The structure of a novel glucuronoyl esterase from Myceliophthora thermophila gives new insights into its role as a potential biocatalyst

The increasing demand for the development of efficient biocatalysts is a consequence of their broad industrial applications. Typical difficulties that are encountered during their exploitation in a variety of processes are interconnected with factors such as temperature, pH, product inhibitors etc. To eliminate these, research has been directed towards the identification of new enzymes that would comply with the required standards. To this end, the recently discovered glucuronoyl esterases (GEs) are an enigmatic family within the carbohydrate esterase (CE) family. Structures of the thermophilic StGE2 esterase from Myceliophthora thermophila (synonym Sporotrichum thermophile), a member of the CE15 family, and its S213A mutant were determined at 1.55 and 1.9 Å resolution, respectively. The first crystal structure of the S213A mutant in complex with a substrate analogue, methyl 4-O-methyl-β-D-glucopyranuronate, was determined at 2.35 Å resolution. All of the three-dimensional protein structures have an α/β-hydrolase fold with a three-layer αβα-sandwich architecture and a Rossmann topology and comprise one molecule per asymmetric unit. These are the first crystal structures of a thermophilic GE both in an unliganded form and bound to a substrate analogue, thus unravelling the organization of the catalytic triad residues and their neighbours lining the active site. The knowledge derived offers novel insights into the key structural elements that drive the hydrolysis of glucuronic acid esters

Cubic lyotropic liquid crystals as drug delivery carriers: Physicochemical and morphological studies

The self-assembly process of amphiphilic molecules into solvents results in different mesophases, such as inverse cubic and hexagonal that both belong to the wider category of lyotropic liquid crystals. The above mesophases can be further exploited upon the formation of liquid crystalline nanoparticles, cubosomes and hexosomes respectively, which may be utilized as drug delivery nanosystems, exhibiting major advantages. In the present study, liquid crystalline nanoparticles were prepared and evaluated in terms of morphology and physicochemical behavior. The goal of this study is to examine the effect of the different formulation parameters, as well as the effect of the different microenvironmental factors (temperature, ionic strength, pH, serum proteins presence) on their behavior. The physicochemical behavior and the morphology of the systems were investigated by X-Ray Diffraction (XRD), cryogenic-Transmission Electron Microscopy (cryo-TEM), fluorescence spectroscopy and a gamut of light scattering techniques. The formulation process was proved to influence strictly the physicochemical behavior of the prepared nanosystems. They presented colloidal stability over time and upon ionic strength increase, but they were affected by the presence of proteins and presented reversible structure alterations upon temperature increase. Their morphological structure and internal microenvironment, reflected by micropolarity and microfluidity, were also influenced by the formulation parameters. © 2018 Elsevier B.V

Proton Translocation via Tautomerization of Asn298 During the S-2-S-3 State Transition in the Oxygen-Evolving Complex of Photosystem II

In biological water oxidation, a redox-active tyrosine residue (D1-Tyr161 or Y-Z) mediates electron transfer between the Mn4CaO5 cluster of the driving the cluster through progressively higher oxidation states S-i (i = 0-4). In contrast to lower S-states (S-0, S-1), in higher S-states (S-2, S-3) of the Mn4CaO5 cluster, Yz cannot be oxidized at cryogenic temperatures due to the accumulation of positive charge in the S-1 -> S-2 transition. However, oxidation of Y-Z by illumination of S-2 at 77-190 K followed by rapid freezing and charge recombination between Yz and the plastoquinone radical Q(A)(center dot-) allows trapping of an S-2 variant, the so-called S-2(trapped) state (S-2(t)), that is capable of forming Y-z(center dot) at cryogenic temperature. To identify the differences between the S-2 and S-2(t) states, we used the (S2Yz center dot)-Y-t intermediate as a probe for the S-2(t) state and followed the (S2Yz center dot)-Y-t/Q(A)(center dot-) recombination kinetics at 10 K using time-resolved electron paramagnetic resonance spectroscopy in H2O and D2O. The results show that while (S2Yz center dot)-Y-t/Q(A)(center dot-) recombination can be described as pure electron transfer occurring in the Marcus inverted region, the S-2(t) -> S-2 reversion depends on proton rearrangement and exhibits a strong kinetic isotope effect. This suggests that Y-Z oxidation in the 521 state is facilitated by favorable proton redistribution in the vicinity of Y-Z, most likely within the hydrogen-bonded Y-Z-His190-Asn298 triad. Computational models show that tautomerization of Asn298 to its imidic acid form enables proton translocation to an adjacent asparagine-rich cavity of water molecules that functions as a proton reservoir and can further participate in proton egress to the lumen

Stimuli-responsive lyotropic liquid crystalline nanosystems with incorporated poly(2-dimethylamino ethyl methacrylate)-b-poly(lauryl methacrylate) amphiphilic block copolymer

