Mapping the Catalytic Cycle of Schistosoma mansoni Thioredoxin Glutathione Reductase by X-ray Crystallography

Schistosomiasis is the second most widespread human parasitic disease. It is principally treated with one drug, praziquantel, that is administered to 100 million people each year; less sensitive strains of schistosomes are emerging. One of the most appealing drug targets against schistosomiasis is thioredoxin glutathione reductase (TGR). This natural chimeric enzyme is a peculiar fusion of a glutaredoxin domain with a thioredoxin selenocysteine (U)-containing reductase domain. Selenocysteine is located on a flexible C-terminal arm that is usually disordered in the available structures of the protein and is essential for the full catalytic activity of TGR. In this study, we dissect the catalytic cycle of Schistosoma mansoni TGR by structural and functional analysis of the U597C mutant. The crystallographic data presented herein include the following: the oxidized form (at 1.9 Å resolution); the NADPH- and GSH-bound forms (2.3 and 1.9 Å, respectively); and a different crystal form of the (partially) reduced enzyme (3.1 Å), showing the physiological dimer and the entire C terminus of one subunit. Whenever possible, we determined the rate constants for the interconversion between the different oxidation states of TGR by kinetic methods. By combining the crystallographic analysis with computer modeling, we were able to throw further light on the mechanism of action of S. mansoni TGR. In particular, we hereby propose the putative functionally relevant conformational change of the C terminus after the transfer of reducing equivalents from NADPH to the redox sites of the enzyme.     

Crystallography, evolution, and the structure of viruses

My undergraduate education in mathematics and physics was a good grounding for graduate studies in crystallographic studies of small organic molecules. As a postdoctoral fellow in Minnesota, I learned how to program an early electronic computer for crystallographic calculations. I then joined Max Perutz, excited to use my skills in the determination of the first protein structures. The results were even more fascinating than the development of techniques and provided inspiration for starting my own laboratory at Purdue University. My first studies on dehydrogenases established the conservation of nucleotide-binding structures. Having thus established myself as an independent scientist, I could start on my most cherished ambition of studying the structure of viruses. About a decade later, my laboratory had produced the structure of a small RNA plant virus and then, in another six years, the first structure of a human common cold virus. Many more virus structures followed, but soon it became essential to supplement crystallography with electron microscopy to investigate viral assembly, viral infection of cells, and neutralization of viruses by antibodies. A major guide in all these studies was the discovery of evolution at the molecular level. The conservation of three-dimensional structure has been a recurring theme, from my experiences with Max Perutz in the study of hemoglobin to the recognition of the conserved nucleotide-binding fold and to the recognition of the jelly roll fold in the capsid protein of a large variety of viruses.     

Current state of aromatics production using yeast: achievements and challenges

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Crithidia spp.: Structural comparison of polysaccharides for taxonomic significance

The chemical structures of polysaccharide components of cells of several Crithidia species have been partially elucidated. The structures have been used as criteria to evaluate evolutionary lines previously proposed for species of Crithidia and Herpetomonas and for Trypanosoma cruzi. In accord with the suggestion that Crithidia and Herpetomonas are closely interrelated, all species investigated synthesize a linear (1→2)-linked β-d-mannopyranan and a heteropolysaccharide. These differ from T. cruzi polysaccharide, which contains α-d-mannopyranosyl structures and likely indicates a separate evolutionary route for this flagellate. Crithidia fasciculata, Crithidia harmosae, and Crithidia luciliae form a closely knit group since they form arabinogalactans with related structures. The similarity is particularly close between arabinogalactans of C. fasciculata and C. harmosae whose ¹³C nuclear magnetic resonance spectra show a high degree of resemblance. An unnamed Crithidia sp. contains polysaccharides with fucose and xylose units, intermediate between those of Crithidia deanei, which gave glucose and fucose on hydrolysis, and Herpetomnas samuelpessoai, which gave glucuronic acid and xylose.     

Current challenges facing one-step production of l-ascorbic acid

l-ascorbic acid (L-AA, vitamin C) is an essential vitamin that is widely used as a nutrient or medicine in the pharmaceuticals, cosmetics, food, beverage and feed additive industries, and accounts for the largest share of the global vitamins market. L-AA is mainly produced by a classic two-step fermentation process that suffers from the use of a multi-step mixed culture system and two rounds of sterilisation, which significantly increases the cost of the final product. One-step fermentation has been attempted, but a method rivalling the efficiency of the two-step process has not yet been achieved on an industrial scale. In this review, based on the current classical two-step fermentation processes and other potential routes for L-AA production, the challenges and pitfalls of a one-step fermentation process are summarised. The prospects for one-step fermentation production of L-AA and how this might be achieved are also discussed.     

