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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 ( Ps CHS) is a processive glycosyltransferase, which can rapidly produce and tightly bind with the highly polymerized chitin. We further demonstrate that Ps CHS is a bifunctional enzyme, which is necessary and sufficient to translocate the synthesized chitin. Ps CHS 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 Ps CHS translocates the newly produced chitin into the lumen of the PLs. We also attempted to resolve the Ps CHS structure of the synthesized chitin-bound state, although it was not successful; the obtained high-resolution structure of the UDP/Mn 2+ -bound state could still assist in describing the characterization of the Ps CHS's transmembrane channel. Consistently, we demonstrate that Ps CHS is indispensable and capable of translocating chitin in a process that is tightly coupled to chitin synthesis.

In order to speculate the three-dimensional structure of the potential binding pocket of the chitin synthase inhibitor, a series of 2,4-diphenyloxazoline derivatives with different lengths of alkyl chains and heteroatoms were designed and synthesized by a homologous strategy. The bioassay results indicate that both the length of the alkyl chains and the type of substituents can affect the acaricidal activity against mite eggs. Compounds containing chloropropyl, alkoxyalkyl, and para-substituted phenoxyalkyl or phenylthioalkyl groups exhibit good activity, while those containing steric hindrance substituents or carbonyl substituents on the benzene ring exhibit reduced activity. Three-dimensional quantitative structure-activity relationship (3D-QSAR) study showed that there may be a narrow hydrophobic region deep in the pocket, and the steric effect plays a more important role than the electrostatic effect. The current work will provide assistance for future molecular design and target binding research.

Plant intracellular nucleotide-binding leucine-rich repeat receptors (NLRs) analyzed to date oligomerize and form resistosomes upon activation to initiate immune responses. Some NLRs are encoded in tightly linked co-regulated head-to-head genes whose products function together as pairs. We uncover the oligomerization requirements for different Arabidopsis paired CHS3-CSA1 alleles. These pairs form resting-state heterodimers that oligomerize into complexes distinct from NLRs analyzed previously. Oligomerization requires both conserved and allele-specific features of the respective CHS3 and CSA1 Toll-like interleukin-1 receptor (TIR) domains. The receptor kinases BAK1 and BIRs inhibit CHS3-CSA1 pair oligomerization to maintain the CHS3-CSA1 heterodimer in an inactive state. Our study reveals that paired NLRs hetero-oligomerize and likely form a distinctive "dimer of heterodimers" and that structural heterogeneity is expected even among alleles of closely related paired NLRs.

Glycosyltransferases (GTs) are a large and diverse group of enzymes responsible for catalyzing the formation of a glycosidic bond between a donor molecule, usually a monosaccharide, and a wide range of acceptor molecules, thus, playing critical roles in various essential biological processes. Chitin and cellulose synthases are two inverting processive integral membrane GTs, belonging to the type-2 family involved in the biosynthesis of chitin and cellulose, respectively. Herein, we report that bacterial cellulose and chitin synthases share an E-D-D-ED-QRW-TK active site common motif that is spatially co-localized. This motif is conserved among distant bacterial evolutionary species despite their low amino acid sequence and structural similarities between them. This theoretical framework offers a new perspective to the current view that bacterial cellulose and chitin synthases are substrate specific and that chitin and cellulose are organism specific. It lays the ground for future in vivo and in silico experimental assessment of cellulose synthase catalytic promiscuity against uridine diphosphate N-acetylglucosamine and chitin synthase against uridine diphosphate glucose, respectively., (© 2023 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

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., (© 2023. Springer Nature Limited.)

Chitin is an aminopolysaccharide present in insects as a major structural component of the cuticle. However, current knowledge on the chitin biosynthetic machinery, especially its constituents and mechanism, is limited. Using three independent binding assays, including co-immunoprecipitation, split-ubiquitin membrane yeast two-hybrid assay, and pull-down assay, we demonstrate that choline transporter-like protein 2 (Ctl2) interacts with krotzkopf verkehrt (kkv) in Drosophila melanogaster. The global knockdown of Ctl2 by RNA interference (RNAi) induced lethality at the larval stage. Tissue-specific RNAi to silence Ctl2 in the tracheal system and in the epidermis of the flies resulted in lethality at the first larval instar. The knockdown of Ctl2 in wings led to shrunken wings containing accumulated fluid. Calcofluor White staining demonstrated reduced chitin content in the first longitudinal vein of Ctl2 knockdown wings. The pro-cuticle, which was thinner compared to wildtype, exhibited a reduced number of chitin laminar layers. Phylogenetic analyses revealed orthologues of Ctl2 in different insect orders with highly conserved domains. Our findings provide new insights into cuticle formation, wherein Ctl2 plays an important role as a chitin-synthase interacting protein., (Copyright © 2021. Published by Elsevier Ltd.)

