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"Chitin"
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Structural basis for directional chitin biosynthesis
Chitin, the most abundant aminopolysaccharide in nature, is an extracellular polymer consisting of
N
-acetylglucosamine (GlcNAc) units
1
. The key reactions of chitin biosynthesis are catalysed by chitin synthase
2
–
4
, a membrane-integrated glycosyltransferase that transfers GlcNAc from UDP-GlcNAc to a growing chitin chain. However, the precise mechanism of this process has yet to be elucidated. Here we report five cryo-electron microscopy structures of a chitin synthase from the devastating soybean root rot pathogenic oomycete
Phytophthora sojae
(
Ps
Chs1). They represent the apo, GlcNAc-bound, nascent chitin oligomer-bound, UDP-bound (post-synthesis) and chitin synthase inhibitor nikkomycin Z-bound states of the enzyme, providing detailed views into the multiple steps of chitin biosynthesis and its competitive inhibition. The structures reveal the chitin synthesis reaction chamber that has the substrate-binding site, the catalytic centre and the entrance to the polymer-translocating channel that allows the product polymer to be discharged. This arrangement reflects consecutive key events in chitin biosynthesis from UDP-GlcNAc binding and polymer elongation to the release of the product. We identified a swinging loop within the chitin-translocating channel, which acts as a ‘gate lock’ that prevents the substrate from leaving while directing the product polymer into the translocating channel for discharge to the extracellular side of the cell membrane. This work reveals the directional multistep mechanism of chitin biosynthesis and provides a structural basis for inhibition of chitin synthesis.
Using cryo-electron microscopy, the directional multiple step mechanism of chitin biosynthesis is revealed.
Journal Article
Chitin Translocation Is Functionally Coupled with Synthesis in Chitin Synthase
Chitin, an extracellular polysaccharide, is synthesized by membrane-embedded chitin synthase (CHS) utilizing intracellular substrates. The mechanism of the translocation of synthesized chitin across the membrane to extracellular locations remains unresolved. We prove that the chitin synthase from Phytophthora sojae (PsCHS) is a processive glycosyltransferase, which can rapidly produce and tightly bind with the highly polymerized chitin. We further demonstrate that PsCHS is a bifunctional enzyme, which is necessary and sufficient to translocate the synthesized chitin. PsCHS was purified and then reconstituted into proteoliposomes (PLs). The nascent chitin is generated and protected from chitinase degradation unless detergent solubilizes the PLs, showing that PsCHS translocates the newly produced chitin into the lumen of the PLs. We also attempted to resolve the PsCHS structure of the synthesized chitin-bound state, although it was not successful; the obtained high-resolution structure of the UDP/Mn2+-bound state could still assist in describing the characterization of the PsCHS’s transmembrane channel. Consistently, we demonstrate that PsCHS is indispensable and capable of translocating chitin in a process that is tightly coupled to chitin synthesis.
Journal Article
Distinct cellular and molecular mechanisms contribute to the specificity of the two Drosophila melanogaster chitin synthases in chitin deposition
Chitin is a major component of arthropod extracellular matrices, including the exoskeleton and the midgut peritrophic matrix. It plays a key role in the development, growth and viability of insects. Beyond the biological importance of this aminopolysaccharide, chitin also receives considerable attention for its practical applications in medicine and biotechnology, as it is a superior biopolymer with excellent physicochemical and mechanical properties. Chitin is synthesised and deposited extracellularly by chitin synthases. Most insects encode two types of chitin synthases: type A, which are presumed to be required for exoskeleton formation, and type B, which are thought to produce the peritrophic matrix. However, the factors that contribute to the specificity of each type of chitin synthase remain unclear. Here, we leverage the advantages of Drosophila melanogaster for functional manipulations to evaluate the mechanisms of activity and the functional requirements of Kkv (Chitin synthase A) and Chs2 (Chitin synthase B). We first demonstrate that Chs2 is expressed and required in a specific region of the larval proventriculus responsible for producing chitin in the peritrophic matrix. We then assess whether the two chitin synthases can functionally substitute for each other. Additionally, we examine their subcellular localisation in different tissues and their ability to deposit chitin in combination with known auxiliary proteins. Our results indicate that these two different chitin synthases are not functionally interchangeable and that they use distinct cellular and molecular mechanisms to deposit chitin. We suggest that the specificity of insect chitin synthases may underlie the production of chitin polymers with different properties, conferring different physiological activities to the extracellular matrices.
Journal Article
Express Method for Isolation of Ready-to-Use 3D Chitin Scaffolds from Aplysina archeri (Aplysineidae: Verongiida) Demosponge
Sponges are a valuable source of natural compounds and biomaterials for many biotechnological applications. Marine sponges belonging to the order Verongiida are known to contain both chitin and biologically active bromotyrosines. Aplysina archeri (Aplysineidae: Verongiida) is well known to contain bromotyrosines with relevant bioactivity against human and animal diseases. The aim of this study was to develop an express method for the production of naturally prefabricated 3D chitin and bromotyrosine-containing extracts simultaneously. This new method is based on microwave irradiation (MWI) together with stepwise treatment using 1% sodium hydroxide, 20% acetic acid, and 30% hydrogen peroxide. This approach, which takes up to 1 h, made it possible to isolate chitin from the tube-like skeleton of A. archeri and to demonstrate the presence of this biopolymer in this sponge for the first time. Additionally, this procedure does not deacetylate chitin to chitosan and enables the recovery of ready-to-use 3D chitin scaffolds without destruction of the unique tube-like fibrous interconnected structure of the isolated biomaterial. Furthermore, these mechanically stressed fibers still have the capacity for saturation with water, methylene blue dye, crude oil, and blood, which is necessary for the application of such renewable 3D chitinous centimeter-sized scaffolds in diverse technological and biomedical fields.
