Structure of the hypothetical protein TTHA1873 from Thermus thermophilus.

The crystal structure of an uncharacterized hypothetical protein, TTHA1873 from Thermus thermophilus, has been determined by X-ray crystallography to a resolution of 1.78 Šusing the single-wavelength anomalous dispersion method. The protein crystallized as a dimer in two space groups: P43212 and P6122. Structural analysis of the hypothetical protein revealed that the overall fold of TTHA1873 has a ß-sandwich jelly-roll topology with nine ß-strands. TTHA1873 is a dimeric metal-binding protein that binds to two Ca2+ ions per chain, with one on the surface and the other stabilizing the dimeric interface of the two chains. A structural homology search indicates that the protein has moderate structural similarity to one domain of cell-surface proteins or agglutinin receptor proteins. Red blood cells showed visible agglutination at high concentrations of the hypothetical protein.

Production and characterization of biodegradable polyhydroxybutyrate by Micrococcus luteus isolated from marine environment.

Marine microorganisms are reported to produce polyhydroxybutyrate (PHB) that has wide range of medical and industrial applications with the advantage of biodegradability. PHBs are synthesized as an energy and carbon storage element under metabolic pressure. The scope of this work is enhancing PHB production using marine microbial isolate, Micrococcus luteus by selectively optimizing various growth conditions such as different media components and growth parameters that influence the cell growth and PHB production were sampled. Micrococcus luteus produced 7.54 g/L of PHB utilizing glucose as a carbon source and ammonium sulphate as a nitrogen source with maximum efficiency. The same optimized operational conditions were further employed in batch fermentation over a time span of 72 h. Interestingly higher cell dry weight of 21.52 g/L with PHB yield of 12.18 g/L and 56.59% polymer content was observed in batch fermentation studies at 64 h. The chemical nature of the extracted polymer was validated with physio-chemical experiments and was at par with the commercially available PHB. This study will spotlight M. luteus as a potential source for large-scale industrial production of PHB with reducing environmental pollutions.

Effect of C/N substrates for enhanced extracellular polymeric substances (EPS) production and Poly Cyclic Aromatic Hydrocarbons (PAHs) degradation.

Extracellular Polymeric Substances (EPS) influenced Poly Cyclic Aromatic Hydrocarbons (PAHs) degrading Klebsiella pneumoniae was isolated from the marine environment. To increase the EPS production by Klebsiella pneumoniae, several physicochemical parameters were tweaked such as different carbon sources (arabinose, glucose, glycerol, lactose, lactic acid, mannitol, sodium acetate, starch, and sucrose at 20 g/L), nitrogen sources (ammonium chloride, ammonium sulphate, glycine, potassium nitrate, protease peptone and urea at 2 g/L), different pH, carbon/nitrogen ratio, temperature, and salt concentration were examined. Maximum EPS growth and biodegradation of Anthracene (74.31%), Acenaphthene (67.28%), Fluorene (62.48%), Naphthalene (57.84%), and mixed PAHs (55.85%) were obtained using optimized conditions such as glucose (10 g/L) as carbon source, potassium nitrate (2 g/L) as the nitrogen source at pH 8, growth temperature of 37 °C, 3% NaCl concentration and 72 h incubation period. The Klebsiella pneumoniae biofilm architecture was studied by confocal laser scanning microscopy (CLSM) and scanning electron microscope (SEM). The present study demonstrates the EPS influenced PAHs degradation of Klebsiella pneumoniae.

In silico Functional Annotation and Characterization of Hypothetical Proteins from Serratia marcescens FGI94.

Serratia marcescens, rod-shaped Gram-negative bacteria is classified as an opportunistic pathogen in the family Enterobacteriaceae. It causes a wide variety of infections in humans, including urinary, respiratory, ocular lens and ear infections, osteomyelitis, endocarditis, meningitis and septicemia. Unfortunately, over the past decade, antibiotic resistance has become a serious health care issue; the effective means to control and dissemination of S. marcescens resistance is the need of hour. The whole genome sequencing of S. marcescens FGI94 strain contains 4434 functional proteins, among which 690 (15.56%) proteins were classified under hypothetical. In the present study, we applied the power of various bioinformatics tools on the basis of protein family comparison, motifs, functional properties of amino acids and genome context to assign the possible functions for the HPs. The pseudo sequences (protein sequence that contain ≤100 amino acid residues) are eliminated from the study. Although we have successfully predicted the function for 483 proteins, we were able to infer the high level of confidence only for 108 proteins. The predicted HPs were classified into various classes such as enzymes, transporters, binding proteins, cell division, cell regulatory and other proteins. The outcome of the study could be helpful to understand the molecular mechanism in bacterial pathogenesis and also provide an insight into the identification of potential targets for drug and vaccine development.

