Possible Steps of the Carboxylation of Ribulose-1,5-biphosphate from Intermediates: 2,3-Enediol versus 1,2-Enol

Ribulose 1,5-bisphosphate (RuBP) undergoes enolization to initiate fixation of atmospheric carbon dioxide in the plant carbon cycle. The known model assumes the binding of RuBP to the Rubisco active site with the subsequent formation of 2,3-enediol (2,3,4-trihydroxypent-2-ene-1,5-diyl diphosphate). In the present study, it is assumed that 1,2-enol (2,3,4-trihydroxypent-1-ene-1,5-diyl diphosphate) can be formed in the enolization step to initiate the carboxylation reaction. We have used Kohn–Sham density functional theory on WB97X-D3/Def2-TZVP levels to compare the reaction barriers in the two ways. We considered the pathways of carboxylation of 1/2-ene (mono/di)ol via the C1 and C2 carbons without taking into account the binding of RuBP to the magnesium ion. Calculations of Gibbs free energies confirm the equal probability of the formation of 2,3-enediol and 1,2-enol. Quantum–chemical modeling of enolization and carboxylation reactions supports the important role of the bridging water molecule and diphosphate groups, which provide proton transfer and lower reaction barriers. The results show that carbon dioxide fixation can occur without a magnesium ion, and binding with C1 can have a lower barrier (~12 kcal/mol) than with C2 (~23 kcal/mol).     

Structural and Biofunctional Insights into the Cyclo(Pro-Pro-Phe-Phe-) Scaffold from Experimental and In Silico Studies: Melanoma and Beyond

Short peptides have great potential as safe and effective anticancer drug leads. Herein, the influence of short cyclic peptides containing the Pro-Pro-Phe-Phe sequence on patient-derived melanoma cells was investigated. Cyclic peptides such as cyclo(Leu-Ile-Ile-Leu-Val-Pro-Pro-Phe-Phe-), called CLA, and cyclo(Pro-homoPro-β³homoPhe-Phe-), called P11, exert the cytotoxic and the cytostatic effects in melanoma cells, respectively. CLA was the most active peptide as it reduced the viability of melanoma cells to 50% of control at about 10 µM, whereas P11 at about 40 µM after 48 h incubation. Interestingly, a linear derivative of P11 did not induce any effect in melanoma cells confirming previous studies showing that cyclic peptides exert better biological activity compared to their linear counterparts. According to in silico predictions, cyclic tetrapeptides show a better pharmacokinetic and toxic profile to humans than CLA. Notably, the spatial structure of those peptides containing synthetic amino acids has not been explored yet. In the Cambridge Structural Database, there is only one such cyclic tetrapeptide, cyclo((R)-β²homoPhe-D-Pro-Lys-Phe-), while in the Protein Data Bank—none. Therefore, we report the first crystal structure of cyclo(Pro-Pro-β³homoPhe-Phe-), denoted as 4B8M, a close analog of P11, which is crucial for drug discovery. Comparative molecular and supramolecular analysis of both structures was performed. The DFT findings revealed that 4B8M is well interpreted in the water solution. The results of complex Hirshfeld surface investigations on the cooperativity of interatomic contacts in terms of electrostatic and energetic features are provided. In short, the enrichment ratio revealed O^…H/H^…O and C^…H/H^…C as privileged intercontacts in the crystals in relation to basic and large supramolecular H-bonding synthon patterns. Furthermore, the ability of self-assemble 4B8M leading to a nanotubular structure is also discussed.     

New Insights into the Structure of Kappa/Beta-Carrageenan: A Novel Potential Inhibitor of HIV-1