The oomycetes are filamentous eukaryotic microorganisms, distinct from true fungi, many of which act as crop or fish pathogens that cause devastating losses in agriculture and aquaculture. Chitin is present in all true fungi, but it occurs in only small amounts in some Saprolegniomycetes and it is absent in Peronosporomycetes. However, the growth of several oomycetes is severely impacted by competitive chitin synthase (CHS) inhibitors. Here, we shed light on the diversity, evolution and function of oomycete CHS proteins. We show by phylogenetic analysis of 93 putative CHSs from 48 highly diverse oomycetes, including the early diverging Eurychasma dicksonii, that all available oomycete genomes contain at least one putative CHS gene. All gene products contain conserved CHS motifs essential for enzymatic activity and form two Peronosporomycete-specific and six Saprolegniale-specific clades. Proteins of all clades, except one, contain an N-terminal microtubule interacting and trafficking (MIT) domain as predicted by protein domain databases or manual analysis, which is supported by homology modelling and comparison of conserved structural features from sequence logos. We identified at least three groups of CHSs conserved among all oomycete lineages and used phylogenetic reconciliation analysis to infer the dynamic evolution of CHSs in oomycetes. The evolutionary aspects of CHS diversity in modern-day oomycetes are discussed. In addition, we observed hyphal tip rupture in Phytophthora infestans upon treatment with the CHS inhibitor nikkomycin Z. Combining data on phylogeny, gene expression, and response to CHS inhibitors, we propose the association of different CHS clades with certain developmental stages., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The alarm is rang for friendly fire; Saccharomyces cerevisiae (S. cerevisiae) newfound as a fungal pathogen with an individual feature. S. cerevisiae has food safety and is not capable of producing infection but, when the host defenses are weakened, there is room for opportunistic S. cerevisiae strains to cause a health issues. Fungal diseases are challenging to treat because, unlike bacteria, the fungal are eukaryotes. Antibiotics only target prokaryotic cells, whereas compounds that kill fungi also harm the mammalian host. Small differences between mammalian and fungal cells regarding genes and proteins sequence and function make finding a drug target more challenging. Recently, Chitin synthase has been considered as a promising target for antifungal drug development as it is absent in mammals. In S. cerevisiae, CHS3, a class IV chitin synthase, produces 90% of the chitin and essential for cell growth. CHS3 from the trans-Golgi network to the plasma membrane requires assembly of the exomer complex (including proteins cargo such as CHS5, CHS6, Bach1, and Arf1). In this work, we performed SELEX (Systematic Evolution of Ligands by EXponential enrichment) as high throughput virtual screening of the RCSB data bank to find an aptamer as potential inhibit of the class IV chitin synthase of S. cerevisiae. Among all the candidates, G-rich VEGF (GVEGF) aptamer (PDB code: 2M53) containing locked sugar parts was observed as potential inhibitor of the assembly of CHS5-CHS6 exomer complex a subsequently block the chitin biosynthesis pathway as an effective anti-fungal. It was suggested from the simulation that an assembly of exomer core should begin CHS5-CHS6, not from CHS5-Bach1. It is notable that secondary structures of CHS6 and Bach1 was observed very similar, but they have only 25% identity at the amino acid sequence that exhibited different features in exomer assembly., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Chitin synthase is responsible for chitin synthesis in the cuticles and cuticular linings of other tissues in insects. We cloned two alternative splicing variants of the chitin synthase 1 gene (SfCHS1) from the white-backed planthopper, Sogatella furcifera. The full-length cDNA of the two variants (SfCHS1a and SfCHS1b) consists of 6408 bp, contains a 4719-bp open reading frame encoding 1572 amino acids, and has 5' and 3' non-coding regions of 283 and 1406 bp, respectively. The two splicing variants occur at the same position in the cDNA sequence between base pairs 4115 and 4291, and consist of 177 nucleotides that encode 59 amino acids but show 74.6% identity at the amino acid level. Analysis in different developmental stages showed that expression of SfCHS1 and SfCHS1a were highest just after molting, whereas SfCHS1b reached its highest expression level 2 days after molting. Further, SfCHS1 and SfCHS1a were mainly expressed in the integument, whereas SfCHS1b was predominately expressed in the gut and fat body. RNAi-based gene silencing inhibited transcript levels of the corresponding mRNAs in S. furcifera nymphs injected with double-stranded RNA of SfCHS1, SfCHS1a, and SfCHS1b, resulted in malformed phenotypes, and killed most of the treated nymphs. Our results indicate that SfCHS1 may be a potential target gene for RNAi-based S. furcifera control.

