activity of Pcal_1032 even after overnight incubation in the presence of 8 M urea. To the best of our knowledge, Pcal_1032 is the most active and highly stable glucokinase characterized to date from archaea, and this is the first description of the characterization of a glucokinase from genus Pyrobaculum.

Pcal_1127, a highly stable and efficient ribose-5-phosphate pyrophosphokinase from Pyrobaculum calidifontis.

Analysis of the genome sequence of Pyrobaculum calidifontis revealed the presence of an open reading frame Pcal_1127 annotated as ribose-5-phosphate pyrophosphokinase. To examine the properties of Pcal_1127 the coding gene was cloned, expressed in Escherichia coli, and the purified gene product was characterized. Pcal_1127 exhibited higher activity when ATP was replaced by dATP as pyrophosphate donor. Phosphate and EDTA activated the enzyme activity and equivalent amount of activity was detected with ATP and dATP in their presence. Recombinant Pcal_1127 could utilize all the four nucleotides as pyrophosphate donors with a marked preference for ATP. Optimum temperature and pH for the enzyme activity were 55 °C and 10.5, respectively. A unique feature of Pcal_1127 was its stability against temperature as well as denaturants. Pcal_1127 exhibited more than 95 % residual activity after heating for 4 h at 90 °C and a half-life of 15 min in the boiling water. The enzyme activity was not affected by the presence of 8 M urea or 4 M guanidinium chloride. Pcal_1127 was a highly efficient enzyme with a catalytic efficiency of 5183 mM-1 s-1. These features make Pcal_1127, a novel and unique ribose-5-phosphate pyrophosphokinase.

Ethnomedicinal uses of plants in the treatment of paediatric geohelminth infections in Kalat district of Northern Balochistan, Pakistan.

ETHNOPHARMACOLOGICAL RELEVANCE: Infection by intestinal parasitic worms (soil-transmitted helminths or geohelminths) is prevalent in many parts of the world, and poses a particular health risk to children. This paper presents findings from a preliminary study with the primary aim to document indigenous knowledge about the use of herbal medicines in the treatment of intestinal worm infections in children among the local communities of Kalat district of northern Balochistan, Pakistan. MATERIALS AND METHODS: Ethnomedicinal data were collected through a triangulation approach, that included participant-observation and rapid appraisal methods. Prior-informed consent (PIC) was obtained from participants before conducting structured and semi-structured interviews and delivering an open ended questionnaire. A total of 94 participants, including 28 men (of whom 7 were traditional healers), and 66 women of four different age groups were interviewed. Results were analyzed using quantitative indices of Use Value citations (UVC) and Disease-Consensus Index (DCI). RESULTS: Fewer men than women agreed to be interviewed, thus overall women in the area appeared to have more ethnomedicinal knowledge. The majority of study participants belonged to the older age group (>55 years). A total of 49 plant species, belonging to 47 genera, distributed in 30 families were reported. The families Asteraceae and Lamiaceae were most frequently represented, with four species each. Trees were the most common life form, with seeds the most frequently cited plant part used (29%). Nearly a third (31%) of plant-based remedies reported in the treatment of intestinal worms were administered as a decoction. The highest UVC and DCI was reported for the species Ferula assa-foetida sL. (UVC 0.51, DCI 0.46). CONCLUSIONS: This study provides previously unreported data on the use of medicinal plants in the treatment of geohelminth infections in children of Kalat. Eight species, Acacia modesta Wall., Asparagus capitatus Baker, Microcephala lamellata (Bunge) Pobed., Nepeta praetervisa Rech.f., Plantago ciliata Desf., Pistacia atlantica Desf., Seriphidium quettense (Podlech) Y.R.Ling and Thymus linearis Benth. are reported here as anthelmintics for the first time. Detailed studies on the anthelmintic activity of chemical constituents of these species are lacking from existing literature. Further phytochemical, pharmacological and toxicity studies are required in order to evaluate the efficacy and safety of these newly reported anthelmintic species. These plants may provide a source of novel anthelmintic drug leads, which are urgently required due to the problem of global anthelmintic resistance.

The endemic medicinal plants of Northern Balochistan, Pakistan and their uses in traditional medicine.