New insights into the structure of the hybrid κ/β-carrageenan (κ/β-CRG) of the red alga Tichocarpus crinitus have been obtained. Carrageenan oligosaccharides were prepared through the chemical and enzymatic depolymerization of κ/β-CRG with κ-carrageenase and its the enzyme-resistant fraction. The composition and distribution of the repetition units of κ/β- CRG were investigated by using the negative ion tandem MALDI-TOFMS and ESIMS method, which made it possible to prove and characterize the hybrid structure of this polysaccharide. An analysis revealed the blockwise distribution of the long β-blocks along the polysaccharide chain, with the inclusion of κ/β, μ/ν-blocks and some ι-blocks. Furthermore, the desulfated κ/β-CRG was shown to contain of –G–D– repeating units up to 3.5 kDa. Previous studies have demonstrated that CRGs suppress the replication of several viruses. Here, we established that κ/β-CRG and its oligosaccharides significantly inhibit the transduction efficiency of replication-defective lentiviral particles pseudotyped with the envelope proteins of three different viruses. We found that the polysaccharide and its oligosaccharides strongly reduced the transduction efficiency of lentiviral particles pseudotyped with GP160—the envelope protein of the human immunodeficiency virus HIV-1—when added to T-lymphocyte Jurkat cells. The CRG oligosaccharides displayed significantly higher antiviral activity.     

Insights into the Structure of the Highly Glycosylated Ffase from Rhodotorula dairenensis Enhance Its Biotechnological Potential

Rhodotorula dairenensis β-fructofuranosidase is a highly glycosylated enzyme with broad substrate specificity that catalyzes the synthesis of 6-kestose and a mixture of the three series of fructooligosaccharides (FOS), fructosylating a variety of carbohydrates and other molecules as alditols. We report here its three-dimensional structure, showing the expected bimodular arrangement and also a unique long elongation at its N-terminus containing extensive O-glycosylation sites that form a peculiar arrangement with a protruding loop within the dimer. This region is not required for activity but could provide a molecular tool to target the dimeric protein to its receptor cellular compartment in the yeast. A truncated inactivated form was used to obtain complexes with fructose, sucrose and raffinose, and a Bis-Tris molecule was trapped, mimicking a putative acceptor substrate. The crystal structure of the complexes reveals the major traits of the active site, with Asn387 controlling the substrate binding mode. Relevant residues were selected for mutagenesis, the variants being biochemically characterized through their hydrolytic and transfructosylating activity. All changes decrease the hydrolytic efficiency against sucrose, proving their key role in the activity. Moreover, some of the generated variants exhibit redesigned transfructosylating specificity, which may be used for biotechnological purposes to produce novel fructosyl-derivatives.     

Experimental Insights into the Structure and Reactivity of Frustrated Lewis Pairs

Frustrated Lewis pairs (FLPs), namely the combination of a Lewis acid (LA) and a Lewis base (LB) that cannot fulfil their natural propensity to form stable Lewis adducts, have been recently exploited to activate small molecules, such as H₂ and CO₂, and have drawn much interest, due to their applicability in metal-free catalytic reactions. This review focuses mainly on the experimental studies of both the structure and reactivity of FLPs providing a starting body of evidence to elucidate the mechanism of their action. Important insights coming from NMR spectroscopy, X-ray diffraction and other experimental techniques are highlighted and discussed. In particular, evidence about the tendency of LA and LB to interact in the absence of a substrate are commented on for both inter- and intramolecular FLPs, underlining the role played by the structure of LA and LB in determining the level of interaction. Next, the effect of the structure of FLPs on the kinetics and thermodynamics of substrate activation is illustrated, with a particular focus on catalytic hydrogenation reactions.     

Insights into Comparative Modeling of VHH Domains

In the particular case of the Camelidae family, immunoglobulin proteins have evolved into a unique and more simplified architecture with only heavy chains. The variable domains of these chains, named V_HHs, have a number of Complementary Determining Regions (CDRs) reduced by half, and can function as single domains making them good candidates for molecular tools. 3D structure prediction of these domains is a beneficial and advantageous step to advance their developability as molecular tools. Nonetheless, the conformations of CDRs loops in these domains remain difficult to predict due to their higher conformational diversity. In addition to CDRs loop diversity, our earlier study has established that Framework Regions (FRs) are also not entirely conformationally conserved which establishes a need for more rigorous analyses of these regions that could assist in template selection. In the current study, V_HHs models using different template selection strategies for comparative modeling using Modeller have been extensively assessed. This study analyses the conformational changes in both CDRs and FRs using an original strategy of conformational discretization based on a structural alphabet. Conformational sampling in selected cases is precisely reported. Some interesting outcomes of the structural analyses of models also draw attention towards the distinct difficulty in 3D structure prediction of V_HH domains.     