Chitin biosynthesis in yeast is accomplished by three chitin synthases (Chs) termed Chs1, Chs2 and Chs3, of which the latter accounts for most of the chitin deposited within the cell wall. While the overall structures of Chs1 and Chs2 are similar to those of other chitin synthases from fungi and arthropods, Chs3 lacks some of the C-terminal transmembrane helices raising questions regarding its structure and topology. To fill this gap of knowledge, we performed bioinformatic analyses and protease protection assays that revealed significant information about the catalytic domain, the chitin-translocating channel and the interfacial helices in between. In particular, we identified an amphipathic, crescent-shaped α-helix attached to the inner side of the membrane that presumably controls the channel entrance and a finger helix pushing the polymer into the channel. Evidence has accumulated in the past years that chitin synthases form oligomeric complexes, which may be necessary for the formation of chitin nanofibrils. However, the functional significance for living yeast cells has remained elusive. To test Chs3 oligomerization in vivo, we used bimolecular fluorescence complementation. We detected oligomeric complexes at the bud neck, the lateral plasma membrane, and in membranes of Golgi vesicles, and analyzed their transport route using various trafficking mutants.

Unlabelled: Chitin synthases (Chs) are responsible for the synthesis of chitin, a key structural cell wall polysaccharide in many organisms. They are essential for growth in certain oomycete species, some of which are pathogenic to diverse higher organisms. Recently, a microtubule interacting and trafficking (MIT) domain, which is not found in any fungal Chs, has been identified in some oomycete Chs proteins. Based on experimental data relating to the binding specificity of other eukaryotic MIT domains, there was speculation that this domain may be involved in the intracellular trafficking of Chs proteins. However, there is currently no evidence for this or any other function for the MIT domain in these enzymes. To attempt to elucidate their function, MIT domains from two Chs enzymes from the oomycete Saprolegnia monoica were cloned, expressed, and characterized. Both were shown to interact strongly with the plasma membrane component, phosphatidic acid, and to have additional putative interactions with proteins thought to be involved in protein transport and localization. Aiding our understanding of these data, the structure of the first MIT domain from a carbohydrate-active enzyme (MIT1) was solved by NMR, and a model structure of a second MIT domain (MIT2) was built by homology modeling. Our results suggest a potential function for these MIT domains in the intracellular transport and/or regulation of Chs enzymes in the oomycetes., Database: Structural data are available in the Biological Magnetic Resonance Bank (BMRB) database under the accession number 19987 and the PDB database under the accession number 2MPK., (© 2016 Federation of European Biochemical Societies.)

Chitin synthase (ChS) plays a critical role in chitin synthesis and excretion. In this study, two ChS genes (LdChSA and LdChSB) were identified in Leptinotarsa decemlineata. LdChSA contains two splicing variants, LdChSAa and LdChSAb. Within the first, second, and third larval instars, the mRNA levels of LdChSAa, LdChSAb, and LdChSB coincide with the peaks of circulating 20-hydroxyecdysone (20E) and juvenile hormone (JH). In vitro culture of midguts and an in vivo bioassay revealed that 20E and an ecdysteroid agonist halofenozide stimulated the expression of the three LdChSs. Conversely, a reduction of 20E by RNA interference (RNAi) of an ecdysteroidogenesis gene LdSHD repressed the expression of these LdChSs, and ingestion of halofenozide by LdSHD RNAi larvae rescued the repression. Moreover, disruption of 20E signaling by RNAi of LdEcR, LdE75, LdHR3, and LdFTZ-F1 reduced the expression levels of these genes. Similarly, in vitro culture and an in vivo bioassay showed that exogenous JH and a JH analog methoprene activated the expression of the three LdChSs, whereas a decrease in JH by RNAi of a JH biosynthesis gene LdJHAMT downregulated these LdChSs. It seems that JH upregulates LdChSs at the early stage of each instar, whereas a 20E pulse triggers the transcription of LdChSs during molting in L. decemlineata., (© 2016 Wiley Periodicals, Inc.)