Bending and branching of calcite laths in the foliated microstructure of pectinoidean bivalves occurs at coherent crystal lattice orientation

Foliated calcite is widely employed by some important pteriomorph bivalve groups as a construction material. It is made from calcite laths, which are inclined at a low angle to the internal shell surface, although their arrangement is different among the different groups. They are strictly ordered into folia in the anomiids, fully independent in scallops, and display an intermediate arrangement in oysters. Pectinids have particularly narrow laths characterized by their ability to change their growth direction by bending or winding, as well as to bifurcate and polyfurcate. Electron backscatter analysis indicates that the c-axes of laths are at a high, though variable, angle to the growth direction, and that the laths grow preferentially along the projection of an intermediate axis between two a-axes, although they can grow in any intermediate direction. Their main surfaces are not particular crystallographic faces. Analyses done directly on the lath surfaces demonstrate that, during the bending/branching events, all crystallographic axes remain invariant. The growth flexibility of pectinid laths makes them an excellent space-filling material, well suited to level off small irregularities of the shell growth surface. We hypothesize that the exceptional ability of laths to change their direction may be promoted by the mode of growth of biogenic calcite, from a precursor liquid phase induced by organic molecules.     

The polysaccharides of red wine: total fractionation and characterization

Ethanol-precipitated red wine polysaccharides were fractionated by a combination of anion-exchange, size-exclusion and affinity chromatography steps. This comprehensive fractionation allowed us to prepare a collection of wine polysaccharides in sufficient amount to permit the determination of their intrinsic properties. Glycosyl-residue composition of each polysaccharide fraction was determined by GC–EI–MS of the per-O-trimethylsilylated methyl glycoside derivatives (TMS), a method that has been recently developed and adapted to suit simultaneous determination of neutral and acidic glycosyl-residue compositions of polysaccharides present in plant-derived products. The results showed that mannoproteins released by yeast during fermentation, and grape derived arabinogalactan-proteins, rhamnogalacturonans I and II are the main wine polysaccharides and accounted for 35, 42, 4 and 19%, respectively, of the total polysaccharides. Structural characterization revealed that rhamnogalacturonan I fractions were linked with xyloglucan-like polysaccharides. This finding represents compelling evidence of the existence of cross-linking between pectin and hemicellulose domains in plant primary cell walls.     

Rasayana properties of Ayurvedic herbs: Are polysaccharides a major contributor

Rasayana constitutes a very important class of Ayurvedic herbs, which acts as rejuvenators and tonic. Despite intensive research on evaluating the medicinal basis for the purported medical benefits of Rasayana herbs, still a specific chemical guiding principle for the characterization of these herbs in one single category is unclear. Here we explore the possibility that polysaccharides play a key role in Rasayana properties exhibited by medicinal plants. Further on this could be a possible field for exploration for a common factor present in some of the Ayurvedic herbs. The present review covers a literature spanning from 1956 to 2011. Some translations of traditional Ayurvedic texts dating back to the first century AD have been referred to as well. In our assessment of the present literature and studies carried out it is presupposed that the presence of benevolent plant polysaccharides must be one of the important features symbolizing a common effectiveness in most of the Ayurvedic Rasayana herbs. Of the many plant metabolites polysaccharides have not so far been considered and studied effectively compared to other secondary metabolites like saponins, alkaloids, etc. Although all the results reported until now suggest a major contribution of polysaccharide towards the maintenance of physiological homeostasis, which is the guiding principle of Rasayana therapy. The present review is an attempt to find a connective link between the concept of Rasayana and well-being; and the role of plant polysaccharides. Lack of clinical information on number of polysaccharides showing promise is a limiting factor for a complete understanding. It is also important to carry out a molecular interaction study to understand the behavior of polysaccharides discussed in the present review.     