Sertraline as a promising antifungal agent: inhibition of growth and biofilm of Candida auris with special focus on the mechanism of action in vitro.

AIM: The aim of this present study was to investigate the antifungal mechanism of sertraline against Candida auris (C. auris) and its effect on biofilm formation. METHODS AND RESULTS: Sertraline, a repurposing drug with a history of human use for the treatment of depression was screened against three different isolates of C. auris, and was found to possess efficient antifungal activity. The antifungal activity of sertraline was further confirmed by killing kinetics assay and post-antifungal effect (PAFE). Sertraline inhibited C. auris yeast to hyphae conversion and further the inhibition of biofilm formation showed 71% inhibition upon treatment. Cell damage caused due to C. auris after treatment with sertraline was observed using SEM and cell membrane damage was ascertained using flow cytometry by Propidium Iodide (PI) uptake assay. The results of sorbitol protection assay and ergosterol effect assay suggested that sertraline did not affect the cell wall and did not act by binding to membrane ergosterol. The mechanism of action of sertraline against C. auris was understood through in silico docking studies that revealed the binding nature of sertraline to the sterol 14 alpha demethylase which is involved in ergosterol biosynthesis. Ergosterol that was quantified from treated cells showed a 5·5-fold decrease in ergosterol production. CONCLUSION: Sertraline displayed promising antifungal activity against C. auris involved in candidiasis infection and the mechanism of action was predicted. SIGNIFICANCE AND IMPACT OF THIS STUDY: The results of this study can encourage for the development of new antifungal agents and can be promising antifungal agent against C. auris infection.

Biological synergy of greener gold nanoparticles by using Coleus aromaticus leaf extract.

Greener nanotechnology plays an important role in developing alternative and effective treatment strategies for various diseases. Biological synthesis of metal nanoparticles (MNPs) has known to possess suitable alternatives than the existing chemical methods. Greener synthesis of MNPs by using plant extracts has become an emerging field due to their safe, eco-friendly and non-toxic nature that are suitable for synergistic biological activities. Hence, the greener method is chosen by the present study. In the present study, the greener synthesis of gold nanoparticles (AuNPs) was successfully done by using Coleus aromaticus leaf extract at three different temperatures (30 °C, 60 °C and 100 °C). The formation of AuNPs was initially monitored by visual observation and then characterized with the help of diverse techniques like UV-Vis spectroscopy (UV-Vis), X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy, Scanning Electron Microscopy (SEM), High Resolution-Transmission Electron Microscopy (HR-TEM) and dynamic light scattering (DLS). Surface plasmon resonance (SPR) peak and crystalline nature of AuNPs were obtained by UV-Vis and XRD spectroscopies respectively. FT-IR analysis shows the different characteristic functional groups in turn responsible for the bio-reduction of gold ions by using leaf extract. The formations of different nano-sized AuNPs with their different morphologies were observed by SEM and HR-TEM analyses. Surface charge and stability of the AuNPs were measured by zeta potential and DLS respectively. The synthesized AuNPs coated with cotton fabric was analyzed by UV-Diffuse Reflectance Spectroscopy (UV-DRS), which revealed excellent UV protection against UV radiation. The AuNPs coated cotton fabric exhibited remarkable antibacterial sensitivity against Staphylococcus epidermidis and Escherichia coli. Further, the synthesized AuNPs showed significant cytotoxicity against human liver cancer (HepG2) cell line. The findings of this study revealed that, AuNPs synthesized using Coleus aromaticus leaf extract could be an alternative, safe, and effective source of UV protection, antibacterial and anticancer agents.

Improved conductivity and antibacterial activity of poly(2-aminothiophenol)-silver nanocomposite against human pathogens.