There is an emerging need to evolve the conventional lyotropic liquid crystalline nanoparticles to advanced stimuli-responsive, therapeutic nanosystems with upgraded functionality. Towards this effort, typically used stabilizers, such as Pluronics®, can be combined or replaced by smart, stimuli-responsive block copolymers. The aim of this study is to incorporate the stimuli-responsive amphiphilic block copolymer poly(2-(dimethylamino)ethyl methacrylate)-b-poly(lauryl methacrylate) (PDMAEMA-b-PLMA) as a stabilizer in lipidic liquid crystalline nanoparticles, in order to provide steric stabilization and simultaneous stimuli-responsiveness. The physicochemical and morphological characteristics of the prepared nanosystems were investigated by light scattering techniques, cryogenic-transmission electron microscopy (cryo-TEM), X-ray diffraction (XRD) and fluorescence spectroscopy. The PDMAEMA-b-PLMA, either individually or combined with Poloxamer 407, exhibited different modes of stabilization depending on the lipid used. Due to the protonation ability of PDMAEMA blocks in acidic pH, the nanoparticles exhibited high positive charge, as well as pH-responsive charge conversion, which can be exploited towards pharmaceutical applications. The ionic strength, temperature and serum proteins influenced the physicochemical behavior of the nanoparticles, while the polymer concentration differentiated their morphology; their micropolarity and microfluidity were also evaluated. The proposed liquid crystalline nanosystems can be considered as novel and attractive pH-responsive drug and gene delivery nanocarriers due to their polycationic content. © 2019 by the authors

Unusual ³¹P Hyperfine Strain Effects in a Conformationally Flexible Cu(II) Complex Revealed by Two-Dimensional Pulse EPR Spectroscopy

Strain effects on g and metal hyperfine coupling tensors, A, are often manifested in Electron Paramagnetic Resonance (EPR) spectra of transition metal complexes, as a result of their intrinsic and/or solvent-mediated structural variations. Although distributions of these tensors are quite common and well understood in continuous-wave (cw) EPR spectroscopy, reported strain effects on ligand hyperfine coupling constants are rather scarce. Here we explore the case of a conformationally flexible Cu(II) complex, [Cu{Ph2P(O)NP(O)Ph2-κ2O,O′}2], bearing P atoms in its second coordination sphere and exhibiting two structurally distinct CuO4 coordination spheres, namely a square planar and a tetrahedrally distorted one, as revealed by X-ray crystallography. The Hyperfine Sublevel Correlation (HYSCORE) spectra of this complex exhibit 31P correlation ridges that have unusual inverse or so-called “boomerang” shapes and features that cannot be reproduced by standard simulation procedures assuming only one set of magnetic parameters. Our work shows that a distribution of isotropic hyperfine coupling constants (hfc) spanning a range between negative and positive values is necessary in order to describe in detail the unusual shapes of HYSCORE spectra. By employing DFT calculations we show that these hfc correspond to molecules showing variable distortions from square planar to tetrahedral geometry, and we demonstrate that line shape analysis of such HYSCORE spectra provides new insight into the conformation-dependent spectroscopic response of the spin system under investigation

Unusual 31P Hyperfine Strain Effects in a Conformationally Flexible Cu(II) Complex Revealed by Two-Dimensional Pulse EPR Spectroscopy

Strain effects on g and metal hyperfine coupling tensors, A, are often manifested in Electron Paramagnetic Resonance (EPR) spectra of transition metal complexes, as a result of their intrinsic and/or solvent-mediated structural variations. Although distributions of these tensors are quite common and well understood in continuous-wave (cw) EPR spectroscopy, reported strain effects on ligand hyperfine coupling constants are rather scarce. Here we explore the case of a conformationally flexible Cu(II) complex, [Cu{Ph2P(O)NP(O)Ph2-κ2O,O′}2], bearing P atoms in its second coordination sphere and exhibiting two structurally distinct CuO4 coordination spheres, namely a square planar and a tetrahedrally distorted one, as revealed by X-ray crystallography. The Hyperfine Sublevel Correlation (HYSCORE) spectra of this complex exhibit 31P correlation ridges that have unusual inverse or so-called "boomerang" shapes and features that cannot be reproduced by standard simulation procedures assuming only one set of magnetic parameters. Our work shows that a distribution of isotropic hyperfine coupling constants (hfc) spanning a range between negative and positive values is necessary in order to describe in detail the unusual shapes of HYSCORE spectra. By employing DFT calculations we show that these hfc correspond to molecules showing variable distortions from square planar to tetrahedral geometry, and we demonstrate that line shape analysis of such HYSCORE spectra provides new insight into the conformation-dependent spectroscopic response of the spin system under investigation. © 2020 American Chemical Society