Journal Article
Architecture of the dynamic fungal cell wall
The fungal cell wall is essential for growth and survival, and is a key target for antifungal drugs and the immune system. The cell wall must be robust but flexible, protective and shielding yet porous to nutrients and membrane vesicles and receptive to exogenous signals. Most fungi have a common inner wall skeleton of chitin and β-glucans that functions as a flexible viscoelastic frame to which a more diverse set of outer cell wall polymers and glycosylated proteins are attached. Whereas the inner wall largely determines shape and strength, the outer wall confers properties of hydrophobicity, adhesiveness, and chemical and immunological heterogeneity. The spatial organization and dynamic regulation of the wall in response to prevailing growth conditions enable fungi to thrive within changing, diverse and often hostile environments. Understanding this architecture provides opportunities to develop diagnostics and drugs to combat life-threatening fungal infections.In this Review, Gow and Lenardon describe how fungal cell walls are organized, focusing on the underlying architectural and mechanical principles that are required to deliver differing and bespoke biochemical and biophysical attributes.
Journal Article
Structure, catalysis, chitin transport, and selective inhibition of chitin synthase
Chitin is one of the most abundant natural biopolymers and serves as a critical structural component of extracellular matrices, including fungal cell walls and insect exoskeletons. As a linear polymer of β-(1,4)-linked N-acetylglucosamine, chitin is synthesized by chitin synthases, which are recognized as targets for antifungal and anti-insect drugs. In this study, we determine seven different cryo-electron microscopy structures of a
Saccharomyces cerevisiae
chitin synthase in the absence and presence of glycosyl donor, acceptor, product, or peptidyl nucleoside inhibitors. Combined with functional analyses, these structures show how the donor and acceptor substrates bind in the active site, how substrate hydrolysis drives self-priming, how a chitin-conducting transmembrane channel opens, and how peptidyl nucleoside inhibitors inhibit chitin synthase. Our work provides a structural basis for understanding the function and inhibition of chitin synthase.
Chitin, the second most abundant natural polysaccharide in nature, is synthesized by chitin synthases, which are recognized as targets for antifungal and anti-insect drugs. Here the authors determine cryo-EM structures of the chitin synthase, which reveal its activation, catalytic and inhibitory mechanisms
Journal Article
A dynamic interplay between chitin synthase and the proteins Expansion/Rebuf reveals that chitin polymerisation and translocation are uncoupled in Drosophila
Chitin is a highly abundant polymer in nature and a principal component of apical extracellular matrices in insects. In addition, chitin has proved to be an excellent biomaterial with multiple applications. In spite of its importance, the molecular mechanisms of chitin biosynthesis and chitin structural diversity are not fully elucidated yet. To investigate these issues, we use Drosophila as a model. We previously showed that chitin deposition in ectodermal tissues requires the concomitant activities of the chitin synthase enzyme Kkv and the functionally interchangeable proteins Exp and Reb. Exp/Reb are conserved proteins, but their mechanism of activity during chitin deposition has not been elucidated yet. Here, we carry out a cellular and molecular analysis of chitin deposition, and we show that chitin polymerisation and chitin translocation to the extracellular space are uncoupled. We find that Kkv activity in chitin translocation, but not in polymerisation, requires the activity of Exp/Reb, and in particular of its conserved Nα-MH2 domain. The activity of Kkv in chitin polymerisation and translocation correlate with Kkv subcellular localisation, and in absence of Kkv-mediated extracellular chitin deposition, chitin accumulates intracellularly as membrane-less punctae. Unexpectedly, we find that although Kkv and Exp/Reb display largely complementary patterns at the apical domain, Exp/Reb activity nonetheless regulates the topological distribution of Kkv at the apical membrane. We propose a model in which Exp/Reb regulate the organisation of Kkv complexes at the apical membrane, which, in turn, regulates the function of Kkv in extracellular chitin translocation.
Journal Article
Chitin and Chitosan - Properties and Applications
This book presents a comprehensive review of the isolation, properties and applications of chitin and chitosan. These promising biomaterials have the potential to be broadly applied and there is a growing market for these biopolymers in areas such as medical and pharmaceutical, packaging, agricultural, textile, cosmetics, nanoparticles and more. The authors - noted experts in the field - explore the isolation, characterization and the physical and chemical properties of chitin and chitosan. They also examine their properties such as hydrogels, immunomodulation and biotechnology, antimicrobial activity and chemical enzymatic modifications. The book offers an analysis of the myriad medical and pharmaceutical applications as well as a review of applications in other areas. In addition, the authors discuss regulations, markets and perspectives for the use of chitin and chitosan.
eBook