A site-saturated mutagenesis study of pentaerythritol tetranitrate reductase reveals that residues 181 and 184 influence ligand binding, stereochemistry and reactivity

We have conducted a site‐specific saturation mutagenesis study of H181 and H184 of flavoprotein pentaerythritol tetranitrate reductase (PETN reductase) to probe the role of these residues in substrate binding and catalysis with a variety of α,β‐unsaturated alkenes. Single mutations at these residues were sufficient to dramatically increase the enantiopurity of products formed by reduction of 2‐phenyl‐1‐nitropropene. In addition, many mutants exhibited a switch in reactivity to predominantly catalyse nitro reduction, as opposed to C?C reduction. These mutants showed an enhancement in a minor side reaction and formed 2‐phenylpropanal oxime from 2‐phenyl‐1‐nitropropene. The multiple binding conformations of hydroxy substituted nitro‐olefins in PETN reductase were examined by using both structural and catalytic techniques. These compounds were found to bind in both active and inhibitory complexes; this highlights the plasticity of the active site and the ability of the H181/H184 couple to coordinate with multiple functional groups. These properties demonstrate the potential to use PETN reductase as a scaffold in the development of industrially useful biocatalysts.     

An Engineered Old Yellow Enzyme that Enables Efficient Synthesis of (4R,6R)‐Actinol in a One‐Pot Reduction System

(4R,6R)‐Actinol can be stereo‐selectively synthesized from ketoisophorone by a two‐step conversion using a mixture of two enzymes: Candida macedoniensis old yellow enzyme (CmOYE) and Corynebacterium aquaticum (6R)‐levodione reductase. However, (4S)‐phorenol, an intermediate, accumulates because of the limited substrate range of CmOYE. To address this issue, we solved crystal structures of CmOYE in the presence and absence of a substrate analogue p‐HBA, and introduced point mutations into the substrate‐recognition loop. The most effective mutant (P295G) showed two‐ and 12‐fold higher catalytic activities toward ketoisophorone and (4S)‐phorenol, respectively, than the wild‐type, and improved the yield of the two‐step conversion from 67.2 to 90.1 %. Our results demonstrate that the substrate range of an enzyme can be changed by introducing mutation(s) into a substrate‐recognition loop. This method can be applied to the development of other favorable OYEs with different substrate preferences.     

Directed Divergent Evolution of a Thermostable D‐Tagatose Epimerase towards Improved Activity for Two Hexose Substrates

Functional promiscuity of enzymes can often be harnessed as the starting point for the directed evolution of novel biocatalysts. Here we describe the divergent morphing of an engineered thermostable variant (Var8) of a promiscuous D ‐tagatose epimerase (DTE) into two efficient catalysts for the C3 epimerization of D ‐fructose to D ‐psicose and of L ‐sorbose to L ‐tagatose. Iterative single‐site randomization and screening of 48 residues in the first and second shells around the substrate‐binding site of Var8 yielded the eight‐site mutant IDF8 (ninefold improved k_cat for the epimerization of D ‐fructose) and the six‐site mutant ILS6 (14‐fold improved epimerization of L ‐sorbose), compared to Var8. Structure analysis of IDF8 revealed a charged patch at the entrance of its active site; this presumably facilitates entry of the polar substrate. The improvement in catalytic activity of variant ILS6 is thought to relate to subtle changes in the hydration of the bound substrate. The structures can now be used to select additional sites for further directed evolution of the ketohexose epimerase.     