Future ocean acidification (OA) will affect physiological traits of marine species, with calcifying species being particularly vulnerable. As OA entails high energy demands, particularly during the rapid juvenile growth phase, food supply may play a key role in the response of marine organisms to OA. We experimentally evaluated the role of food supply in modulating physiological responses and biomineralization processes in juveniles of the Chilean scallop, Argopecten purpuratus, that were exposed to control (pH ~ 8.0) and low pH (pH ~ 7.6) conditions using three food supply treatments (high, intermediate, and low). We found that pH and food levels had additive effects on the physiological response of the juvenile scallops. Metabolic rates, shell growth, net calcification, and ingestion rates increased significantly at low pH conditions, independent of food. These physiological responses increased significantly in organisms exposed to intermediate and high levels of food supply. Hence, food supply seems to play a major role modulating organismal response by providing the energetic means to bolster the physiological response of OA stress. On the contrary, the relative expression of chitin synthase, a functional molecule for biomineralization, increased significantly in scallops exposed to low food supply and low pH, which resulted in a thicker periostracum enriched with chitin polysaccharides. Under reduced food and low pH conditions, the adaptive organismal response was to trade-off growth for the expression of biomineralization molecules and altering of the organic composition of shell periostracum, suggesting that the future performance of these calcifiers will depend on the trajectories of both OA and food supply. Thus, incorporating a suite of traits and multiple stressors in future studies of the adaptive organismal response may provide key insights on OA impacts on marine calcifiers., (© 2015 John Wiley & Sons Ltd.)

The critical role of chitin synthases in oomycete hyphal tip growth has been established. A microtubule interacting and trafficking (MIT) domain was discovered in the chitin synthases of the oomycete model organism, Saprolegnia monoica. MIT domains have been identified in diverse proteins and may play a role in intracellular trafficking. The structure of the Saprolegnia monoica chitin synthase 1 (SmChs1) MIT domain has been recently determined by our group. However, although our in vitro assay identified increased strength in interactions between the MIT domain and phosphatidic acid (PA) relative to other phospholipids including phosphatidylcholine (PC), the mechanism used by the MIT domain remains unknown. In this work, the adsorption behavior of the SmChs1 MIT domain on POPA and POPC membranes was systematically investigated by molecular dynamics simulations. Our results indicate that the MIT domain can adsorb onto the tested membranes in varying orientations. Interestingly, due to the specific interactions between MIT residues and lipid molecules, the binding affinity to the POPA membrane is much higher than that to the POPC membrane. A binding hotspot, which is critical for the adsorption of the MIT domain onto the POPA membrane, was also identified. The lower binding affinity to the POPC membrane can be attributed to the self-saturated membrane surface, which is unfavorable for hydrogen-bond and electrostatic interactions. The present study provides insight into the adsorption profile of SmChs1 and additionally has the potential to improve our understanding of other proteins containing MIT domains.

The triangle sail mussel, Hyriopsis cumingii, is the most important freshwater pearl mussel in China. However, the mechanisms underlying its chitin-mediated shell and nacre formation remain largely unknown. Here, we characterized a chitin synthase (CS) gene (HcCS1) in H. cumingii, and analyzed its possible physiological function. The complete ORF sequence of HcCS1 contained 6903 bp, encoding a 2300-amino acid protein (theoretical molecular mass = 264 kDa; isoelectric point = 6.22), and no putative signal peptide was predicted. A myosin motor head domain, a CS domain, and 12 transmembrane domains were found. The predicted spatial structures of the myosin head and CS domains were similar to the electron microscopic structure of the heavy meromyosin subfragment of chicken smooth muscle myosin and the crystal structure of bacterial cellulose synthase, respectively. This structural similarity indicates that the functions of these two domains might be conserved. Quantitative reverse transcription PCR results showed that HcCS1 was present in all detected tissues, with the highest expression levels detected in the mantle. The HcCS1 transcripts in the mantle were upregulated following shell damage from 12 to 24 h post-damage, and they peaked (approximately 1.5-fold increase) at 12 h after shell damage. These findings suggest that HcCS1 was involved in shell regeneration, and that it might participate in shell and nacre formation in this species via chitin synthesis. HcCS1 might also dynamically regulate chitin deposition during the process of shell and nacre formation with the help of its conserved myosin head domain.

Complex carbohydrates perform essential functions in life, including energy storage, cell signaling, protein targeting, quality control, as well as supporting cell structure and stability. Extracellular polysaccharides (EPS) represent mainly structural polymers and are found in essentially all kingdoms of life. For example, EPS are important biofilm and capsule components in bacteria, represent major constituents in cell walls of fungi, algae, arthropods and plants, and modulate the extracellular matrix in vertebrates. Different mechanisms evolved by which EPS are synthesized. Here, we review the structures and functions of membrane-integrated processive glycosyltransferases (GTs) implicated in the synthesis and secretion of chitin, alginate, hyaluronan and poly-N-acetylglucosamine (PNAG)., (Copyright © 2015. Published by Elsevier Ltd.)