白术多糖对小鼠免疫功能调节的研究

目的将白术多糖作为抗原刺激小鼠免疫系统,探讨其对免疫功能调节作用,为多糖在免疫调节功能方面的应用开发及在药物鉴定方面的应用提供科学依据。方法分别给予小鼠自术多糖、可溶性淀粉多糖、伤寒多糖抗原刺激,检测小鼠血清中的相应抗体及交叉抗体,探讨补益类水溶性多糖对小鼠免疫功能的调节作用。结果多糖抗原均能刺激机体产生特异性IgG类抗体（P〈0.05）;也能在一定程度上激发非特异性IgG类抗体即交叉抗体的产生;但不激发病理性抗体产生（RF阴性）。结论补益类水溶性多糖能激发免疫反应与其他多糖可能有共同的途径和机制。     

Biological chemistry of immunomodulation by zwitterionic polysaccharides

Capsular polysaccharides isolated from pathogenic bacteria are comprised typically of many repeating units from one to eight or more monosaccharides in length. These polysaccharides stimulate the murine humoral immune system to elicit primarily IgM antibody responses. Studies conducted primarily in the mouse have characterized these polymers as T cell-independent antigens. These mouse studies and the relatively poor immunogenicity of polysaccharides in human hosts have led to the design of vaccines by coupling these polysaccharides to protein carriers to stimulate a T cell-dependent response. However, a newly described class of bacterial polysaccharides has been characterized that have the ability to modulate the cellular immune system. They are structurally diverse, but all share a zwitterionic charge motif that allows them to directly interact with T cells and antigen-presenting cells to initiate an immunomodulatory T cell response. These polymers, termed zwitterionic polysaccharides (ZPSs), elicit T cell-derived chemokines and cytokines that influence the immune response governing at least one classic host response to bacterial infection: abscess formation. This review will describe the biological and structural aspects of ZPSs that convey these activities.     

Microbial production of propionic acid from propionibacteria: Current state,challenges and perspectives

Propionic acid (PA) is an important building block chemical and finds a variety of applications in organic synthesis, food, feeding stuffs, perfume, paint and pharmaceutical industries. Presently, PA is mainly produced by petrochemical route. With the continuous increase in oil prices, public concern about environmental pollution, and the consumers’ desire for bio-based natural and green ingredients in foods and pharmaceuticals, PA production from propionibacteria has attracted considerable attention, and substantial progresses have been made on microbial PA production. However, production of PA by propionibacteria is facing challenges such as severe inhibition of end-products during cell growth and the formation of by-products (acetic acid and succinic acid). The integration of reverse metabolic engineering and systematic metabolic engineering provides an opportunity to significantly improve the acid tolerance of propionibacteria and reduce the formation of by-products, and makes it feasible to strengthen the commercial competition of biotechnological PA production from propionibacteria to be comparable to the petrochemical route.     

Recent advances in Agaricus bisporus polysaccharides: Extraction,purification, physicochemical characterization and bioactivities

Agaricus bisporus (A. bisporus), known as a cultivated mushroom or button mushroom, is a very important edible and medicinal basidiomycete fungus. The numerous health benefits of A. bisporus may be related to their polysaccharides, which have significant dietary value and bioactivity, including immunity stimulation and high antioxidant, anticancer, hepatoprotection, anti-inflammation and anti-obesity functions. In general, the extraction method of A. bisporus polysaccharides (ABPs) is relatively simple, and the yield from enzyme-assisted extraction is the highest among various extraction methods. The monosaccharide composition analysis revealed that ABPs mainly consist of glucose, galactose, fucose and xylose, which each have a backbone composed of (1→6)- and (1→4)-linked α-glucan or alternating (1→4)- and (1→6)-linked β-glucan. The biological activity of ABPs may vary significantly depending on their source, composition, structural properties, and purity, and it is highly correlated with molecular weight (MW) and the monosaccharide components. Therefore, this review aims to introduce the extraction methods, chemical structure, and biological activity of ABPs which may provide a theoretical basis for the further development and utilization of polysaccharides and have important reference value for the future study of the relationship between structural features and biological activities.     

Gut microbiota,host health,and polysaccharides

The intestinal microbiota is a complicated ecosystem that influences many aspects of host physiology (i.e. diet, disease development, drug metabolism, and regulation of the immune system). It also exhibits spatial patterning and temporal dynamics. In this review, the effects of internal and external (environmental) factors on intestinal microbiota are discussed. We describe the roles of the gut microbiota in maintaining intestinal and immune system homeostasis and the relationship between gut microbiota and diseases. In particular, the contributions of polysaccharides, as the most abundant diet components in intestinal microbiota and host health are presented. Finally, perspectives for research avenues relating to gut microbiota are also discussed.     