The Structure of Glycerol Trinitrate Reductase NerA from Agrobacterium radiobacter Reveals the Molecular Reason for Nitro‐ and Ene‐Reductase Activity in OYE Homologues

In recent years, Old Yellow Enzymes (OYEs) and their homologues have found broad application in the efficient asymmetric hydrogenation of activated C?C bonds with high selectivities and yields. Members of this class of enzymes have been found in many different organisms and are rather diverse on the sequence level, with pairwise identities as low as 20 %, but they exhibit significant structural similarities with the adoption of a conserved (αβ)₈‐barrel fold. Some OYEs have been shown not only to reduce C?C double bonds, but also to be capable of reducing nitro groups in both saturated and unsaturated substrates. In order to understand this dual activity we determined and analyzed X‐ray crystal structures of NerA from Agrobacterium radiobacter, both in its apo form and in complex with 4‐hydroxybenzaldehyde and with 1‐nitro‐2‐phenylpropene. These structures, together with spectroscopic studies of substrate binding to several OYEs, indicate that nitro‐containing substrates can bind to OYEs in different binding modes, one of which leads to C?C double bond reduction and the other to nitro group reduction.     

Inhibition of Leishmania infantum Trypanothione Reductase by Azole‐Based Compounds: a Comparative Analysis with Its Physiological Substrate by X‐ray Crystallography

Herein we report a study aimed at discovering a new class of compounds that are able to inhibit Leishmania donovani cell growth. Evaluation of an in‐house library of compounds in a whole‐cell screening assay highlighted 4‐((1‐(4‐ethylphenyl)‐2‐methyl‐5‐(4‐(methylthio)phenyl)‐1H‐pyrrol‐3‐yl)methyl)thiomorpholine (compound 1 ) as the most active. Enzymatic assays on Leishmania infantum trypanothione reductase (LiTR, belonging to the Leishmania donovani complex) shed light on both the interaction with, and the nature of inhibition by, compound 1 . A molecular modeling approach based on docking studies and on the estimation of the binding free energy aided our rationalization of the biological data. Moreover, X‐ray crystal structure determination of LiTR in complex with compound 1 confirmed all our results: compound 1 binds to the T(SH)₂ binding site, lined by hydrophobic residues such as Trp21 and Met113, as well as residues Glu18 and Tyr110. Analysis of the structure of LiTR in complex with trypanothione shows that Glu18 and Tyr110 are also involved in substrate binding, according to a competitive inhibition mechanism.     

Electrospinning of Continuous Aligning Yarns with a ‘Funnel’ Target

A new electrospinning set‐up is described for continuous aligning of poly(L ‐lactide) yarn. It comprises a slowly rotating grounded ‘funnel’ target and a winder placed right up to the funnel. A charged polymer jet is ejected from a nozzle. The electrospun fibers are first accumulated on the mouth plane of the funnel to form a web. The web is then pulled upward and guided to a winder on which twisted fibers are continuously wound as a bundle. Each filament of the bundle is slanted at about 45° relative to the collecting direction, and its average diameter is 6.0 ± 1.9 µm. The drawn fiber shows improved tensile properties as a result of the increased molecular orientation. The morphologies of the twisted fibers before and after drawing are compared.

     

Effects of an oxygen‐ion beam (O+7, 100 MeV) and γ irradiation on polypyrrole films

Nanostructured polypyrrole films doped with para‐toluene sulfonic acid were prepared by an electrochemical process, and a comparative study of the effects of swift heavy ions and γ‐ray irradiation on the structural and optical properties of the polypyrrole was carried out. Oxygen‐ion (energy = 100 MeV, charge state = +7) fluence varied from 1 × 10¹⁰ to 3 × 10¹² ions/cm², and the γ dose varied from 6.8 to 67 Gy. The polymer films were characterized by X‐ray diffraction, ultraviolet–visible spectroscopy, and scanning electron microscopy. The X‐ray diffraction pattern showed that after irradiation, the crystallinity improved with increasing fluence because of an increase in the crystalline regions dispersed in an amorphous phase. The ultraviolet–visible spectra showed a shift in the absorbance edge toward higher wavelengths, which indicated a significant decrease in the band gap of the polypyrrole film after irradiation. The scanning electron microscopy study showed a systematic change in the surface of the polymer. A similar pattern was observed with the γ irradiation. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2010     

Binding to Large Enzyme Pockets: Small‐Molecule Inhibitors of Trypanothione Reductase