Plasmodiophora brassicae causes clubroot, a major disease of Brassica oil and vegetable crops worldwide. P. brassicae is a Plasmodiophorid, obligate biotrophic protist in the eukaryotic kingdom of Rhizaria. Here we present the 25.5 Mb genome draft of P. brassicae, developmental stage-specific transcriptomes and a transcriptome of Spongospora subterranea, the Plasmodiophorid causing powdery scab on potato. Like other biotrophic pathogens both Plasmodiophorids are reduced in metabolic pathways. Phytohormones contribute to the gall phenotypes of infected roots. We report a protein (PbGH3) that can modify auxin and jasmonic acid. Plasmodiophorids contain chitin in cell walls of the resilient resting spores. If recognized, chitin can trigger defense responses in plants. Interestingly, chitin-related enzymes of Plasmodiophorids built specific families and the carbohydrate/chitin binding (CBM18) domain is enriched in the Plasmodiophorid secretome. Plasmodiophorids chitin synthases belong to two families, which were present before the split of the eukaryotic Stramenopiles/Alveolates/Rhizaria/Plantae and Metazoa/Fungi/Amoebozoa megagroups, suggesting chitin synthesis to be an ancient feature of eukaryotes. This exemplifies the importance of genomic data from unexplored eukaryotic groups, such as the Plasmodiophorids, to decipher evolutionary relationships and gene diversification of early eukaryotes.

The cell wall is a protective and versatile structure distributed in all fungi. The component responsible for its rigidity is chitin, a product of chitin synthase (Chsp) enzymes. There are seven classes of chitin synthase genes (CHS) and the amount and type encoded in fungal genomes varies considerably from one species to another. Previous Chsp sequence analyses focused on their study as individual units, regardless of genomic context. The identification of blocks of conserved genes between genomes can provide important clues about the interactions and localization of chitin synthases. On the present study, we carried out an in silico search of all putative Chsp encoded in 54 full fungal genomes, encompassing 21 orders from five phyla. Phylogenetic studies of these Chsp were able to confidently classify 347 out of the 369 Chsp identified (94%). Patterns in the distribution of Chsp related to taxonomy were identified, the most prominent being related to the type of fungal growth. More importantly, a synteny analysis for genomic blocks centered on class IV Chsp (the most abundant and widely distributed Chsp class) identified a putative cell wall metabolism gene cluster in members of the genus Aspergillus, the first such association reported for any fungal genome.

Chitin synthases (CHS) produce chitin, an essential component of the fungal cell wall. The molecular mechanism of processive chitin synthesis is not understood, limiting the discovery of new inhibitors of this enzyme class. We identified the bacterial glycosyltransferase NodC as an appropriate model system to study the general structure and reaction mechanism of CHS. A high throughput screening-compatible novel assay demonstrates that a known inhibitor of fungal CHS also inhibit NodC. A structural model of NodC, on the basis of the recently published BcsA cellulose synthase structure, enabled probing of the catalytic mechanism by mutagenesis, demonstrating the essential roles of the DD and QXXRW catalytic motifs. The NodC membrane topology was mapped, validating the structural model. Together, these approaches give insight into the CHS structure and mechanism and provide a platform for the discovery of inhibitors for this antifungal target., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Even though chitin is one of the most abundant biopolymers in nature, current knowledge on chitin formation is largely based only on data from fungi and insects. This study reveals unanticipated broad taxonomic distribution and extensive diversification of chitin synthases (CSs) in Metazoa, shedding new light on the relevance of chitin in animals and suggesting unforeseen complexity of chitin synthesis in many groups. We uncovered robust orthologs to insect type CSs in several representatives of deuterostomes, which generally are not thought to possess chitin. This suggests a broader distribution and function of chitin in this branch of the animal kingdom. We characterize a new CS type present not only in basal metazoans such as sponges and cnidarians but also in several bilaterian representatives. The most extensive diversification of CSs took place during emergence of lophotrochozoans, the third large group of protostomes next to arthropods and nematodes, resulting in coexistence of up to ten CS paralogs in molluscs. Independent fusion to different kinds of myosin motor domains in fungi and lophotrochozoans points toward high relevance of CS interaction with the cytoskeleton for fine-tuned chitin secretion. Given the fundamental role that chitin plays in the morphology of many animals, the here presented CS diversification reveals many evolutionary complexities. Our findings strongly suggest a very broad and multifarious occurrence of chitin and question an ancestral role as cuticular component. The molecular mechanisms underlying regulation of animal chitin synthesis are most likely far more complex and diverse than existing data from insects suggest.

