Improving the Thermostability of a Fungal GH11 Xylanase via Fusion of a Submodule (C2) from Hyperthermophilic CBM9_1-2

Xylanases have been applied in many industrial fields. To improve the activity and thermostability of the xylanase CDBFV from Neocallimastix patriciarum (GenBank accession no. KP691331), submodule C2 from hyperthermophilic CBM9_1-2 was inserted into the N- and/or C-terminal regions of the CDBFV protein (producing C2-CDBFV, CDBFV-C2, and C2-CDBFV-C2) by genetic engineering. CDBFV and the hybrid proteins were successfully expressed in Escherichia coli BL21 (DE3). Enzymatic property analysis indicates that the C2 submodule had a significant effect on enhancing the thermostability of the CDBFV. At the optimal temperature (60.0 °C), the half-lives of the three chimeras C2-CDBFV, CDBFV-C2, and C2-CDBFV-C2 are 1.5 times (37.5 min), 4.9 times (122.2 min), and 3.8 times (93.1 min) longer than that of wild-type CDBFV (24.8 min), respectively. More importantly, structural analysis and molecular dynamics (MD) simulation revealed that the improved thermal stability of the chimera CDBFV-C2 was on account of the formation of four relatively stable additional hydrogen bonds (S42-S462, T59-E277, S41-K463, and S44-G371), which increased the protein structure’s stability. The thermostability characteristics of CDBFV-C2 make it a viable enzyme for industrial applications.

Resistivity Testing of Palladium Dilution Limits in CoPd Alloys for Hydrogen Storage

Palladium satisfies most of the requirements for an effective hydrogen storage material with two major drawbacks: it has a relatively low gravimetric hydrogen density and is prohibitively expensive for large scale applications. Pd-based alloys should be considered as possible alternatives to a pure Pd. The question is how much one can dilute the Pd concentration in a variety of candidate materials while preserving the hydrogen absorption capability. We demonstrate that the resistivity measurements of thin film alloy samples can be used for a qualitative high-throughput screening and study of the hydrogen absorbing properties over the entire range of palladium concentrations. Contrary to palladium-rich alloys where additional hydrogen scattering indicates a degree of hydrogen content, the diluted alloy films respond by a decrease in resistance due to their thickness expansion. Evidence of significant hydrogen absorption was found in thin CoPd films diluted to just 20% of Pd.

Generation of Iminoxyl Radicals via Photoredox Catalysis Enables Oxidant-Free Hydroxygenation of β,γ-Unsaturated Oximes

A visible-light photocatalytic generation of iminoxyl radicals has been accomplished under oxidant-free condition. This approach offers a mild, atom-economical and redox-neutral synthesis of 5-methyl isoxazolines via radical hydroxygenation of β,γ-unsaturated oximes in the absence of additional hydrogen source.

Increased neutralization of SARS-CoV-2 Delta variant by nanobody (Nb22) and the structural basis

Delta variant, also known as B.1.617.2, has become a predominant circulating variant in many countries since it first emerged in India in December 2020. Delta variant is less sensitive to serum neutralization from COVID-19 convalescent individuals or vaccine recipients, relative to Alpha strains. It was also resistant to neutralization by some anti-receptor binding domain (RBD) and anti-N-terminal domain (NTD) antibodies in clinics. Previously, we reported the discovery of nanobodies isolated from an alpaca immunized with spike protein, exhibiting ultrahigh potency against SARS-CoV-2 and its mutated variants, where a novel inhalable bispecific Nb15 protected SARS-CoV-2 infection in hACE2 mice. Here, we found that Nb22-Fc, among our previously reported nanobodies, exhibited 8.4-fold increased neutralization potency against Delta variant with an IC50 value of 0.41 ng/ml (5.13 pM) relative to Alpha variant. Furthermore, our crystal structural analysis reveals that the binding of Nb22 on SARS-CoV-2 RBD effectively blocks the binding of RBD to ACE2 during virus infection. Furthermore, the L452R mutation in RBD of Delta variant forms an additional hydrogen bond with the hydroxy group of T30 of Nb22, leading to the increased neutralization potency of Nb22 against Delta variant. Thus, Nb22 is a potential therapeutic agent against SARS-CoV-2, especially the highly contagious Delta variant.

Physical and Mechanical Properties of the Road Surface when Replacing the Finely Crushed Mineral Part of Asphalt Concrete with Porcelain Stoneware Production Waste

The possibility of using waste porcelain stoneware - a high-strength non-porous, dense material as a finely crushed mineral part of asphalt concrete with 0-5 mm fraction has been established. The adhesive additive Bitaden content was revealed, that intensifies porcelain stoneware interaction with bitumen due to the additional hydrogen bonds formation, the particles’ contact plane activation with the action of van der Waals forces. The conditions for obtaining asphalt concrete mixtures of type B, grade 1 and G, grade 2 of the optimal grain composition with a reduced content of BND 60/90 bitumen have been developed. Physical and mechanical properties are characterized by an increase in the water resistance of asphalt concrete samples based on porcelain stoneware, compressive strength and shear resistance.