Insights into Molecular Structure of Pterins Suitable for Biomedical Applications

Pterins are an inseparable part of living organisms. Pterins participate in metabolic reactions mostly as tetrahydropterins. Dihydropterins are usually intermediates of these reactions, whereas oxidized pterins can be biomarkers of diseases. In this review, we analyze the available data on the quantum chemistry of unconjugated pterins as well as their photonics. This gives a comprehensive overview about the electronic structure of pterins and offers some benefits for biomedicine applications: (1) one can affect the enzymatic reactions of aromatic amino acid hydroxylases, NO synthases, and alkylglycerol monooxygenase through UV irradiation of H₄pterins since UV provokes electron donor reactions of H₄pterins; (2) the emission properties of H₂pterins and oxidized pterins can be used in fluorescence diagnostics; (3) two-photon absorption (TPA) should be used in such pterin-related infrared therapy because single-photon absorption in the UV range is inefficient and scatters in vivo; (4) one can affect pathogen organisms through TPA excitation of H₄pterin cofactors, such as the molybdenum cofactor, leading to its detachment from proteins and subsequent oxidation; (5) metal nanostructures can be used for the UV-vis, fluorescence, and Raman spectroscopy detection of pterin biomarkers. Therefore, we investigated both the biochemistry and physical chemistry of pterins and suggested some potential prospects for pterin-related biomedicine.     

Novel Insights into Epigenetic Regulation of IL6 Pathway: In Silico Perspective on Inflammation and Cancer Relationship

IL-6 pathway is abnormally hyperactivated in several cancers triggering tumor cell growth and immune system inhibition. Along with genomic mutation, the IL6 pathway gene expression can be affected by DNA methylation, microRNAs, and post-translational modifications. Computational analysis was performed on the Cancer Genome Atlas (TCGA) datasets to explore the role of IL6, IL6R, IL6ST, and IL6R transmembrane isoform expression and their epigenetic regulation in different cancer types. IL6 was significantly modulated in 70% of tumor types, revealing either up- or down-regulation in an approximately equal number of tumors. Furthermore, IL6R and IL6ST were downregulated in more than 10 tumors. Interestingly, the correlation analysis demonstrated that only the IL6R expression was negatively affected by the DNA methylation within the promoter region in most tumors. Meanwhile, only the IL6ST expression was extensively modulated by miRNAs including miR-182-5p, which also directly targeted all three genes. In addition, IL6 upregulated miR-181a-3p, mirR-214-3p, miR-18a-5p, and miR-938, which in turn inhibited the expression of IL6 receptors. Finally, the patients’ survival rate was significantly affected by analyzed targets in some tumors. Our results suggest the relevance of epigenetic regulation of IL6 signaling and pave the way for further studies to validate these findings and to assess the prognostic and therapeutic predictive value of these epigenetic markers on the clinical outcome and survival of cancer patients.     

Studies into the Internal Structure of Gibbsite Agglomerates

This paper outlines techniques that have been developed or adapted to characterise the internal structure of gibbsite agglomerates and their application to selected laboratory gibbsite sample. The specific structural properties investigated include the quantification of gibbsite voidage (Floc Density Analyser), determination of relative changes in gibbsite crystal orientation (Raman spectroscopy and electron backscattered diffraction) and location of the cement binding agglomerated gibbsite particles (Raman spectroscopy, charge contrast and orientation contrast imaging). This paper also describes the use of Kikuchi patterns for investigating crystal orientation at a point and as a tool to assess if a particle is a single crystal or an agglomerate or a multi‐fold twin.     

Insights into the correlation between strain and electrostrictive coefficient of potassium sodium niobate based ceramics from relaxor structure