Chs3, the catalytic subunit of chitin synthase III in Saccharomyces cerevisiae, is a complex polytopic membrane protein whose plasma membrane expression is tightly controlled: export from the ER requires interaction with Chs7; exit from the Golgi is dependent on the exomer complex, and precise bud neck localization relies on endocytosis. Moreover, Chs3 is efficiently recycled from endosomes to the TGN in an AP-1-dependent manner. Here we show that the export of Chs3 requires the cargo receptor Erv14, in a step that is independent of Chs7. Chs3 oligomerized in the ER through its N-terminal cytosolic region. However, the truncated (Δ126)Chs3 was still exported by Erv14, but was sent back from the Golgi to the ER in a COPI- and Rer1-dependent manner. A subset of the oligomerization-deficient Chs3 proteins evaded Golgi quality control and reached the plasma membrane, where they were enzymatically active but poorly endocytosed. This resulted in high CSIII levels, but calcofluor white resistance, explained by the reduced intercalation of calcofluor white between nascent chitin fibres. Our data show that the oligomerization of Chs3 through its N-terminus is essential for proper protein trafficking and chitin synthesis and is therefore monitored intracellularly., (© 2013 John Wiley & Sons Ltd.)

This work demonstrates that chitin is an important structural component within the skeletal fibers of the freshwater sponge Spongilla lacustris. Using a variety of analytical techniques ((13)C solid state NMR, FT-IR, Raman, NEXAFS, ESI-MS, Morgan-Elson assay and Calcofluor White Staining); we show that this sponge chitin is much closer to α-chitin, known to be present in other animals, than to β-chitin. Genetic analysis confirmed the presence of chitin synthases, which are described for the first time in a sponge. The presence of chitin in both marine (demosponges and hexactinellids) and freshwater sponges indicates that this important structural biopolymer was already present in their common ancestor., (Copyright © 2013 Elsevier Inc. All rights reserved.)

A holdfast is a root- or basal plate-like structure of principal importance that anchors aquatic sessile organisms, including sponges, to hard substrates. There is to date little information about the nature and origin of sponges' holdfasts in both marine and freshwater environments. This work, to our knowledge, demonstrates for the first time that chitin is an important structural component within holdfasts of the endemic freshwater demosponge Lubomirskia baicalensis. Using a variety of techniques (near-edge X-ray absorption fine structure, Raman, electrospray ionization mas spectrometry, Morgan-Elson assay and Calcofluor White staining), we show that chitin from the sponge holdfast is much closer to α-chitin than to β-chitin. Most of the three-dimensional fibrous skeleton of this sponge consists of spicule-containing proteinaceous spongin. Intriguingly, the chitinous holdfast is not spongin-based, and is ontogenetically the oldest part of the sponge body. Sequencing revealed the presence of four previously undescribed genes encoding chitin synthases in the L. baicalensis sponge. This discovery of chitin within freshwater sponge holdfasts highlights the novel and specific functions of this biopolymer within these ancient sessile invertebrates.

Exomer is a cargo adaptor that mediates the sorting of specific plasma membrane proteins into vesicles at the trans-Golgi network. Cargo adaptors must bind to multiple partners, including their cargo, regulatory proteins, and the membrane surface. During biogenesis of a vesicle, the membrane makes a transition from a relatively flat surface to one of high curvature, requiring cargo adaptors to somehow maintain protein-protein and protein-membrane interactions on a changing membrane environment. Here, we present the crystal structure of a tetrameric Chs5/Bch1 exomer complex and use small-angle X-ray scattering to demonstrate its flexibility in solution. The structural data suggest that the complex flexes primarily around the dimeric N-terminal domain of the Chs5 subunits, which adopts a noncanonical β sandwich fold. We propose that this flexible hinge domain enables exomer to maintain interactions in the context of a dynamic membrane environment., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

In this study, we identified seven chitin synthase-encoding genes in the genome of the dimorphic yeast Yarrowia lipolytica. Three encoded chitin synthases with myosin motor-like domains at their N-termini, and we designated these CSM1 to CSM3, whereas four were identified as CHS1 to CHS4. To investigate the functions of these seven genes, we constructed and characterized their deletion mutants. The chs2Δ mutant formed chained cells in which daughter cells were connected with mother cells and had abnormally thick septa at the bud neck. The chs4Δ mutant showed remarkably reduced chitin content in its cell wall. The chs2Δ, csm1Δ, and csm2Δ mutants were found to be highly sensitive to chitin binding dyes, calcofluor white (CFW) and Congo red, whereas the chs4Δ mutant was resistant to CFW. These results suggest that Chs2 and Chs4 play major roles in septum formation and cell wall chitin synthesis respectively, whereas Csm1 and Csm2 are involved in the maintenance of cell wall architecture and/or cell wall integrity. The populations of filamentous cells, a type of cell population that are defined by the lengths of the cellular long and short axes, decreased in the chs3Δ mutant, suggesting that Chs3 is involved in cellular morphogenesis.

