Non-native ligands define the active site of Pennisetum glaucum (L.) R. Br dehydroascorbate reductase.

Dehydroascorbate reductase (DHAR), a member of the glutathione-S-transferase (GST) family, reduces dehydroascorbate (DHA) to ascorbate (AsA; Vitamin-C) in a glutathione (GSH)-dependent manner and in doing so, replenishes the critical AsA pool of the cell. To understand the enzyme mechanism in detail, we determined the crystal structure of a plant DHAR from Pennisetum glaucum (PgDHAR) using Iodide-Single Anomalous Dispersion (SAD) and Molecular replacement methods, in two different space groups. Here, we show PgDHAR in complex with two non-native ligands, viz. an acetate bound at the G-site, which resembles the γ-carboxyl moiety of GSH, and a glycerol at the H-site, which shares the backbone of AsA. We also show that, in the absence of bound native substrates, these non-native ligands help define the critical 'hook points' in the DHAR enzyme active site. Further, our data suggest that these non-native ligands can act as the logical bootstrapping points for iterative design of inhibitors/analogs for DHARs.

Dengue NS3, an RNAi suppressor, modulates the human miRNA pathways through its interacting partner.

RNAi acts as a host immune response against non-self molecules, including viruses. Viruses evolved to neutralize this response by expressing suppressor proteins. In the present study, we investigated dengue virus non structural protein 3 (dvNS3), for its RNAi-suppressor activity in human cell lines. Dengue virus (DV) NS3 reverts the GFP expression in GFP-silenced cell lines. Pull-down assays of dvNS3 revealed that it interacts with the host factor human heat shock cognate 70 (hHSC70). Down-regulation of hHSC70 resulted in accumulation of dengue viral genomic RNA. Also, the interaction of dvNS3 with hHSC70 perturbs the formation of RISC (RNA-induced silencing complex)-loading complex (RLC), by displacing TRBP (TAR RNA-binding protein) and possibly impairing the downstream activity of miRNAs. Interestingly, some of these miRNAs have earlier been reported to be down-regulated upon DV infection in Huh7 cells. Further studies on the miRNA-mRNA relationship along with mRNA profiling of samples overexpressing dvNS3 revealed up-regulation of TAZ (tafazzin) and SYNGR1 (synaptogyrin 1), known dengue viral host factors (DVHFs). Importantly, overexpression of dvNS3 in human embryonic kidney (HEK) 293T cells resulted in modulation of both mature and precursor miRNAs in human cell lines. Subsequent analysis suggested that dvNS3 induced stage-specific down-regulation of miRNAs. Taken together, these results suggest that dvNS3 affects biogenesis and function of host miRNAs to regulate DVHFs for favouring DV replication.

Molecular and biochemical characterization of dehydroascorbate reductase from a stress adapted C4 plant, pearl millet [Pennisetum glaucum (L.) R. Br].

KEY MESSAGE: PgDHAR was isolated from Pennisetum glaucum. PgDHAR responded to abiotic stress and exhibited enzyme activity at broad ranges of temperature, pH and substrate concentrations suggesting its role in stress tolerance. ABSTRACT: Dehydroascorbate reductase (EC 1.8.5.1) is a crucial enzyme actively involved in the recycling of ascorbate redox pool in the cellular environment. In this study, the full-length cDNA coding for DHAR polypeptide and its corresponding gene was isolated from Pennisetum glaucum (PgDHAR). PgDHAR encodes a polypeptide of 213 amino acids with a predicted molecular mass of 23.4 kDa and shares 80-75 % sequence homology with DHAR from other plants. The heterologously expressed recombinant PgDHAR protein exhibited activity in a wide range of substrate concentrations. The recombinant PgDHAR is thermostable and retains its activity over a broad pH range. Furthermore, transcript level of PgDHAR is quantitatively up-regulated in response to temperature. On the whole, PgDHAR alone or in combination with other genes of ascorbate-glutathione cycle can be used for the development of stress tolerant as well as nutritionally improved food crop with enhanced ascorbic acid content.

Biochemical and molecular analyses of copper-zinc superoxide dismutase from a C4 plant Pennisetum glaucum reveals an adaptive role in response to oxidative stress.

Superoxide dismutases (SODs) form the foremost line of defense against ROS in aerobes. Pennisetum glaucum cDNA library is constructed to isolate superoxide dismutase cDNA clone (PgCuZnSOD) of 798 bp comprising 5'UTR (111 bp), an ORF (459 bp) and 3'UTR (228 bp). Deduced protein of 152 amino acids (16.7 kDa) with an estimated isoelectric point of 5.76 shared highest homology to cytoplasmic CuZnSODs from monocots i.e., maize, rice. Predicted 3D model reveals a conserved eight-stranded ß-barrel with active site held between barrel and two surface loops. Purified recombinant protein is relatively thermo-stable with maximal activity at pH 7.6 and shows inhibition with H(2)O(2) (4.3 mM) but not with azide (10 mM). In Pennisetum seedlings, abiotic stress induced PgCuZnSOD transcript up-regulation directly correlates to high protein and activity induction. Overexpression of PgCuZnSOD confers comparatively enhanced tolerance to methyl viologen (MV) induced oxidative stress in bacteria. Results imply that PgCuZnSOD plays a functional role in conferring oxidative stress tolerance to prokaryotic system and may hold significant potential to impart oxidative stress tolerance in higher plants through transgenic approach.

