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Pyrrolidone carboxypeptidases (Pcps) (E.C. 3.4.19.3) can cleave the peptide bond adjacent to pyro-glutamic acid (pGlu), an N-terminal modification observed in some proteins that provides protection against common proteases. Pcp derived from extremely thermophilic Fervidobacterium islandicum AW-1 (FiPcp), that belongs to the cysteine protease family, is involved in keratin utilization under stress conditions. Although an irreversible oxidative modification of active cysteine to its sulfonic acid derivative (Cys-SO3H) renders the enzyme inactive, the molecular details for the sulfonic acid modification in inactive Pcp remain unclear. Here, we determined the crystal structure of FiPcp at 1.85 A, revealing the oxidized form of cysteine sulfonic acid (C156–SO3H) in the catalytic triad (His-Cys-Glu), which participates in the hydrolysis of pGlu residue containing peptide bond. The three oxygen atoms of cysteine sulfonic acid were stabilized by hydrogen bonds with H180, carbonyl backbone of Q83, and water molecules, resulting in inactivation of FiPcp. Furthermore, FiPcp demonstrated a unique 139KKKK142 motif involved in inter-subunit electrostatic interactions whose mutation significantly affects the thermostability of tetrameric FiPcp. Thus, our high-resolution structure of the first inactive FiPcp with irreversible oxidative modification of active cysteine provides not only the molecular basis of the redox-dependent catalysis of Pcp, but also the structural features of its thermostability.

Extensive usage of synthetic chemical pesticides have collateral effect in harming the health, environment and development of resistance in insect pests. Scorpion produces variety of molecules that are specific to insects, mammals and to both. Insect specific molecules act as potential candidature as an alternative to synthetic chemical pesticides. We have successfully expressed and purified recombinant Scorpion Leiurus quinquestriatus quinquestriaus β-BUTX-Lqq1a toxin in bacterial system. Cytotoxic activity assay with the help of insect cell line Sf-21 from Spodoptera frugiperda reveals that mean IC50 1.72–3.0 μg ml −1 significantly reduced the cell proliferation when compared with control. Microscopic examination of treated Sf-21 cell lines also showed changes in the cell morphology such as cell membrane blebbing, cell shrinkage and granulated apoptotic bodies. When β-BUTX-Lqq1a was hemocoelly injected with various doses, significant reduction in survival of Helicoverpa armigera (LC50 = 0.13 μg insect−1) and Spodoptera litura (LC50 = 0.147 μg insect −1) were noticeable with immediate paralysis, and reduced feeding when compared with control. Toxicity with purified recombinant β-BUTX-Lqq1a protein towards insect cell line Sf-21 and major agricultural pest was demonstrated by various bioassays. Cytotoxicity and insect bioassay demonstrated the potential use of β-BUTX-Lqq1a protein as an effective insecticide against lepidopteran insects. These results strongly suggest that the development of rational insecticidal molecule against with significant promise.

Chromium-induced toxicity and mechanisms of cell death involved in plants are yet to be fully elucidated. To understand the events of these processes, the stress response of the soybean plant using trivalent and hexavalent chromium compounds, namely, basic chromium sulphate (BCS) and potassium dichromate (K2Cr2O7) was investigated. The leaf surface morphology for stomatal aperture, wax deposition and presence of trichomes for chromium accumulation was examined by SEM-EDAX and light microscopy. The leaf mesophyll cell integrity was identified by trypan blue staining; chlorophyll autofluorescence, ROS generation and mitochondrial function were studied by fluorescence microscopy using different dyes. Isolated chloroplasts were analysed for micronutrients and total chromium content by AAS. Elevated Cr level and decreased Fe, Cu and Zn content in chloroplast revealed the active transportation of highly soluble Cr6+ species resulting in poor absorption of micronutrients. Cr accumulation as Cr(V) in chloroplast was noticed at g = 1.98 of electron paramagnetic resonance signal. Plants grown in Cr(VI) amended soil showed chemical modification of biological macromolecules in the chloroplast as observed from fourier transform infra-red (FTIR) spectra; the chloroplast DNA damage was confirmed by DAPI staining. Cr(VI)-treated plants showed significant reduction in the levels of various biochemical parameters. The results altogether clearly indicate that Cr(VI)-induced reactive oxygen species (ROS) production leads to oxidative stress-associated changes in the organelles, particularly in chloroplast, resulting in cell death.

Comparative genomics of the keratin-degrading extremophilic eubacterium Fervidobacterium islandicum AW-1 and the closely related Fervidobacterium nodosum with no keratinolytic activity suggested that the FIAW1_1600 gene encoding a carboxypeptidase (CP) plays an important role in keratin degradation. The presumptive 489 amino acid sequence of the gene showed a conserved HEXXH motif with low levels of sequence identity (

Chromium toxicity is one of the major causes of environmental pollution due to its heavy discharge in industrial wastewaters. Chromate reduction is a viable method to detoxify hexavalent chromium to nontoxic trivalent species mediated by enzymes and metabolites. A new Bacillus methylotrophicus strain was isolated from tannery sludge and was an efficient candidate for chromate reduction. An initial chromate reductase activity of 212.84 U/mg protein was obtained at 48 h in a low-cost defined medium formulation with 0.25 mM chromate. The extracellular enzyme was inducible at 12h substrate addition with 312.99 U/mg at 48 h. Reduced glutathione was required for enhanced specific activity of 356.48 U/mg. Enzyme activity was optimum at pH 7.0 and at 30 °C, and was stable in the presence of EDTA, DTT and metal ions. The enzyme exhibited a Vmax of 59.89 μM/min/mg protein and a Km of 86.5 μM, suggesting feasibility of the reaction with K₂Cr₂O₇ as substrate. Application of the crude reductase in tannery effluent resulted in 91.3% chromate reduction at 48 h. An enzyme-mediated chromate reduction process has therefore been developed for bioremediation of toxic chromium sp. in industrial effluents.