A novel esterase from Bacillus subtilis (RRL 1789): purification and characterization of the enzyme

An esterase (EC 3.1.1.1) produced by an isolated strain of Bacillus subtilis RRL 1789 exhibited moderate to high enantioselectivity in the kinetic resolution of several substrates like aryl carbinols, hydroxy esters, and halo esters. The enzyme named as B. subtilis esterase (BSE), was purified to >95% purity with a specific activity of 944 U/mg protein and 12% overall yield. The purified enzyme is approximately 52 kDa monomer, maximally activity at 37 degrees C and pH 8.0 and fairly stable up to 55 degrees C. The enzyme does not exhibit the phenomenon of interfacial activation with tributyrin and p-nitrophenyl butyrate beyond the saturation concentration. The enzyme showed preference for triacyglycerols and esters of p-nitrophenol with short chain fatty acid. Presence of Ca2+ ions increases the activity of enzyme by approximately 20% but its presence does not have any influence on the thermostability of the enzyme. The enzyme is not a metalloprotein and belongs to the family of serine proteases. The N-terminal amino acid sequence of BSE determined, as Met-Thr-Pro-Glu-Iso-Val-Thr-Thr-Glu-Tyr-Gly- revealed similarity with the N-terminal amino acid sequence of p-nitrobenzylesterase of B. subtilis.     

Predicted binding of certain antifilarial compounds with glutathione-S-transferase of human Filariids

Glutathione-S-transferase is a major phase-II detoxification enzyme in parasitic helminthes. Previous research highlights the importance of GSTs in the establishment of chronic infections in cytotoxic microenvironments. Filarial nematodes depend on these detoxification enzymes for their survival in the host. GST plays an important role in filariasis and other diseases. GST from W.bancrofti and B.malayi are very much different from human GST. This structural difference makes GST potential chemotherapeutic targets for antifilarial treatment. In this study we have checked the efficacy of some well known antifilarial compounds against GST from B.malayi and W.bancrofti. The structure of BmGST was modeled using modeller9v10 and was submitted to PMDB. Molecular docking study reveals arbindazole to be the most potent compounds against GST from both the filarial parasites. Role of some residues playing important role in the binding of compounds within the active site of GST has also been revealed in the present study. The BmGST and WbGST structural information and docking studies could aid in screening new antifilarials or selective inhibitors for chemotherapy against filariasis.

Abbreviations

GST - Glutathione-S-transferase, Bm - Brugia malayi, Wb - Wuchereria bancrofti.     

Conformation and thermodynamic stability of pre-molten and molten globule states of mammalian cytochromes-c

Proteins in cells fold via a number of intermediates. These intermediates are quite important as they guide the protein to attain its unique native conformation. To solve the immensely difficult problem of protein folding, it is necessary to characterize intermediates which will unravel the mystery of the steps involved in the proper folding of proteins. Cytochromes-c (cyts-c) have played an important role in studies of the earliest events and intermediates in protein folding. They have always been considered as model proteins for protein folding studies due to their intrinsic properties that can be measured by multiple probes. A large number of different solvent conditions have been employed to obtain equilibrium intermediates of cyts-c. These intermediates show structural heterogeneity which is mainly due to the different solvent conditions used to induce them. In this review we present results of conformational and thermodynamic characterization of equilibrium intermediates (molten globules and pre-molten globules) of the mammalian cyts-c under different solvent conditions.     

Phosphoproteome sequence analysis and significance: Mining association patterns around phosphorylation sites utilizing MAPRes

Phosphorylation, one of the most common protein post‐translational modifications (PTMs) on hydroxyl groups of S/T/Y is catalyzed by kinases and involves the presence or absence of certain amino acid residues in the vicinity of the phosphorylation sites. Using MAPRes, we have analyzed the substrate proteins of Phospho.ELM 7.0 and found that there are both general and specific requirements for the presence or absence of particular amino acids in the vicinity of phosphorylated S/T/Y for both of the phosphorylation data, whether or not kinase information was taken into account. Patterns extracted by MAPRes for kinase‐specific data have been utilized to find the consensus sequence motifs for various kinases required to catalyze the process of phosphorylation on S/T/Y. These consensus sequences for different kinase groups, families, and individual members are consistent with those described earlier with some novel consensus reported for the first time. A comparison study for the patterns mined by MAPRes with the results of existing prediction methods was performed by searching for these patterns in the vicinity of phosphorylation sites predicted by different available method. This comparison resulted in 87–98% conformity with the results of the predictions by available methods. Additionally, the patterns mined by MAPRes for substrate sites included 61 kinases, the highest number analyzed so far. J. Cell. Biochem. 108: 64–74, 2009. © 2009 Wiley‐Liss, Inc.     