The causative agents of the parasitic disease human African trypanosomiasis belong to the family of trypanosomatids. These parasitic protozoa exhibit a unique thiol redox metabolism that is based on the flavoenzyme trypanothione reductase (TR). TR was identified as a potential drug target and features a large active site that allows a multitude of possible ligand orientations, which renders rational structure‐based inhibitor design highly challenging. Herein we describe the synthesis, binding properties, and kinetic analysis of a new series of small‐molecule inhibitors of TR. The conjunction of biological activities, mutation studies, and virtual ligand docking simulations led to the prediction of a binding mode that was confirmed by crystal structure analysis. The crystal structures revealed that the ligands bind to the hydrophobic wall of the so‐called “mepacrine binding site”. The binding conformation and potency of the inhibitors varied for TR from Trypanosoma brucei and T. cruzi.     

Monitoring Binding of HIV‐1 Capsid Assembly Inhibitors Using 19F Ligand‐and 15N Protein‐Based NMR and X‐ray Crystallography: Early Hit Validation of a Benzodiazepine Series

The emergence of resistance to existing classes of antiretroviral drugs underlines the need to find novel human immunodeficiency virus (HIV)‐1 targets for drug discovery. The viral capsid protein (CA) represents one such potential target. Recently, a series of benzodiazepine inhibitors was identified via high‐throughput screening using an in vitro capsid assembly assay (CAA). Here, we demonstrate how a combination of NMR and X‐ray co‐crystallography allowed for the rapid characterization of the early hits from this inhibitor series. Ligand‐based ¹⁹F NMR was used to confirm inhibitor binding specificity and reversibility as well as to identify the N‐terminal domain of the capsid (CA_NTD) as its molecular target. Protein‐based NMR (¹H and ¹⁵N chemical shift perturbation analysis) identified key residues within the CA_NTD involved in inhibitor binding, while X‐ray co‐crystallography confirmed the inhibitor binding site and its binding mode. Based on these results, two conformationally restricted cyclic inhibitors were designed to further validate the possible binding modes. These studies were crucial to early hit confirmation and subsequent lead optimization.     

Self‐welding 1‐butene/ethylene copolymers from metallocene catalysts: Structure,morphology, and mechanical properties

Samples of random copolymers consisting of 1‐butene modified with a low ethylene content (4, 5, 8% by weight) produced with metallocene catalysts were studied to elucidate the polymorphic behavior of this new class of materials and to characterize them from a structural, morphological, and mechanical point of view. The samples cooled down from the melt are in amorphous phase and crystallize in a mixture of form I and I′ or in pure form I′ with aging time, according to the C2 content. Infrared and nuclear magnetic resonance spectroscopy, X‐ray diffraction and microscopic techniques were used to follow the changes of the material with aging time and to correlate the structural and morphological behavior with the peculiar mechanical properties that differentiate the samples with increasing C2 content. The presence, in the aged samples with higher C2 content, of the pure form I′ induces the peculiar ability to self‐welding and these copolymers combine high flexibility with good elasticity and ductility and can be processed directly or used as modifying agents in polymers. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 40119.     

Crystal Structures of a Bioactive 6′‐Hydroxy Variant of Sisomicin Bound to the Bacterial and Protozoal Ribosomal Decoding Sites

Parasitic infections recognized as neglected tropical diseases are a source of concern for several regions of the world. Aminoglycosides are potent antimicrobial agents that have been extensively studied by biochemical and structural studies in prokaryotes. However, the molecular mechanism of their potential antiprotozoal activity is less well understood. In the present study, we have examined the in vitro inhibitory activities of some aminoglycosides with a 6′‐hydroxy group on ring I and highlight that one of them, 6′‐hydroxysisomicin, exhibits promising activity against a broad range of protozoan parasites. Furthermore, we have conducted X‐ray analyses of 6′‐hydroxysisomicin bound to the target ribosomal RNA A‐sites in order to understand the mechanisms of both its antibacterial and antiprotozoal activities at the molecular level. The unsaturated ring I of 6′‐hydroxysisomicin can directly stack on G1491, which is highly conserved in bacterial and protozoal species, through π–π interaction and fits closer to the guanidine base than the typically saturated and hydroxylated ring I of other structurally related aminoglycosides. Consequently, the compound adopts a lower energy conformation within the bacterial and protozoal A‐sites and makes pseudo pairs to either A or G at position 1408. The A‐site‐selective binding mode strongly suggests that 6′‐hydroxysisomicin is a potential lead for the design of next‐generation aminoglycosides targeting a wide variety of infectious diseases.     