A rapid and simple chemical synthesis of poly(2-aminothiophenol)­silver (P2ATP-Ag) nanocomposite using conductive and electroactive silver nanoparticles (AgNPs) is reported. The AgNPs was synthesized by chemical reduction method using tri­sodium citrate as reducing agent and poly(N-vinyl-2-pyrrolidone) (PVP) as stabilizing agent. P2ATP-Ag nanocomposite was synthesized by using potassium peroxodisulphate as oxidant and the samples were characterized. The presence of AgNPs in the composite was confirmed from UV-Vis, FTIR and X-ray diffraction studies. Morphology of the P2ATP and its composite were investigated by SEM. HR-TEM images show spherical, trigonal and rod like morphologies with sizes of Ag nanoparticles and its composite. Thermal analysis revealed that the thermal stability of the P2ATP-Ag nanocomposite is improved when compared with pure P2ATP. The synthesized AgNPs, pure P2ATP and P2ATP-Ag nanocomposite were screened for antibacterial activity test against human pathogen such as Gram positive (Bacillus subtilis, ATCC-6051) and Gram negative (Vibrio cholerae, ATCC-14035), carried out by agar-well diffusion method at micro molar concentration. The result shows that P2ATP-Ag nanocomposite has excellent antibacterial activity due to the presence of Ag nanoparticles. The electrical conductivity of the P2ATP-Ag nanocomposite is better than that of pure P2ATP. The reported nanocomposite will be a potential material for electrocatalysis, sensors and biomedical applications.

Structural basis for the hydrolysis of ATP by a nucleotide binding subunit of an amino acid ABC transporter from Thermus thermophilus.

ATP-binding cassette (ABC) transporters are a major family of small molecule transporter proteins, and their deregulation is associated with several diseases, including cancer. Here, we report the crystal structure of the nucleotide binding domain (NBD) of an amino acid ABC transporter from Thermus thermophilus (TTHA1159) in its apo form and as a complex with ADP along with functional studies. TTHA1159 is a putative arginine ABC transporter. The apo-TTHA1159 was crystallized in dimeric form, a hitherto unreported form of an apo NBD. Structural comparison of the apo and ADP-Mg(2+) complexes revealed that Phe14 of TTHA1159 undergoes a significant conformational change to accommodate ADP, and that the bound ADP interacts with the P-loop (Gly40-Thr45). Modeling of ATP-Mg(2+):TTHA1159 complex revealed that Gln86 and Glu164 are involved in water-mediated hydrogen bonding contacts and Asp163 in Mg(2+) ion-mediated hydrogen bonding contacts with the γ-phosphate of ATP, consistent with the findings of other ABC transporters. Mutational studies confirmed the necessity of each of these residues, and a comparison of the apo/ADP Mg(2+):TTHA1159 with its ATP-complex model suggests the likelihood of a key conformational change to the Gln86 side chain for ATP hydrolysis.

Crystal structure analysis of L-fuculose-1-phosphate aldolase from Thermus thermophilus HB8 and its catalytic action: as explained through in silico.

Fuculose phosphate aldolase catalyzes the reversible cleavage of fuculose-1-phosphate to dihydroxyacetone phosphate and L-lactaldehyde. A tetramer by nature, this enzyme from Thermus thermophilus HB8 represents the group of Class II aldolases. The structure was solved in two different space groups using the crystals obtained from slow evaporation vapour-diffusion and microbatch techniques. The detailed crystallization description has been reported previously. In this study, the structural features of fuculose phosphate aldolase from T. thermophilus have been explored extensively through sequence and structure comparisons with fuculose phosphate aldolases of different species. Finally, an in silico analysis using induced fit docking was attempted to deduce the binding mode of fuculose phosphate aldolase with its natural substrate fuculose-1-phosphate along with a substrate analog dihydroxyacetone phosphate and phosphoglycolohydroxymate--a potential aldolase inhibitor. The results show the mechanism of action may be similar to that of Escherichia coli fuculose aldolase.

catena-Poly[[[aqua-(glycine-κO)lithium]-µ-glycine-κ(2) O:O'] bromide].

In the title coordination polymer, {[Li(C2H5NO2)2(H2O)]Br} n , the Li(+) cation is coordinated by three carboxyl-ate O atoms of zwitterionic glycine mol-ecules and by a water mol-ecule, forming a distorted tetra-hedral geometry. One of the two glycine mol-ecules bridges neighbouring complexes, forming an infinite chain parallel to the c axis. Polymeric chains are further linked by extensive hydrogen bonds involving the Br(-) anions and glycine and water mol-ecules, producing a three-dimensional network.

Impact of protein binding cavity volume (PCV) and ligand volume (LV) in rigid and flexible docking of protein-ligand complexes.