Eco-friendly (K, Na)NbO₃ (KNN)-based electrostrictive materials have attracted increasing attention as potential candidates for high-precision displacement actuators. Although a series of breakthroughs have increased the electrostrictive coefficient of KNN-based materials with relaxor behaviour (Q₃₃ > 0.0450 m⁴/C²), the electrostrictive strain is still low (<0.1%), making the improvement of the electrostrictive strain a crucial next step. Here, a KNN-based relaxor ceramic of 0.96K_0.48Na_0.52Nb_1-xSb_xO₃-0.04Bi_0.5Na_0.5ZrO₃-0.3%Fe₂O₃ (KNNS_x-BNZ) was designed to simultaneously achieve high electrostrictive strain and Q₃₃. The phase structure transformed from the T phase to the C phase with increasing Sb concentration, which also introduced fine grains and domains. A high electrostrictive strain (～0.102%) and Q₃₃ (～0.0461 m⁴/C²) were obtained at x = 0.09 through a small adjustment of the structure of the relaxor, while an electrostrictive strain with low hysteresis (<10.5%) and an outstanding temperature stability (≥95%) were achieved in the broadened temperature range of 20–180 °C, representing properties superior to those of previous KNN-based and typical PZT-based materials. Our results will help researchers understand how to balance the strain and electrostrictive coefficient in lead-free materials, and thereby contribute toward accelerating the application of KNN-based electrostrictive materials in actuators.     

Molecular Characterization of Four Alkaline Chitinases from Three Chitinolytic Bacteria Isolated from a Mudflat

Four chitinases were cloned and characterized from three strains isolated from a mudflat: Aeromonas sp. SK10, Aeromonas sp. SK15, and Chitinibacter sp. SK16. In SK10, three genes, Chi18A, Pro2K, and Chi19B, were found as a cluster. Chi18A and Chi19B were chitinases, and Pro2K was a metalloprotease. With combinatorial amplification of the genes and analysis of the hydrolysis patterns of substrates, Chi18A and Chi19B were found to be an endochitinase and exochitinase, respectively. Chi18A and Chi19B belonged to the glycosyl hydrolase family 18 (GH18) and GH19, with 869 and 659 amino acids, respectively. Chi18C from SK15 belonged to GH18 with 864 amino acids, and Chi18D from SK16 belonged to GH18 with 664 amino acids. These four chitinases had signal peptides and high molecular masses with one or two chitin-binding domains and, interestingly, preferred alkaline conditions. In the activity staining, their sizes were determined to be 96, 74, 95, and 73 kDa, respectively, corresponding to their expected sizes. Purified Chi18C and Chi18D after pET expression produced N,N′-diacetylchitobiose as the main product in hydrolyzing chitooligosaccharides and colloidal chitin. These results suggest that Chi18A, Chi18C, and Chi18D are endochitinases, that Chi19B is an exochitinase, and that these chitinases can be effectively used for hydrolyzing natural chitinous sources.     

The Structure and Dynamics of BmR1 Protein from Brugia malayi: In Silico Approaches

Brugia malayi is a filarial nematode, which causes lymphatic filariasis in humans. In 1995, the disease has been identified by the World Health Organization (WHO) as one of the second leading causes of permanent and long-term disability and thus it is targeted for elimination by year 2020. Therefore, accurate filariasis diagnosis is important for management and elimination programs. A recombinant antigen (BmR1) from the Bm17DIII gene product was used for antibody-based filariasis diagnosis in “Brugia Rapid”. However, the structure and dynamics of BmR1 protein is yet to be elucidated. Here we study the three dimensional structure and dynamics of BmR1 protein using comparative modeling, threading and ab initio protein structure prediction. The best predicted structure obtained via an ab initio method (Rosetta) was further refined and minimized. A total of 5 ns molecular dynamics simulation were performed to investigate the packing of the protein. Here we also identified three epitopes as potential antibody binding sites from the molecular dynamics average structure. The structure and epitopes obtained from this study can be used to design a binder specific against BmR1, thus aiding future development of antigen-based filariasis diagnostics to complement the current diagnostics.     

Insights into the thermo-mechanical properties of films cast from emulsion terpolymers

Emulsion polymerization is among the most used processes at the industrial level for the production of a wide range of polymers. The films cast from such emulsions are widely used in coatings industry and their thermo-mechanical properties are extremely important. In this work, n-butyl acrylate was copolymerized with different monomers (styrene, methyl methacrylate and acrylonitrile) in emulsion polymerization, using reaction conditions feasible to be applied at the industrial level. The films cast from the emulsions were characterized in terms of their chemical structure and the structure/thermo-mechanical properties relationships were studied in detail using different thermal analysis techniques. Both the thermal stability and the thermo-mechanical behaviour showed to be highly dependent on the type of monomers used during the emulsion polymerization. The films showed no melting temperature, indicating that the copolymers formed were amorphous. The elastic modulus and glass transition temperature showed to be dependent on the different percentages of the monomers used in the formulation and it was clearly shown that small changes on composition have strong impact on the mechanical and thermal properties of cast films. To the best of our knowledge, this work presents the first study towards the understanding of the properties of the films obtained from cast acrylic emulsions.     