Effect of Ordering on the Microwave Dielectric Properties of Spinel‐Structured (Zn1−x(Li2/3Ti1/3)x)2TiO4 Ceramics

Spinel‐structured (Zn_1?x(Li_2/3Ti_1/3)_x)₂TiO₄ (x = 0–1) microwave dielectric ceramics were manufactured via a conventional mixed‐oxide method. The X‐ray diffraction and Raman spectra revealed that a disordered face‐centered cubic phase was found in the composition range of x < 0.5, and an ordered primitive cubic spinel solid solution was achieved as x was beyond 0.5. Such a disorder–order transition near x = 0.5 was accompanied by the variation of composition‐induced cation occupancy. The Q × f value first kept increasing up to ~160 000 (GHz) in disordered ceramics, and then sharply decreased as an ordered structure appeared at x ≥ 0.5. An obvious decrease in τ_f value was also accompanied by the appearance of an ordered structure. The minimum τ_f value (~ ?20 ppm/°C) was obtained in the x = 0.75 sample with the highest structural order degree. These results demonstrated that microwave dielectric properties of current spinel ceramics could be successfully modified by adjusting their structural order degree, which could be appropriately adopted for the design of spinel‐structured materials with favorable properties.     

Synthesis and Properties of Branched Oligo(2‐thienyl)‐ and Oligo(2,2′‐bithien‐5‐yl)‐Substituted Pyridine Derivatives

Starting from easily accessible precursors we describe the preparation of a series of branched oligo(2‐thienyl)‐ and oligo(2,2′‐bithienyl)‐substituted pyridine derivatives. With palladium‐catalyzed cross‐coupling reactions of pyridyl nonaflates/triflates as key steps we synthesized 2,6‐di(2‐thienyl)pyridines bridged by thiophene or benzene rings. By selective bromination of 2,6‐di(2‐thienyl)pyridine and 2,4,6‐tri(2‐thienyl)pyridine and subsequent coupling reactions an access to oligo(2,2′‐bithien‐5‐yl)‐substituted pyridine derivatives was gained. The constitution and solid state conformation of 2,6‐bis(2,2’‐bithien‐5‐yl)pyridine was determined by X‐ray analysis. This series of new pyridine‐thiophene conjugates was systematically investigated by UV/vis spectroscopy. Large Stokes shifts in the neutral and protonated form were observed. The electrochemical oxidation of two typical compounds was studied, however, these oxidations were irreversible forming a polymeric film at the anode. As a selected example, a thiophene‐bridged 2,6‐di(2‐thienyl)pyridine derivative was also investigated by scanning tunneling microscopy showing an interesting self‐assembly into a highly ordered monolayer on highly oriented pyrolytic graphite.

     

Controlling the surface wettability of poly(sulfone) films by UV‐assisted treatment: benefits in relation to plasma treatment

Hydrophilic and superhydrophilic surfaces of poly(sulfone) (PSU) thin films were prepared by UV irradiation in the presence of O₂ or acrylic acid (AA) vapor. Treated surfaces were then investigated by water contact angle measurements, Fourier transformed IR spectroscopy in attenuated total reflectance mode (FTIR‐ATR), X‐ray photoelectron spectroscopy (XPS), near‐edge X‐ray absorption fine structure (NEXAFS) and AFM. Water contact angle values of treated PSU films using either O₂ or AA vapor as the reactive atmosphere reached about 6° after more than 120 min of irradiation. FTIR‐ATR, XPS and NEXAFS analysis showed incorporation of oxygenated groups onto the surface that led to its hydrophilic characteristics. In addition, when AA vapor was used as the reactive atmosphere, a photopolymerization process of poly(acrylic acid) onto the surface of the PSU was observed. AFM analysis showed a very low level of roughness after the treatments. A comparison of UV‐assisted surface modifications of PSU films with traditional plasma treatments showed excellent qualitative agreement between the two techniques. Our results show that UV‐assisted treatments in the presence of AA vapor or O₂ are efficient ways of controlling the surface wettability and functionalities grafted on the surface of PSU films. This treatment can be considered as a permanent dry grafting method that resists aging and uses a simple experimental setup. © 2012 Society of Chemical Industry     