Biochemical and molecular characterization of stress-induced ß-carbonic anhydrase from a C(4) plant, Pennisetum glaucum.

Genes encoding for many ß-carbonic anhydrases and their functions in various developmental processes are well established in lower plants, however, similar studies are limited in higher plants. We report the cloning and characterization of cDNA encoding for a ß-carbonic anhydrase (PgCA) from Pennisetum glaucum, a C(4) crop plant. cDNA encoding 249 amino acids and its deduced amino acid sequence analysis revealed that is related to other plant ß-CA family members with an over all conserved architecture of a typical ß-CA protein. Phylogenetic analysis revealed that PgCA is evolutionarily very close to chloroplast ß-CA isoform. Signal sequence predicting programs identify a N-terminus putative chloroplast targeting sequence. Heterologous Escherichia coli expression system was utilized to overexpress recombinant PgCA, which showed high thermostability, an alkaline pH optima and dual activity with both reversible CO(2) hydration and esterase activities. The ß-CAs studied so far possessed only CO(2) hydration activity with no detectable esterase activity. Recombinant PgCA esterase activity is inhibited by standard CA inhibitors acetazolamide, methazolamide and azide. Subcellular immunostaining studies revealed a chloroplastic localization of PgCA protein. Expression of PgCA transcript is differentially up regulated in response to various abiotic stresses wherein its accumulation in Pennisetum leaves positively correlated with the intensity and duration of stress. Biochemical and transcript analyses suggest that PgCA may play a significant role in plant's adaptation to different abiotic stresses in addition to the previously recognized role of replenishing the CO(2) supply within plant cells.

Molecular cloning and characterization of genes encoding Pennisetum glaucum ascorbate peroxidase and heat-shock factor: interlinking oxidative and heat-stress responses.

The recent genetic and biochemical studies reveal a considerable overlap among cellular processes in response to heat and oxidative stress stimuli in plants suggesting an intimate relationship between the heat-shock response and oxidative stress responses. Pennisetum glaucum (Pg) seedlings were exposed to heat stress (42 degrees C for 0.5, 1.0 and 24h) and a mixture of RNA from all the heat stressed seedlings was used to prepare cDNA. Full-length cDNA clones encoding for cytoplasmic ascorbate peroxidase 1 (PgAPX1) and heat-shock factor (PgHSF) were isolated by screening heat stress-specific cDNA library using corresponding EST sequences as radioactive probes. These full-length cDNAs were expressed in E. coli and their recombinant proteins were purified to near homogeneity. The recombinant PgAPX1 preferred ascorbate but did not accept guaiacol as a reducing substrate. Over-expression of PgAPX1 protects E. coli cells against methyl viologen-induced oxidative stress. Sequence analysis of PgAPX1 promoter identified a number of putative stress regulatory cis-elements including a heat-shock element (HSE). Heat-shock transcription factors (HSFs) play a central role in mediating these overlapping cellular processes. Gel shift analysis and competition with specific and non-specific unlabeled DNA probes showed a specific interaction between HSE of PgAPX1 and the PgHSF protein. Expression analysis of PgHSF in Pennisetum showed maximum increase in transcript level in response to heat stress within 30 min of exposure and slowed down at subsequent time points of heat stress, indicating a typical characteristic of HSF in terms of early responsiveness. Expression of PgAPX1 significantly increased under heat-stress condition; however, the maximum expression observed at 24h of heat stress. In gel activity of PgAPX1 in Pennisetum plants also showed an increase in response to heat stress (42 degrees C) being maximum at 24h and these trends are in conformity with the expression pattern of PgAPX1. Expression patterns and interactive specificity of HSF with HSE (PgAPX1) suggest a probable vital interlink in heat and oxidative stress signaling pathways that plays a significant role in comprehending the underlying mechanisms in plant abiotic stress tolerance.

A high-throughput genome-walking method and its use for cloning unknown flanking sequences.

We developed a PCR-based high-throughput genome-walking protocol. The novelty of this protocol is in the random introduction of unique walker primer binding sites into different regions of the genome efficiently by taking advantage of the rolling circle mode of DNA synthesis by Phi29 DNA polymerase after annealing the partially degenerate primers to the denatured genomic DNA. The inherent strand-displacement activity of the Phi29 DNA polymerase displaces the 5' ends of downstream strands and DNA synthesis continues, resulting in a large number of overlapping fragments that cover the whole genome with the unique walker adapter attached to the 5' end of all the genomic DNA fragments. The directional genome walking can be performed using a locus-specific primer and the walker primer and Phi29 DNA polymerase-amplified genomic DNA fragments as template. The locus-specific primer will determine the position and direction of the genome walk. Two rounds of successive PCR amplifications by locus-specific and walker primers and their corresponding nested primers effectively amplify the flanking DNA fragments. The desired PCR fragment can be either cloned or sequenced directly using another nested, locus-specific primer. We successfully used this protocol to isolate and sequence 5' flanking regions/promoters of selected plant genes.