微生物发酵产生苝醌类化合物和甘露醇的工艺研究

目的:研究产生艹北醌类化合物和甘露醇的菌寄生菌属真菌(AscomycetesHypocreaceaeHypomyces(Fr.)Tul.sp.)的发酵工艺。方法:不同固体培养基、温度、固体培养基与水质量比、pH值、氮源、光及碳源对艹北醌类化合物和甘露醇含量的影响。结果:筛选出一条好的发酵工艺。结论:艹北醌类化合物含量提高0.05%～0.1%,甘露醇含量提高0.5%。     

Simultaneous elimination of two species of algae from a contaminated water through ozonation process: mechanism and destruction intermediates

An ozonation process was used for in situ removal of two species of green algae namely Oocystis sp. and Cosmarium sp. from a dam. According to the results, under the optimum conditions of the ozonation process 85.18% and 58% of Oocystis sp. and Cosmarium sp. were removed, respectively. Furthermore, the obtained results confirmed the main role of ozone molecules, and active hydroxyl radicals during the degradation mechanism. Also, chlorophyll-a concentration, SEM images, GC–MS technique proved the effectiveness of ozonation process for the complete and in situ removal of algal species.     

Insights into the Structure and Protein Composition of Moorella thermoacetica Spores Formed at Different Temperatures

The bacterium Moorella thermoacetica produces the most heat-resistant spores of any spoilage-causing microorganism known in the food industry. Previous work by our group revealed that the resistance of these spores to wet heat and biocides was lower when spores were produced at a lower temperature than the optimal temperature. Here, we used electron microcopy to characterize the ultrastructure of the coat of the spores formed at different sporulation temperatures; we found that spores produced at 55 °C mainly exhibited a lamellar inner coat tightly associated with a diffuse outer coat, while spores produced at 45 °C showed an inner and an outer coat separated by a less electron-dense zone. Moreover, misarranged coat structures were more frequently observed when spores were produced at the lower temperature. We then analyzed the proteome of the spores obtained at either 45 °C or 55 °C with respect to proteins putatively involved in the spore coat, exosporium, or in spore resistance. Some putative spore coat proteins, such as CotSA, were only identified in spores produced at 55 °C; other putative exosporium and coat proteins were significantly less abundant in spores produced at 45 °C. Altogether, our results suggest that sporulation temperature affects the structure and protein composition of M. thermoacetica spores.     

Computational Insights into the Deleterious Impacts of Missense Variants on N-Acetyl-d-glucosamine Kinase Structure and Function

An enzyme of the mammalian amino-sugar metabolism pathway, N-acetylglucosamine kinase (NAGK), that synthesizes N-acetylglucosamine (GlcNAc)-6-phosphate, is reported to promote dynein functions during mitosis, axonal and dendritic growth, cell migration, and selective autophagy, which all are unrelated to its enzyme activity. As non-enzymatic structural functions can be altered by genetic variation, we made an effort in this study aimed at deciphering the pathological effect of nonsynonymous single-nucleotide polymorphisms (nsSNPs) in NAGK gene. An integrated computational approach, including molecular dynamics (MD) simulation and protein–protein docking simulation, was used to identify the damaging nsSNPs and their detailed structural and functional consequences. The analysis revealed the four most damaging variants (G11R, G32R, G120E, and A156D), which are highly conserved and functional, positioned in both small (G11R and G32R) and large (G120E and A156D) domains of NAGK. G11R is located in the ATP binding region, while variants present in the large domain (G120E and A156D) were found to induce substantial alterations in the structural organizations of both domains, including the ATP and substrate binding sites. Furthermore, all variants were found to reduce binding energy between NAGK and dynein subunit DYNLRB1, as revealed by protein–protein docking and MM-GBSA binding energy calculation supporting their deleteriousness on non-canonical function. We hope these findings will direct future studies to gain more insight into the role of these variants in the loss of NAGK function and their role in neurodevelopmental disorders.