Crystal Structure of Caryolan-1-ol Synthase, a Sesquiterpene Synthase Catalyzing an Initial Anti-Markovnikov Cyclization Reaction.

In a continuing effort to understand reaction mechanisms of terpene synthases catalyzing initial anti-Markovnikov cyclization reactions, we solved the X-ray crystal structure of (+)-caryolan-1-ol synthase (CS) from Streptomyces griseus , with and without an inactive analog of the FPP substrate, 2-fluorofarnesyl diphosphate (2FFPP), bound in the active site of the enzyme. The CS-2FFPP complex was solved to 2.65 Å resolution and showed the ligand in a linear, elongated orientation, incapable of undergoing the initial cyclization event to form a bond between carbons C1 and C11. Intriguingly, the apo CS structure (2.2 Å) also had electron density in the active site, in this case density that was well fit with a curled-up tetraethylene glycol molecule presumably recruited from the crystallization medium. The density was also well fit by a molecule of farnesene suggesting that the structure may mimic an intermediate along the reaction coordinate. The curled-up conformation of tetraethylene glycol was accompanied by dramatic rotamer shifts among active-site residues. Most notably, W56 was observed to undergo a 90° rotation between the 2FFPP complex and apo-enzyme structures, suggesting that it contributes to steric interactions that help curl the tetraethylene glycol molecule in the active site, and by extension perhaps also a derivative of the FPP substrate in the normal course of the cyclization reaction. In support of this proposal, the CS W56L variant lost the ability to cyclize the FPP substrate and produced only the linear terpene products farnesol and α- and ß-farnesene.

A General Mechanism for the General Stress Response in Bacteria.

The General Stress Response promotes survival of bacteria in adverse conditions, but how sensor proteins transduce species-specific signals to initiate the response is not known. The serine/threonine phosphatase RsbU initiates the General Stress Response in B. subtilis upon binding a partner protein (RsbT) that is released from sequestration by environmental stresses. We report that RsbT activates RsbU by inducing otherwise flexible linkers of RsbU to form a short coiled-coil that dimerizes and activates the phosphatase domains. Importantly, we present evidence that related coiled-coil linkers and phosphatase dimers transduce signals from diverse sensor domains to control the General Stress Response and other signaling across bacterial phyla. These results additionally resolve the mystery of how shared sensory domains control serine/threonine phosphatases, diguanylate cyclases and histidine kinases, revealing a common coiled-coil linker transduction mechanism. We propose that this provides bacteria with a modularly exchangeable toolkit for the evolution of diverse signaling pathways.

Transverse instability in nonparaxial systems with four-wave mixing.

We present a two-dimensional coupled nonlinear Schrödinger-like system with spatial diffractions, degree of birefringence, and four-wave mixing. This system describes two physical contexts: optical pulse propagation beyond the paraxial approximation in a weakly birefringence waveguide and light propagation near exciton-polariton resonance in semiconductor superlattice materials. We find that such systems naturally support different types of diffraction profiles, including spherical, ellipsoidal, and hyperbolic structures. We then study the transverse instability of the two-dimensional system caused by an infinitesimal perturbation-induced continuous-wave solution. Also, we find out how various physical parameters, such as nonparaxiality, degree of birefringence, power, and four-wave mixing, affect the modulational instability (MI) process, in particular. We explore the existence of bright solitary wave solutions for the proposed system as the influence of MI is closely related to the latter in a nutshell.

Synthesis, structure and luminescence properties of bifunctional KCaF3 phosphor influenced by incorporating Eu3+ ions for solid state lighting and TL dosimetry applications.