The importance of protein binding cavity volume (PCV) and ligand volume (LV) in rigid and flexible docking has been studied in 48 protein-ligand complexes belonging to eight protein families. In continuation of our earlier study on protein flexibility in relationship to PCV and LV, this study analyzes the importance of PCV and LV in the scoring and ranking of ligands in docking experiments. Crystal structures of protein-ligand complexes with varied PCV were chosen for docking ligands of varied volume in each protein family. Docking and scoring accuracy have been evaluated by self and cross docking of ligands to the given protein conformation. Effect of PCV and LV in rigid and flexible docking has been studied both in apo and holo proteins. Rigid docking has performed well when appropriate protein conformation is used. Selecting the proteins with appropriate PCV based on the LV information is suggested for better results in ensemble docking.

A new pentameric structure of rotavirus NSP4 revealed by molecular replacement.

The region spanning residues 95-146 of the rotavirus nonstructural protein NSP4 from the asymptomatic human strain ST3 has been purified and crystallized and diffraction data have been collected to a resolution of 2.6 Å. Several attempts to solve the structure by the molecular-replacement method using the available tetrameric structures of this domain were unsuccessful despite a sequence identity of 73% to the already known structures. A more systematic approach with a dimer as the search model led to an unexpected pentameric structure using the program Phaser. The various steps involved in arriving at this molecular-replacement solution, which unravelled a case of subtle variation between different oligomeric states unknown at the time of solving the structure, are presented in this paper.

1-(2-Naphth-yl)-3-phenyl-3-(4,5,6,7-tetra-hydro-1,2,3-benzoselenadiazol-4-yl)propan-1-one.

In the title compound, C(25)H(22)N(2)OSe, the fused six-membered cyclo-hexene ring of the 4,5,6,7-tetra-hydro-1,2,3-benzoselenadiazole group adopts a near half-chair conformation and the five-membered 1,2,3-selenadiazole ring is essentially planar (r.m.s. deviation = 0.004 Å). There are weak inter-molecular C-H⋯O and C-H⋯π inter-actions in the crystal structure. Inter-molecular π-π stacking is also observed between the naphthyl units, with a centroid-centroid distance of 3.529 (15) Å.

3-(4-Methyl-phen-yl)-1-phenyl-3-(4,5,6,7-tetra-hydro-1,2,3-benzoselenadiazol-4-yl)propan-1-one.

In the title compound, C(22)H(22)N(2)OSe, the fused six-membered ring of the 4,5,6,7-tetra-hydro-benzo[d][1,2,3] selenadiazole group adopts a near to envelope (E form) conformation and the five-membered 1,2,3-selenadiazole ring is essentially planar (r.m.s. deviation = 0.0059 Å). In the crystal, adjacent mol-ecules are inter-linked through weak inter-molecular C-H⋯π inter-actions.

4-{(4-Chloro-phen-yl)[4-(4-methyl-phen-yl)-1,2,3-selenadiazol-5-yl]meth-yl}-4,5,6,7-tetra-hydro-1,2,3-benzoselenadiazole.

In the title compound, C(22)H(19)ClN(4)Se(2), the mean plane of the non-fused selenadiazole ring forms dihedral angles of 54.20 (16)° and 70.48 (11)°, respectively, with the essentially planar [maximum deviations of 0.025 (5) and 0.009 (2) Å, respectively] methyl-phenyl and chloro-phenyl substituents. The tetra-hydro-1,2,3-benzoselenadiazole group is disordered over two sets of sites with a refined occupancy ratio of 0.802 (5):0.198 (5). In the crystal, weak inter-molecular C-H⋯N inter-actions are observed.

Recognition of active and inactive catalytic triads: A template based approach.

It is well established that a sequence template along with the database is a powerful tool for identifying the biological function of proteins. Here, we describe a method for predicting the catalytic nature of certain proteins among the several protein structures deposited in the Protein Data Bank (PDB). For the present study, we considered a catalytic triad template (Ser-His-Asp) found in serine proteases. We found that a geometrically optimized active site template can be used as a highly selective tool for differentiating an active protein among several inactive proteins, based on their Ser-His-Asp interactions. For any protein to be proteolytic in nature, the bond angle between Ser O(gamma)-Ser H(gamma)...His N(epsilon2) in the catalytic triad needs to be between 115 degrees and 140 degrees. The hydrogen bond distance between Ser H(gamma)...His N(epsilon2) is more flexible in nature and it varies from 2.0 A to 2.7 A while in the case of His H(delta1)...Asp O(delta1), it is from 1.6A to 2.0 A. In terms of solvent accessibility, most of the active proteins lie in the range of 10-16 A(2), which enables easy accessibility to the substrate. These observations hold good for most catalytic triads and they can be employed to predict proteolytic nature of these catalytic triads.