A single mutation induces molten globule formation and a drastic destabilization of wild-type cytochrome c at pH 6.0

Amino acid sequences of seven subfamilies of cytochromes c show that other than heme binding residues there are only four positions which are conserved in all subfamilies: Gly/Ala6, Phe/Tyr10, Leu/Val/Phe94, and Tyr/Trp/Phe97. These residues are 90% conserved in all sequences reported and are also considered to be involved in a common folding nucleus. To determine the importance of conserved interactions offered by the side chain of Leu94, we made an L94G mutant of horse cytochrome c. Characterization of this mutant by the far-UV, near-UV, and Soret circular dichroism, intrinsic and 1-Anilino-8-naphthalene sulfonate fluorescence, and dynamic light scattering measurements led to the conclusion that the L94G mutant has all the common structural characteristics of a molten globule at pH 6.0 and 25 °C. NaCl induces a cooperative transition between the acid-denatured state and a state of L94G having all the common structural characteristics of a pre-molten-globule state at pH 2 and 25 °C. Thermal denaturation studies showed that the midpoint of denaturation of the mutant is 28 °C less than that of the wild-type protein. Interestingly, the structure analysis using the coordinates given in the Protein Data Bank (1hrc) also suggested that the L94G mutant would be less stable than the wild-type protein.     

Phytochemical and Biological Activities of Pseudocalymma elegans: A False Garlic

Evaluation of phytochemical constituents and antioxidant and antimicrobial activities of hexane (PELH), dichloromethane (PELDCM), ethyl acetate (PELEA), and MeOH (PELM) extracts of young leaves of Pseudocalymma elegans have been carried out. Moreover, extracts have also been explored for the presence of sulphur containing compounds, 1,2‐dithiolane ( 33 ), diallyl disulfide ( 35 ), 3‐vinyl‐1,2‐dithiacyclohex‐5‐ene ( 37 ), and diallyl trisulfide ( 38 ) responsible for the garlic like smell of P. elegans. All the extracts were found to be antioxidant and showed potent inhibition with IC₅₀ values of 0.168 ± 0.001, 0.128 ± 0.002, 0.221 ± 0.011, and 0.054 ± 0.001, respectively, as compared to standard drugs ascorbic acid (AA) and butylated hydroxytoluene (BHT). The ethyl acetate extract (PELE) showed excellent activities against few Gram‐positive and Gram‐negative bacteria and some fungi as compared with standard drug ceftriaxone (3rd generation cephalosporin) and nystatin, respectively. Chemical constituents of hexane, dichloromethane, and ethyl acetate extracts were identified by gas chromatography‐mass spectrometry and mass spectral library search. Over all 55 chemical constituents were first time identified from the leaves which included branched and n‐hydrocarbons, fatty acids, fatty acid methyl esters, fatty alcohols, terpenes, alkaloid, vitamins, glycosides, aromatic compounds, and sulfur containing compounds. Two known chemical constituents, ursolic acid ( 1 ) and β‐amyrin ( 2 ), were also purified for the first time from the MeOH extract. To elucidate the structures of these compounds, UV, IR, EI‐MS, ¹H‐ and ¹³C‐NMR spectroscopy were used.     