Europium doped KCaF3 phosphors (KCaF3:Eu3+) were prepared using various concentrations of Eu3+ by conventional solid-state reaction process. The X-ray diffraction (XRD) studies confirmed the formation of orthorhombic structured KCaF3:Eu3+ phosphors. Scanning Electron Microscopy (SEM) image of the synthesized phosphor exhibits agglomerated particles with irregular shapes. The composition of the synthesized sample was determined by Energy Dispersive Spectroscopy (EDS) spectrum and elemental mapping showed the homogeneous dispersion of Eu3+ ions into the synthesized KCaF3:Eu3+ phosphor. The emission peak intensity at 594 nm from photoluminescence (PL) spectra was found to increase with the increase of Eu3+ concentrations from 0.02 mol% to 0.06 mol% and decreased with the further increase of Eu3+ concentration up to 0.1 mol%. CIE1931 chromaticity diagram coordinates (x, y) of KCaF3:(0.06 mol%) Eu3+ phosphors were positioned in the reddish-orange region (x = 0.5736, y = 0.4224). Photoluminescence property confirms the suitability of KCaF3:Eu3+ phosphors for Solid state lighting application. X-ray induced luminescence (radioluminescence, RL) is recorded for KCaF3:Eu3+ phosphors showing the characteristic emission of Eu2+ and Eu3+. ESR study on KCaF3:Eu3+ phosphors confirm the presence of Eu2+ ions. Beta irradiated thermoluminescence (TL) glow curve of Eu3+ doped KCaF3 phosphors is measured and deconvoluted using Gaussian fitting. TL kinetic parameters like activation energy (Ea) and frequency factor (s) are calculated for all the deconvoluted peaks using peak shape method which shows the synthesized KCaF3:Eu3+ phosphors is suitable for dosimetry application.

Role of π conjugation in n-hexylphenothiazine dyes for solar cell-a density functional theory approach.

The N-hexylphenothiazine-based organic sensitizers are designed for Dye Sensitized Solar Cell (DSSC). The different π spacer (thiophene and cyanovinyl) groups were substituted in third and seventh position N-hexylphenothiazine. From the structural modifications, the π spacer effect was analyzed. The optoelectronic properties of the dyes were tuned by structural modifications. The optimized geometry, highest occupied molecular orbital and lowest unoccupied molecular orbital energy level, and absorption spectra were calculated. The natural bond orbital analysis gives the net electron transfer from the donor to acceptor. The electrochemical properties and light-harvesting efficiency of the designed dye sensitizers were calculated. The π spacer increase resulted in the redshift of the absorption peak. Based on the density functional theory and time dependant density functional theory calculations, the designed dye molecules are evaluated for DSSC application.

The influence of π-linkers configuration on properties of 10-hexylphenoxazine donor-based sensitizer for dye-sensitized solar cell application - Theoretical approach.

10-Hexylphenoxazine based dyes with A-(π)n-D-(π)n-A architecture is designed and investigated systematically for dye-sensitized solar cell (DSSC) application by Density Functional Theory (DFT) and Time-dependent Density Functional Theory (TD-DFT). The designed sensitizers consist of 10-Hexylphenoxazine as electron donor and cyanoacrylic acid as an acceptor, connected by the Thiophene and Cyanovinyl π-spacers configurations with symmetrical and asymmetrical form. The effect of π-spacers configurations on the electronic and optical properties of the dyes is also investigated. The optimized structure, electronic properties and absorption characteristics of A-(π)n-D-(π)n-A dyes were investigated. The charge separation and polarization properties are analyzed by co-planarity, natural bond orbital (NBO), dipole moment and linear polarizability studies. The free energy change of electron injection and dye regeneration of the sensitizers are also calculated. The addition of a greater number of π-spacers improves the electronic, spectroscopic, optical, and free energy properties of the designed sensitizer. The DFT studies also reveal that the position of the π-spacers plays an important role in the electronic and spectroscopic properties.

Mechanism Underlying Anti-Markovnikov Addition in the Reaction of Pentalenene Synthase.

Most terpene synthase reactions follow Markovnikov rules for formation of high-energy carbenium ion intermediates. However, there are notable exceptions. For example, pentalenene synthase (PS) undergoes an initial anti-Markovnikov cyclization reaction followed by a 1,2-hydride shift to form an intermediate humulyl cation with positive charge on the secondary carbon C9 atom of the farnesyl diphosphate substrate. The mechanism by which these enzymes stabilize and guide the regioselectivity of secondary carbocations has not heretofore been elucidated. In an effort to better understand these reactions, we grew crystals of apo-PS, soaked them with the nonreactive substrate analogue 12,13-difluorofarnesyl diphosphate, and determined the X-ray structure of the resulting complex at 2.2 Å resolution. The most striking feature of the active site structure is that C9 is perfectly positioned to make a C-H···π interaction with the side chain benzene ring of residue F76; this would enhance hyperconjugation to stabilize a developing cation at C10 and thus support the anti-Markovnikov regioselectivity of the cyclization. The benzene ring is also positioned to catalyze the migration of H to C10 and stabilize a C9 carbocation. On the opposite face of C9, further cation stabilization is possible via interactions with the main chain carbonyl of I177 and the neighboring intramolecular C6═C7 bond. Mutagenesis experiments also support a role for residue 76 in these interactions, but most interesting is the F76W mutant, whose crystal structure clearly shows C9 and C10 centered above the fused benzene and pyrrole rings of the indole side chain, respectively, such that a carbocation at either position could be stabilized in this complex, and two anti-Markovnikov products, pentalenene and humulene, are formed. Finally, we show that there is a rough correlation (although not absolute) of an aromatic side chain (F or Y) at position 76 in related terpene synthases from Streptomyces that catalyze similar anti-Markovnikov addition reactions.