Ion pairs in non-redundant protein structures.

Ion pairs contribute to several functions including the activity of catalytic triads, fusion of viral membranes, stability in thermophilic proteins and solvent-protein interactions. Furthermore, they have the ability to affect the stability of protein structures and are also a part of the forces that act to hold monomers together. This paper deals with the possible ion pair combinations and networks in 25% and 90% non-redundant protein chains. Different types of ion pairs present in various secondary structural elements are analysed. The ion pairs existing between different subunits of multisubunit protein structures are also computed and the results of various analyses are presented in detail. The protein structures used in the analysis are solved using X-ray crystallography, whose resolution is better than or equal to 1.5 A and R-factor better than or equal to 20%. This study can, therefore, be useful for analyses of many protein functions. It also provides insights into the better understanding of the architecture of protein structure.

Third calcium ion found in an inhibitor-bound phospholipase A2.

The lipolytic enzyme phospholipase A2 plays a crucial role in lipid metabolism and catalyzes hydrolysis of the fatty-acid ester bond at the sn-2 position of phospholipids. Here, the crystal structure (1.7 A resolution) of the triple mutant (K53,56,121M) of bovine pancreatic phospholipase A2 complexed with an organic molecule, p-methoxybenzoic acid (anisic acid), is reported. Residues 60-70 (the surface-loop residues) are ordered and adopt conformations which are different from those normally found in structures in which a second calcium ion is present. It is interesting to note that for the first time a third calcium ion has been identified. In addition, four Tris (2-amino-2-hydroxymethyl-1,3-propanediol) molecules were located. It is believed that one of the Tris molecules plays a role in clamping the third calcium ion and that another is involved in controlling the dynamics of the surface loop through hydrogen bonds.

Structure of Mycobacterium smegmatis single-stranded DNA-binding protein and a comparative study involving homologus SSBs: biological implications of structural plasticity and variability in quaternary association.

The structure of Mycobacterium smegmatis single-stranded DNA-binding protein (SSB) has been determined using three data sets collected from related crystals. The structure is similar to that of its homologue from Mycobacterium tuberculosis, indicating that the clamp arrangement that stabilizes the dimer and the ellipsoidal shape of the tetramer are characteristic features of mycobacterial SSBs. The central OB fold is conserved in mycobacterial SSBs as well as those from Escherichia coli, Deinococcus radiodurans and human mitochondria. However, the quaternary structure exhibits considerable variability. The observed plasticity of the subunit is related to this variability. The crystal structures and modelling provide a rationale for the variability. The strand involved in the clamp mechanism, which leads to higher stability of the tetramer, appears to occur in all high-G+C Gram-positive bacteria. The higher stability is perhaps required by these organisms. The mode of DNA binding of mycobacterial SSBs is different from that of E. coli SSB partly on account of the difference in the shape of the tetramers. Another difference between the two modes is that the former contains additional ionic interactions and is more susceptible to salt concentration.

Structure of Mycobacterium tuberculosis single-stranded DNA-binding protein. Variability in quaternary structure and its implications.

Single-stranded DNA-binding protein (SSB) is an essential protein necessary for the functioning of the DNA replication, repair and recombination machineries. Here we report the structure of the DNA-binding domain of Mycobacterium tuberculosis SSB (MtuSSB) in four different crystals distributed in two forms. The structure of one of the forms was solved by a combination of isomorphous replacement and anomalous scattering. This structure was used to determine the structure of the other form by molecular replacement. The polypeptide chain in the structure exhibits the oligonucleotide binding fold. The globular core of the molecule in different subunits in the two forms and those in Escherichia coli SSB (EcoSSB) and human mitochondrial SSB (HMtSSB) have similar structure, although the three loops exhibit considerable structural variation. However, the tetrameric MtuSSB has an as yet unobserved quaternary association. This quaternary structure with a unique dimeric interface lends the oligomeric protein greater stability, which may be of significance to the functioning of the protein under conditions of stress. Also, as a result of the variation in the quaternary structure the path adopted by the DNA to wrap around MtuSSB is expected to be different from that of EcoSSB.