Water-soluble polysaccharides from Angelica sinensis (Oliv.) Diels: Preparation, characterization and bioactivity

Crude water-soluble polysaccharides (ASP) were separated from Angelica sinensis (Oliv.) Diels by hot water extraction. They were fractionated into neutral and acidic polysaccharides by anion-exchange chromatography. The neutral polysaccharide (ASP1) was rich in glucose, galactose, and arabinose suggesting a mixture of glucan and arabinogalactan. The acidic polysaccharide (ASP2, ASP3) consisted mainly of galacturonic acid along with rhamnose, arabinose, and galactose indicating a pectic polysaccharide. The degree of esterification of ASP and ASP3 were 54.06% and 47.14% for the crude and purified sample, respectively. ASP3, with a molecular weight of 3.4 × 10⁴ Da determined by high-performance size-exclusion chromatography (HPSEC), was the major constituent for the crude extracts. The radioprotective effect of the pectic polysaccharide ASP3 was studied in murine models. ASP3 pretreated mice exhibited a significant decrease of apoptosis (P < 0.05, dosage of 200 mg/kg d body weight) in peripheral lymphocytes compared to the irradiated control. The results showed that ASP3 can protect leucocytes and lymphocytes of mice against radiation induced damage, which has potential radioprotective effect on acute radiation injured mice.     

In vitro synthesis of artificial polysaccharides by glycosidases and glycosynthases

Artificial polysaccharides produced by in vitro enzymatic synthesis are new biomaterials with defined structures that either mimic natural polysaccharides or have unnatural structures and functionalities. This review summarizes recent developments in the in vitro polysaccharide synthesis by endo-glycosidases, grouped in two major strategies: (a) native retaining endo-glycosidases under kinetically controlled conditions (transglycosylation with activated glycosyl donors), and (b) glycosynthases, engineered glycosidases devoid of hydrolase activity but with high transglycosylation activity. Polysaccharides are obtained by enzymatic polymerization of simple glycosyl donors by repetitive condensation. This approach not only provides a powerful methodology to produce polysaccharides with defined structures and morphologies as novel biomaterials, but is also a valuable tool to analyze the mechanisms of polymerization and packing to acquire high-order molecular assemblies.     

Rational design of new materials using recombinant structural proteins: Current state and future challenges

Sequence-definable polymers are seen as a prerequisite for design of future materials, with many polymer scientists regarding such polymers as the holy grail of polymer science. Recombinant proteins are sequence-defined polymers. Proteins are dictated by DNA templates and therefore the sequence of amino acids in a protein is defined, and molecular biology provides tools that allow redesign of the DNA as required. Despite this advantage, proteins are underrepresented in materials science. In this publication we investigate the advantages and limitations of using proteins as templates for rational design of new materials.     

Antiangiogenic activities of polysaccharides isolated from medicinal fungi

Extracted polysaccharides from medicinal fungi, including Antrodia cinnamomea, Antrodia malicola, Antrodia xantha, Antrodiella liebmannii, Agaricus murrill, and Rigidoporus ulmarius, were investigated for their effects on vascular endothelial growth factor (VEGF)-induced tube formation in endothelial cells (ECs). Chemical analysis revealed that myo-inositol, sorbitol, fucose, galactosamine, glucosamine, galactose, glucose, and mannose were the neutral sugars in these polysaccharides. These fungal polysaccharides showed no toxicity to ECs. For the inhibition of endothelial tube formation, extracted polysaccharides from A. xantha and R. ulmarius were shown to produce greater inhibition compared to those from other fungi. Fucose, glucose and mannose were the predominant monosaccharides from these two fungi. These results suggest that monosaccharides may play a role in the inhibitory effect of these fungi on endothelial tube formation. In contrast to the inhibition on tube formation from polysaccharides of A. cinnamomea and A. malicola, polysaccharides from A. xantha and R. ulmarius, with molecular weight between 2693-2876 and 304-325 kDa, were critical for this inhibitory activity. Our results show that polysaccharides isolated from A. xantha and R. ulmarius provide greater antiangiogenesis than those from commercialized A. murrill (Brazilian mushroom) and A. cinnamomea. These studies provide a basis for the potential development of these polysaccharides for antiangiogenesis usage.