Abiological Catalysis by Myoglobin Mutant with a Genetically Incorporated Unnatural Amino Acid

To inculcate biocatalytic activity in the oxygen-storage protein myoglobin (Mb), a genetically engineered myoglobin mutant H64DOPA (DOPA = L-3,4-dihydroxyphenylalanine) has been created. Incorporation of unnatural amino acids has already demonstrated their ability to accomplish many non-natural functions in proteins efficiently. Herein, the presence of redox-active DOPA residue in the active site of mutant Mb presumably stabilizes the compound I in the catalytic oxidation process by participating in an additional hydrogen bonding (H-bonding) as compared to the WT Mb. Specifically, a general acid-base catalytic pathway was achieved due to the availability of the hydroxyl moieties of DOPA. The reduction potential values of WT (E° = - 260 mV) and mutant Mb (E° = - 300 mV), w.r.t. Ag/AgCl reference electrode, in the presence of hydrogen peroxide, indicated an additional H-bonding in the mutant protein, which is responsible for the peroxidase activity of the mutant Mb. We observed that in the presence of 5 mM H2O2, H64DOPA Mb oxidizes thioanisole and benzaldehyde with a 10 and 54 folds higher rate, respectively, as opposed to WT Mb. Based on spectroscopic, kinetic, and electrochemical studies, we deduce that DOPA residue, when present within the distal pocket of mutant Mb, alone serves the role of His/Arg-pair of peroxidases.

Crystal structure of (E)-doxepin hydrochloride, C19H22NOCl

The crystal structure of (E)-doxepin hydrochloride has been solved and refined using synchrotron X-ray powder diffraction data, and optimized using density functional techniques. (E)-doxepin hydrochloride crystallizes in space group P21/a (#14) with a = 13.78488(7), b = 8.96141(7), c = 14.30886(9) Å, β = 96.5409(5)°, V = 1756.097(12) Å3, and Z = 4. There is a strong discrete hydrogen bond between the protonated nitrogen atom and the chloride anion. There are six C–H⋯Cl hydrogen bonds between the methyl groups and the chloride, as well as additional hydrogen bonds from methylene groups and the vinyl proton. The hydrogen bonds are important in determining the solid-state conformation of the cation. The compound is essentially isostructural to amitriptyline hydrochloride. The powder pattern is included in the Powder Diffraction File™ as entry 00-066-1613.

The basis of a more contagious 501Y.V1 variant of SARS-COV-2

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) is causing a world-wide pandemic. A variant of SARS-COV-2 (20I/501Y.V1) recently discovered in the United Kingdom has a single mutation from N501 to Y501 within the receptor binding domain (Y501-RBD), of the Spike protein of the virus. This variant is much more contagious than the original version (N501-RBD). We found that this mutated version of RBD binds to human Angiotensin Converting Enzyme 2 (ACE2) a ~10 times more tightly than the native version (N501-RBD). Modeling analysis showed that the N501Y mutation would allow a potential aromatic ring-ring interaction and an additional hydrogen bond between the RBD and ACE2. However, sera from individuals immunized with the Pfizer-BioNTech vaccine still efficiently block the binding of Y501-RBD to ACE2 though with a slight compromised manner by comparison with their ability to inhibit binding to ACE2 of N501-RBD. This may raise the concern whether therapeutic anti-RBD antibodies used to treat COVID-19 patients are still efficacious. Nevertheless, a therapeutic antibody, Bamlanivimab, still binds to the Y501-RBD as efficiently as its binds to N501-RBD.

In crystallo screening for proline analog inhibitors of the proline cycle enzyme PYCR1

Pyrroline-5-carboxylate reductase 1 (PYCR1) catalyzes the biosynthetic half-reaction of the proline cycle by reducing Δ1-pyrroline-5-carboxylate (P5C) to proline through the oxidation of NAD(P)H. Many cancers alter their proline metabolism by up-regulating the proline cycle and proline biosynthesis, and knockdowns of PYCR1 lead to decreased cell proliferation. Thus, evidence is growing for PYCR1 as a potential cancer therapy target. Inhibitors of cancer targets are useful as chemical probes for studying cancer mechanisms and starting compounds for drug discovery; however, there is a notable lack of validated inhibitors for PYCR1. To fill this gap, we performed a small-scale focused screen of proline analogs using X-ray crystallography. Five inhibitors of human PYCR1 were discovered: l-tetrahydro-2-furoic acid, cyclopentanecarboxylate, l-thiazolidine-4-carboxylate, l-thiazolidine-2-carboxylate, and N-formyl l-proline (NFLP). The most potent inhibitor was NFLP, which had a competitive (with P5C) inhibition constant of 100 μm. The structure of PYCR1 complexed with NFLP shows that inhibitor binding is accompanied by conformational changes in the active site, including the translation of an α-helix by 1 Å. These changes are unique to NFLP and enable additional hydrogen bonds with the enzyme. NFLP was also shown to phenocopy the PYCR1 knockdown in MCF10A H-RASV12 breast cancer cells by inhibiting de novo proline biosynthesis and impairing spheroidal growth. In summary, we generated the first validated chemical probe of PYCR1 and demonstrated proof-of-concept for screening proline analogs to discover inhibitors of the proline cycle.

Crystal structure of posnjakite formed in the first crystal water-cooling line of the ANSTO Melbourne Australian Synchrotron MX1 Double Crystal Monochromator

Exceptionally large crystals of posnjakite, Cu4SO4(OH)6(H2O), formed during corrosion of a Swagelock(tm) Snubber copper gasket within the MX1 beamline at the ANSTO-Melbourne, Australian Synchrotron. The crystal structure was solved using synchrotron radiation to R 1 = 0.029 and revealed a structure based upon [Cu4(OH)6(H2O)O] sheets, which contain Jahn–Teller-distorted Cu octahedra. The sulfate tetrahedra are bonded to one side of the sheet via corner sharing and linked to successive sheets via extensive hydrogen bonds. The sulfate tetrahedra are split and rotated, which enables additional hydrogen bonds.