Direct Evidence of an Enzyme-Generated LPP Intermediate in (+)-Limonene Synthase Using a Fluorinated GPP Substrate Analog.

Linalyl diphosphate (LPP) is the postulated intermediate in the enzymatic cyclization of monoterpenes catalyzed by terpene synthases. LPP is considered an obligate intermediate due to the conformationally restrictive trans-C2-C3 double bond of the substrate, geranyl diphosphate (GPP), which precludes the proper positioning of carbons C1 and C6 to enable cyclization. However, because of the complexity of potential carbocation-mediated rearrangements in these enzymatic reactions, it has proven difficult to directly demonstrate the formation of LPP despite significant efforts. Here we synthesized a fluorinated substrate analog, 8,9-difluorogeranyl diphosphate (DFGPP), which is designed to allow initial ionization/isomerization and form the fluorinated equivalent of LPP (DFLPP) while preventing the subsequent ionization/cyclization to produce the α-terpinyl cation. Steady-state kinetic studies with the model enzyme (+)-limonene synthase (LS) under catalytic conditions show that the cyclization of DFGPP is completely blocked and a single linear product, difluoromyrcene, is produced. When crystals of apo-LS are soaked with DFGPP under conditions limiting turnover of the enzyme, we show, using X-ray crystallography, that DFLPP is produced in the enzyme active site and trapped in the crystals. Clear electron density is observed in the active site of the enzyme, but it cannot be appropriately fit with a model for the DFGPP substrate analog, whereas it can accommodate an extended conformation of DFLPP. This result supports the current model for monoterpene cyclization by providing direct evidence of LPP as an intermediate.

Growth, Hirshfeld surfaces, spectral, quantum chemical calculations, photoconductivity and chemical etching analyses of nonlinear optical p-toluidine p-toluenesulfonate single crystal.

A single crystal of p-toluidine p-toluenesulfonate (PTPT) has been grown by slow evaporation solution technique (SEST) at room temperature. Single crystal X-ray analysis confirms that grown crystal belongs to the monoclinic structure with space group P21. Intermolecular interactions and fingerprint plots of PTPT molecules are executed by Hirshfeld surface analysis. It was found that H···H (44.2%) contacts have maximum intermolecular interactions contributions in the total Hirshfeld surface area. The characteristic absorption band occurs at below 290 nm. The functional groups were identified using FTIR and FT-Raman analyses and compared with theoretical values. The title molecule contains fourteen CH bonds and three OH bonds. The calculated HOMO and LUMO energy values are -6.125 eV and -1.157 eV, respectively. The chemical potential (µ) and electronegativity (χ) values are estimated to be -3.4938 eV and 3.4938 eV, respectively. The strongest negative hyperconjugation occurs due to the charge transfer from the occupied orbital (σ) to the unoccupied orbital (π*) which is calculated for the σ(N20-C21) → π*(N20-O18). The green and red lines in the total density of states (TDOS) spectrum indicate the occupied orbital and virtual orbital levels, respectively. Photoconductivity studies have been done for the grown crystal. It is observed that the dark current is greater than photocurrent. It shows negative photoconductivity nature of PTPT crystal. The etching analysis was executed on (001) plane of PTPT crystal. It has rectangular shape etch pits patterns.

Donor functionalized quinoline based organic sensitizers for dye sensitized solar cell (DSSC) applications: DFT and TD-DFT investigations.