ETHNOPHARMACOLOGICAL RELEVANCE: The highlands of Northern Balochistan are the hot spots of medicinal and endemic plant in Pakistan. These plants are still commonly used for medicinal purposes by local people in their daily lives. This study first documented the information about the medicinal uses of endemic species of Balochistan-province Pakistan. MATERIALS AND METHODS: A survey was performed using open ended questionnaires, free listening and personal observations with 152 informants (54% female, 46% male). In addition, the use value (MUV), use report (UR), fidelity level (FL), frequency citation (FC), relative frequency citation (RFC), family importance value (FIV) of species were determined and the informant consensus factor (ICF) was calculated for the medicinal plants included in the study. RESULTS: A total of 24 endemic plants belonging to 19 genera and 14 families were used by the local inhabitants to treat 12 categories of various diseases. The most common families of endemic plant species as depicted by its number of species (6 species) and FIV (9.9) was Fabaceae as the dominant family. The endemic plant species comprised perennial herbs (30%), annual herbs (25%), shrubs (29%) and under shrubs (16% each), no endemic tree species was reported in the study area. The highest number of species were used in the treatment of gastrointestinal diseases (12 species). The main route of administration is oral injection (62%) while the most frequently used form of external administration of herbal medicine was paste (5.4%) and the most commonly applied methods of preparation are powder (48.2%). Highest use report were calculated for Allium baluchistanicum and Viola makranica, (8 UR each), and least use report were calculated for two species Heliotropium remotiflorum and Tetracme stocksii (1 UR for each). Use values of the recorded plant species have been calculated which showed a highest use value of (0.73) for A. baluchistanicum and (0.56) for Berberis baluchistanica while the lowest UVs were attained for T. stocksii (0.13). Highest RFC value were calculated for Achillea millefolium (0.19) and least RFC were calculated for Blepharis sindica (0.02). The endemic species with 100% fidelity level was calculated for two plant species i.e. Seriphidium quettense and B. baluchistanica. CONCLUSIONS: The Balochistan is rich in endemic and other medicinal plants, still needs more exploration and study. Thus, it is important to document and reconstitute the remainders of the ancient medical practices which exist in Balochistan and other areas of the world, and preserve this knowledge for future generations. The endemic species which are used in traditional medicine in the region lacks phototherapeutic evidence. It is necessary to perform phytochemical or pharmacological studies to explore the potential of plants used for medicinal purposes. Overgrazing, urbanization and unsustainable harvesting of such rare and endemic medicinal plants in this region is facing severe threats of extinction. It is thus recommended that cultivation techniques be formulated, especially for the most important endemic plant medicinal species of the region.

Ethnobotany of medicinal plants among the communities of Alpine and Sub-alpine regions of Pakistan.

ETHNO-PHARMACOLOGICAL RELEVANCE: To best of our knowledge it is first quantitative ethno-botanical study from Alpine and Sub-alpine, Western Himalaya of Pakistan. The study aims to report, compare the uses and highlight the ethno-botanical significance of medicinal plants for treatment of various diseases. METHODS: A total of 290 (278 males and 12 females) informants including 14 Local Traditional Healers (LTHs) were interviewed. Information was collected using semi-structured interviews, analyzed and compared by quantitative ethno-botanical indices such as Informant Consensus Factor (ICF), Relative frequency of citation (RFC), use value (UV), Fidelity Level (FL) and Jaccard index (JI). RESULTS: A total of 125 plant species (Gymnosperms 7 species, Monocotyledons 2 and 116 Di-cotyledons) belonging to 41 families are collected, identified and ethno-botanically assessed. The most dominant family is Ranunculaceae (20 species) followed by Rosaceae (14 species). In diseases treated, gastrointestinal tract (GIT) diseases have highest proportion (27.5%) followed by respiratory diseases (20%) in the mountain communities. The most dominant life form of plants used is herbs (78%) followed by shrubs (19%) while the most commonly used plant parts are leaves (44 reports) followed by underground part, the roots (37 reports). The highest ICF (0.68) is found for ear, nose and eye disease category followed by respiratory disorders (0.46). There are 15 medicinal plants having 100% FL. Use value (UV) and Relative frequency of citation (RFC) range from 0.03 to 0.53 and 0.04 to 0.23 respectively. In comparison, maximum similarity index is found in the studies with JI 19.52 followed by 17.39. Similarity percentage of plant uses range from 1.69% to 19.52% while dissimilarity percentage varies from 0% to 20%. CONCLUSIONS: The Alpine and Sub-alpine regions of Pakistan are rich in medicinal plants and still need more research exploration. On the other hand, ethno-botanical knowledge in study areas is decreasing day by day due to high emigration rates resulting from after effects of wars during the last few decades. Historically developed ethno-botanical heritage should be preserved and promoted on global level for analyzing phytochemical, pharmaceutical and other biological activities for future drug discovery.

Ethnobotany of medicinal plants in district Mastung of Balochistan province-Pakistan.

ETHNOPHARMACOLOGICAL RELEVANCE: The aim of this study was to document the medicinal uses of plants in district Mastung of Balochistan province, Pakistan. The ethnobotanical results contain quantitative information on medicinal plants diversity documented for the first time in the area. MATERIALS AND METHODS: The information was collected through semi-structured interviews, rapid appraisal approach, open ended questionnaire and personal observations. Results were analyzed using quantitative indices of information consent factor (ICF), fidelity level (FL), use value (UV), frequency citation (FC) and relative frequency citation (RFC). RESULTS: In total of 102 plant species belonging to 47 families were reported for the medicinal purposes. Asteraceae was found to be dominant family in terms of species in the area with 11 species. The whole plant and leaves were noted as most frequently used parts (24%). Decoction (31% with 40 species) was the most commonly used preparation method. Highest ICF value (1) was recorded for antidote category. 100% fidelity level was found for four plant species i.e. Achillea welhemsii, Caralluma tuberculata, Citrullus colocynthis, and Seripidium quettense. The highest use value was reported for the Acroptilon repens (0.5) while highest RFC value was calculated for Berberis balochistanica and Citrullus colocynthis (0.18). Highest use report was calculated for Caralluma tuberculata, Citrullus colocynthis, Malva neglecta and Mentha longifolia with five use reports for each. CONCLUSIONS: The area is rich in medicinal plants and these plants are still commonly used for medicinal purposes among the people in their daily lives. However, there is a gradual loss of traditional knowledge about these plants in new generation. This study provides basis for the conservation of the local flora, its use as food and medicine. It also provides various socio-economic dimensions associated with the common people.