Immunogenicity of mitochondrial DNA modified by hydroxyl radical

Mitochondria consume about 90 percent of oxygen used by the body, and are a particularly rich source of reactive oxygen species (ROS). In this research communication mitochondrial DNA (mtDNA) was isolated from fresh goat liver and modified in vitro by hydroxyl radical generated from UV irradiation (254 nm) of hydrogen peroxide. As a consequence of hydroxyl radical modification, mtDNA showed hyperchromicity and sensitivity to nuclease S1 digestion as compared to control mtDNA. Animals immunized with mtDNA and ROS-modified mtDNA induced antibodies as detected by direct binding and competition ELISA. The data suggest that immunogenicity of mtDNA got augmented after treatment with hydroxyl radical. IgG isolated from immune sera showed specificity for respective immunogen and cross-reaction with other nucleic acids. Binding of induced antibodies with array of antigens clearly indicates their polyspecific nature. Moreover, the polyspecificity exhibited by induced antibodies is unique in view of similar multiple antigen binding properties of naturally occurring anti-DNA antibodies derived from SLE patients.     

Different molten globule-like folding intermediates of hen egg white lysozyme induced by high pH and tertiary butanol

We have provided evidence that hen egg white lysozyme (HEWL) existed in alpha helical and beta structure dominated molten globule (MG) states at high pH and in the presence of tertiary butanol, respectively. Circular dichroism (CD), intrinsic fluorescence, ANS binding and acrylamide-induced fluorescence quenching techniques have been used to investigate alkali-induced unfolding of HEWL and the effect of tertiary butanol on the alkaline-induced state. At pH 12.75, HEWL existed as molten globule like intermediate. The observed MG-like intermediate was characterized by (i) retention of 77% of the native secondary structure, (ii) enhanced binding of ANS (approximately 5 times) compared to native and completely unfolded state, (iii) loss of the tertiary structure as indicated by the tertiary structural probes (near-UV, CD and Intrinsic fluorescence) and (iv) acrylamide quenching studies showed that MG state has compactness intermediate between native and completely unfolded states. Moreover, structural properties of the protein at isoelectric point (pI) and denatured states have also been described. We have also shown that in the presence of 45% tertiary butanol (t-butanol), HEWL at pH 7.0 and 11.0 (pI 11.0) existed in helical structure without much affecting tertiary structure. Interestingly, MG state of HEWL at pH 12.7 transformed into another MG state (MG2) at 20% t-butanol (v/v), in which secondary structure is mainly beta sheets. On further increasing the t-butanol concentration alpha helix was found to reform. We have proposed that formation of both alpha helical and beta sheet dominated intermediate may be possible in the folding pathway of alpha + beta protein.     

Heterogeneity of Equilibrium Molten Globule State of Cytochrome c Induced by Weak Salt Denaturants under Physiological Condition

While many proteins are recognized to undergo folding via intermediate(s), the heterogeneity of equilibrium folding intermediate(s) along the folding pathway is less understood. In our present study, FTIR spectroscopy, far- and near-UV circular dichroism (CD), ANS and tryptophan fluorescence, near IR absorbance spectroscopy and dynamic light scattering (DLS) were used to study the structural and thermodynamic characteristics of the native (N), denatured (D) and intermediate state (X) of goat cytochorme c (cyt-c) induced by weak salt denaturants (LiBr, LiCl and LiClO₄) at pH 6.0 and 25°C. The LiBr-induced denaturation of cyt-c measured by Soret absorption (Δε ₄₀₀) and CD ([θ]₄₀₉), is a three-step process, N ↔ X ↔ D. It is observed that the X state obtained along the denaturation pathway of cyt-c possesses common structural and thermodynamic characteristics of the molten globule (MG) state. The MG state of cyt-c induced by LiBr is compared for its structural and thermodynamic parameters with those found in other solvent conditions such as LiCl, LiClO₄ and acidic pH. Our observations suggest: (1) that the LiBr-induced MG state of cyt-c retains the native Met80-Fe(III) axial bond and Trp59-propionate interactions; (2) that LiBr-induced MG state of cyt-c is more compact retaining the hydrophobic interactions in comparison to the MG states induced by LiCl, LiClO₄ and 0.5 M NaCl at pH 2.0; and (3) that there exists heterogeneity of equilibrium intermediates along the unfolding pathway of cyt-c as highly ordered (X1), classical (X2) and disordered (X3), i.e., D ↔ X3 ↔ X2 ↔ X1 ↔ N.