Structure and characterization of poly[(vinylidene fluoride)‐co‐hexafluoropropene]–(dibutyl phthalate) blends

Blends of poly[(vinylidene fluoride)‐co‐hexafluoropropene] with dibutyl phthalate were examined by wide‐ and small‐angle X‐ray scattering, differential scanning calorimetry and dynamic mechanical spectroscopy, in order to study the influence of amount of plasticizer and the crystallization rate on the crystallinity and lamellar morphology of the copolymer. The dibutyl phthalate seems, at least for the cooling and heating rates used, simply to dilute the crystalline phase without affecting the amount of polymer that is able to crystallize. Furthermore, the small‐angle X‐ray scattering technique points out that the plasticizer mostly enters the amorphous phase either outside or inside the lamellar stacks. © 2001 Society of Chemical Industry     

Metal Fluorocarbon Pyrolants,XVI : Theoretical and Experimental Investigation of Poly[bis(2,2,2‐trifluoroethoxy)phosphazene] (PTFEP) as Oxidizer in Magnesium Based Pyrolants

Consolidated magnesium/poly[bis(2,2,2‐trifluoroethoxy)phosphazene] (MPZ) pyrolants display a stable combustion regime between ξ(Mg) 30–83 wt‐%. The combustion rate increases with increasing magnesium content. Mixtures containing between 15–29 wt‐% Mg do ignite with a butane torch but extinguish rapidly thereafter. The combustion flames for each stoichiometric range exhibit particular features such as luminous carbon particles, after burn luminescence of Mg and burning Mg‐rich ejecta. UV/Vis spectra of the combustion flames show a distinct continuum superimposed from signals for transient CF₂, Mg, MgF, MgO, P₂⁺, PH, PN, and PO. The thermochemical calculation using NASA‐CEA indicate the highest adiabatic temperatures 1889 K at ξ(Mg)=35 wt‐%. The aerobic combustion of MPZ yields similar temperatures as common magnesium/polytetrafluoroethylene pyrolants.     

Effect of substituting CaO with BaO on the viscosity and structure of CaO‐BaO‐SiO2‐MgO‐Al2O3 slags

In this study, the effect of CaO and BaO substitution on the viscosity and structure of CaO‐BaO‐SiO₂‐MgO‐Al₂O₃ slags was investigated. The results showed that the viscosity increased with an increase in the BaO substitution concentration, which was correlated to an increase in the degree of polymerization (DOP) of the slag structural units as the activation energy increased from 207.9 to 263.8 kJ/mol for viscous flow. Deconvolution and area integration of the Raman spectrum of the slag revealed that the ratio of Q³/Q² (Qⁱ, i is the number of O⁰ in a [SiO₄]‐tetrahedral unit) increased and NBO/Si (nonbridging oxygen per unit silicon atom) decreased with higher BaO content. It was also observed from the ²⁷Al magic angles pinning nuclear magnetic resonance (²⁷Al MAS‐NMR) spectrum that the relative proportion of Al^IV increased, while that of Al^V decreased because of the decrease in the percentage of nonbridging oxygen (O^?), indicating the polymerization of the slag. O_1s X‐ray photoelectron spectroscopy (XPS) was also carried out to semi‐quantitatively analyze the various types of oxygen anions present in the slag. The XPS results correlated well with the results obtained from the analysis of the Raman and ²⁷Al MAS‐NMR spectra of the slags and its viscous behavior.