The influence of different donor groups in quinoline based novel sensitizers for dye sensitized solar cell (DSSC) applications is analyzed by using density functional theory (DFT) and time dependent density functional theory (TD-DFT). Quinoline and donor functionalized quinoline based novel organic sensitizers have been designed with different π-spacers for DSSC applications. The ground state molecular structure of novel organic sensitizers is fully optimized by DFT calculation in both gas and chloroform phases. Electronic absorption characteristics are predicted by the TD-DFT calculation in both gas and chloroform phases. The polarizable continuum model is used for solvent phase optimization. The net electron transfer from the donor to acceptor is calculated from natural bond orbital (NBO) analysis. The injection energy and dye regeneration energy values are also calculated. Different donor groups are substituted in quinoline, and these substituted quinoline donors are used as the donor group. Cyanovinyl and thiophene groups act as π-spacers and cyanoacrylic acid acts as an acceptor. DFT and TD-DFT studies of the quinoline and donor functionalized quinoline sensitizers show that the coumarin based and N-hexyltetrahydroquinoline donors are more efficient for DSSC application.

The effect of different π-bridge configuration on bi-anchored triphenylamine and phenyl modified triphenylamine based dyes for dye sensitized solar cell (DSSC) application: A theoretical approach.

Twenty eight bi-anchored triphenylamine (TH-1 to TH-14) and phenyl modified triphenylamine (PH-TH-1 to PH-TH-14) based metal free organic dyes are designed for DSSC application. The electronic effect of different π-bridge configurations in donor-π-bridge-acceptor (D-π-A)2 structure was theoretically simulated and verified using density functional theory (DFT) and time dependent density functional theory (TD-DFT). The triphenylamine and phenyl modified triphenylamine groups are used as donor and cyanoacrylic acid group is used as acceptor. Thiophene and cyanovinyl groups are used as π-bridge. The ground state molecular structure was optimized by density functional theory and the electronic absorption spectra were calculated by time dependent density functional theory. The light harvesting efficiency (LHE), dye regeneration energy (ΔGreg) and electron injection energy (ΔGinject) are determined by computational examination. It is observed that, when the number of π-bridge increases, the band gap of the dye decreases. Also the absorption maximum and molar extinction coefficient of the dyes are increased. Theoretical result shows that the thiophene-cyanovinyl and thiophene-thiophene-cyanovinyl-cyanovinyl configurations give broader and red shifted absorption spectrum compared to other configurations. Also the results of phenyl modified triphenylamine (PH-TH) dyes clearly show better absorption and dye regeneration energy compared to TH dyes.

Structure and monomer/dimer equilibrium for the guanylyl cyclase domain of the optogenetics protein RhoGC.

RhoGC is a fusion protein from the aquatic fungus Blastocladiella emersonii, combining a type I rhodopsin domain with a guanylyl cyclase domain. It has generated excitement as an optogenetics tool for the manipulation of cyclic nucleotide signaling pathways. To investigate the regulation of the cyclase activity, we isolated the guanylyl cyclase domain from Escherichia coli with (GCwCCRho) and without (GCRho) the coiled-coil linker. Both constructs were constitutively active but were monomeric as determined by size-exclusion chromatography and analytical ultracentrifugation, whereas other class III nucleotidyl cyclases are functional dimers. We also observed that crystals of GCRho have only a monomer in an asymmetric unit. Dimers formed when crystals were grown in the presence of the non-cyclizable substrate analog 2',3'-dideoxyguanosine-5'-triphosphate, MnCl2, and tartrate, but their quaternary structure did not conform to the canonical pairing expected for class III enzymes. Moreover, the structure contained a disulfide bond formed with an active-site Cys residue required for activity. We consider it unlikely that the disulfide would form under intracellular reducing conditions, raising the possibility that this unusual dimer might have a biologically relevant role in the regulation of full-length RhoGC. Although we did not observe it with direct methods, a functional dimer was identified as the active state by following the dependence of activity on total enzyme concentration. The low affinity observed for GCRho monomers is unusual for this enzyme class and suggests that dimer formation may contribute to light activation of the full-length protein.

Purification and Characterization of RhoPDE, a Retinylidene/Phosphodiesterase Fusion Protein and Potential Optogenetic Tool from the Choanoflagellate Salpingoeca rosetta.