Chitin is a major cell wall component of many filamentous fungi. Among the eight chitin synthase genes of Aspergillus nidulans, csmA and csmB encode a myosin motor-like domain (MMD) and a chitin synthase domain (CSD) at their N- and C-termini respectively. In our previous reports, we suggested that CsmA and CsmB play compensatory roles essential for polarized hyphal growth although their functions do not completely overlap, and that their MMDs are essential for their functions. In the present study, we constructed chimeric csm genes by swapping N-terminal MMD-encoding halves of csmA and csmB and studied them to identify functional differences in the MMDs. Expression of the chimeric gene encoding the MMD-including half of CsmA (MA) and the CSD-including half of CsmB thoroughly suppressed the phenotypic defects of the ΔcsmB mutant, whereas the chimeric gene encoding the MMD-including half of CsmB (MB) and the CSD-including half of CsmA did not fully suppress the defects of the ΔcsmA mutant, suggesting that MA suffices for the function of MB while MB is not functionally equivalent to MA.

During anti-Malassezia screening of plants by CLSI broth microdilution method, Cladia aggregata (Swartz) Nyl. (family Cladoniaceae), a fruticose lichen from Sikkim (northeast Himalayan region), has been found effective at minimum inhibitory concentrations (mg/mL) of 2.72, 0.63, and 1.28 against yeast-like fungi namely, M. furfur, M. globosa and M. sympodialis, respectively. These test pathogens are responsible for pityriasis versicolor (PV) and seborrheic dermatitis (SD) in humans. We tried to establish the reason for variable MICs against various Malassezia spp. using bioinformatical tools, thereby reducing the cost of the experimentation. This is the first report on anti-Malassezia activity of C. aggregata and thus can serve as a potential source for the development of cosmaceuticals.

Aspergillus fumigatus has two chitin synthases (CSMA and CSMB) with a myosin motor-like domain (MMD) arranged in a head-to-head configuration. To understand the function of these chitin synthases, single and double csm mutant strains were constructed and analyzed. Although there was a slight reduction in mycelial growth of the mutants, the total chitin synthase activity and the cell wall chitin content were similar in the mycelium of all of the mutants and the parental strain. In the conidia, chitin content in the ΔcsmA strain cell wall was less than half the amount found in the parental strain. In contrast, the ΔcsmB mutant strain and, unexpectedly, the ΔcsmA/ΔcsmB mutant strain did not show any modification of chitin content in their conidial cell walls. In contrast to the hydrophobic conidia of the parental strain, conidia of all of the csm mutants were hydrophilic due to the presence of an amorphous material covering the hydrophobic surface-rodlet layer. The deletion of CSM genes also resulted in an increased susceptibility of resting and germinating conidia to echinocandins. These results show that the deletion of the CSMA and CSMB genes induced a significant disorganization of the cell wall structure, even though they contribute only weakly to the overall cell wall chitin synthesis.

The chitin synthase that makes the primary septum during cell division in budding yeasts is an important therapeutic target with an unknown activation mechanism. We previously found that the C2-domain of the Saccharomyces cerevisiae Inn1 protein plays an essential but uncharacterised role at the cleavage site during cytokinesis. By combining a novel degron allele of INN1 with a point mutation in the C2-domain, we screened for mutations in other genes that suppress the resulting defect in cell division. In this way, we identified 22 dominant mutations of CHS2 (chitin synthase II) that map to two neighbouring sites in the catalytic domain. Chs2 in isolated cell membranes is normally nearly inactive (unless protease treatment is used to bypass inhibition); however, the dominant suppressor allele Chs2-V377I has enhanced activity in vitro. We show that Inn1 associates with Chs2 in yeast cell extracts. It also interacts in a yeast two-hybrid assay with the N-terminal 65% of Chs2, which contains the catalytic domain. In addition to compensating for mutations in the Inn1 C2-domain, the dominant CHS2 alleles suppress cytokinesis defects produced by the lack of the Cyk3 protein. Our data support a model in which the C2-domain of Inn1 acts in conjunction with Cyk3 to regulate the catalytic domain of Chs2 during cytokinesis. These findings suggest novel approaches for developing future drugs against important fungal pathogens.