RhoPDE is a type I rhodopsin/phosphodiesterase gene fusion product from the choanoflagellate Salpingoeca rosetta. The gene was discovered around the time that a similar type I rhodopsin/guanylyl cyclase fusion protein, RhoGC, was shown to control phototaxis of an aquatic fungus through a cGMP signaling pathway. RhoPDE has potential as an optogenetic tool catalyzing the hydrolysis of cyclic nucleotides. Here we provide an expression and purification system for RhoPDE, as well as a crystal structure of the C-terminal phosphodiesterase catalytic domain. We show that RhoPDE contains an even number of transmembrane segments, with N- and C-termini both located on the cytoplasmic surface of the cell membrane. The purified protein exhibits an absorption maximum at 490 nm in the dark state, which shifts to 380 nm upon exposure to light. The protein acts as a cGMP-selective phosphodiesterase. However, the activity does not appear to be modulated by light. The protein is also active with cAMP as a substrate, but with a roughly 5-7-fold lower kcat. A truncation consisting solely of the phosphodiesterase domain is also active with a kcat for cGMP roughly 6-9-fold lower than that of the full-length protein. The isolated PDE domain was crystallized, and the X-ray structure showed the protein to be a dimer similar to human PDE9. We anticipate that the purification system introduced here will enable further structural and biochemical experiments to improve our understanding of the function and mechanism of this unique fusion protein.

Functional and Structural Characterization of a (+)-Limonene Synthase from Citrus sinensis.

Terpenes make up the largest and most diverse class of natural compounds and have important commercial and medical applications. Limonene is a cyclic monoterpene (C10) present in nature as two enantiomers, (+) and (-), which are produced by different enzymes. The mechanism of production of the (-)-enantiomer has been studied in great detail, but to understand how enantiomeric selectivity is achieved in this class of enzymes, it is important to develop a thorough biochemical description of enzymes that generate (+)-limonene, as well. Here we report the first cloning and biochemical characterization of a (+)-limonene synthase from navel orange (Citrus sinensis). The enzyme obeys classical Michaelis-Menten kinetics and produces exclusively the (+)-enantiomer. We have determined the crystal structure of the apoprotein in an "open" conformation at 2.3 Å resolution. Comparison with the structure of (-)-limonene synthase (Mentha spicata), which is representative of a fully closed conformation (Protein Data Bank entry 2ONG ), reveals that the short H-α1 helix moves nearly 5 Å inward upon substrate binding, and a conserved Tyr flips to point its hydroxyl group into the active site.

Structural Characterization of Early Michaelis Complexes in the Reaction Catalyzed by (+)-Limonene Synthase from Citrus sinensis Using Fluorinated Substrate Analogues.

The stereochemical course of monoterpene synthase reactions is thought to be determined early in the reaction sequence by selective binding of distinct conformations of the geranyl diphosphate (GPP) substrate. We explore here formation of early Michaelis complexes of the (+)-limonene synthase [(+)-LS] from Citrus sinensis using monofluorinated substrate analogues 2-fluoro-GPP (FGPP) and 2-fluoroneryl diphosphate (FNPP). Both are competitive inhibitors for (+)-LS with KI values of 2.4 ± 0.5 and 39.5 ± 5.2 µM, respectively. The KI values are similar to the KM for the respective nonfluorinated substrates, indicating that fluorine does not significantly perturb binding of the ligand to the enzyme. FGPP and FNPP are also substrates, but with dramatically reduced rates (kcat values of 0.00054 ± 0.00005 and 0.00024 ± 0.00002 s-1, respectively). These data are consistent with a stepwise mechanism for (+)-LS involving ionization of the allylic GPP substrate to generate a resonance-stabilized carbenium ion in the rate-limiting step. Crystals of apo-(+)-LS were soaked with FGPP and FNPP to obtain X-ray structures at 2.4 and 2.2 Å resolution, respectively. The fluorinated analogues are found anchored in the active site through extensive interactions involving the diphosphate, three metal ions, and three active-site Asp residues. Electron density for the carbon chains extends deep into a hydrophobic pocket, while the enzyme remains mostly in the open conformation observed for the apoprotein. While FNPP was found in multiple conformations, FGPP, importantly, was in a single, relatively well-defined, left-handed screw conformation, consistent with predictions for the mechanism of stereoselectivity in the monoterpene synthases.