Cargo adaptors control intracellular trafficking of transmembrane proteins by sorting them into membrane transport carriers. The COPI, COPII, and clathrin cargo adaptors are structurally well characterized, but other cargo adaptors remain poorly understood. Exomer is a specialized cargo adaptor that sorts specific proteins into trans-Golgi network (TGN)-derived vesicles in response to cellular signals. Exomer is recruited to the TGN by the Arf1 GTPase, a universally conserved trafficking regulator. Here, we report the crystal structure of a tetrameric exomer complex composed of two copies each of the Chs5 and Chs6 subunits. The structure reveals the FN3 and BRCT domains of Chs5, which together we refer to as the FBE domain (FN3-BRCT of exomer), project from the exomer core complex. The overall architecture of the FBE domain is reminiscent of the appendage domains of other cargo adaptors, although it exhibits a distinct topology. In contrast to appendage domains, which bind accessory factors, we show that the primary role of the FBE domain is to bind Arf1 for recruitment of exomer to membranes.

The exomer complex is a putative vesicle coat required for the direct transport of a subset of cargoes from the trans-Golgi network (TGN) to the plasma membrane. Exomer comprises Chs5p and the ChAPs family of proteins (Chs6p, Bud7p, Bch1p, and Bch2p), which are believed to act as cargo receptors. In particular, Chs6p is required for the transport of the chitin synthase Chs3p to the bud neck. However, how the ChAPs associate with Chs5p and recognize cargo is not well understood. Using domain-switch chimeras of Chs6p and Bch2p, we show that four tetratricopeptide repeats (TPRs) are involved in interaction with Chs5p. Because these roles are conserved among the ChAPs, the TPRs are interchangeable among different ChAP proteins. In contrast, the N-terminal and the central parts of the ChAPs contribute to cargo specificity. Although the entire N-terminal domain of Chs6p is required for Chs3p export at all cell cycle stages, the central part seems to predominantly favor Chs3p export in small-budded cells. The cargo Chs3p probably also uses a complex motif for the interaction with Chs6, as the C-terminus of Chs3p interacts with Chs6p and is necessary, but not sufficient, for TGN export.

The transition from unicellular to multicellular life forms requires the development of a specialized structural component, the extracellular matrix (ECM). In Metazoans, there are two main supportive systems, which are based on chitin and collagen/hyaluronan, respectively. Chitin is the major constituent of fungal cell walls and arthropod exoskeleton. However, presence of chitin/chitooligosaccharides has been reported in lower chordates and during specific stages of vertebrate development. In this study, the occurrence of chitin synthases (CHSs) was investigated with a bioinformatics approach in the cephalochordate Branchiostoma floridae, in which the presence of chitin was initially reported in the skeletal rods of the pharyngeal gill basket. Twelve genes coding for proteins containing conserved amino acid residues of processive glycosyltransferases from GT2 family were found and 10 of them display mosaic structures with novel domains never reported previously in a chitin synthase. In particular, the presence of a discoidin (DS) and a sterile alpha motif (SAM) domain was found in nine identified proteins. Sequence analyses and homology modelling suggest that these domains might interact with the extracellular matrix and mediate protein-protein interaction. The multi-domain putative chitin synthases from B. floridae constitute an emblematic example of the explosion of domain innovation and shuffling which predate Metazoans., (Copyright © 2012. Published by Elsevier Ltd.)

Pneumocystis pneumonia remains an important complication of immune suppression. The cell wall of Pneumocystis has been demonstrated to potently stimulate host inflammatory responses, with most studies focusing on β-glucan components of the Pneumocystis cell wall. In the current study, we have elaborated the potential role of chitins and chitinases in Pneumocystis pneumonia. We demonstrated differential host mammalian chitinase expression during Pneumocystis pneumonia. We further characterized a chitin synthase gene in Pneumocystis carinii termed Pcchs5, a gene with considerable homolog to the fungal chitin biosynthesis protein Chs5. We also observed the impact of chitinase digestion on Pneumocystis-induced host inflammatory responses by measuring TNFα release and mammalian chitinase expression by cultured lung epithelial and macrophage cells stimulated with Pneumocystis cell wall isolates in the presence and absence of exogenous chitinase digestion. These findings provide evidence supporting a chitin biosynthetic pathway in Pneumocystis organisms and that chitinases modulate inflammatory responses in lung cells. We further demonstrate lung expression of chitinase molecules during Pneumocystis pneumonia.

How cell cycle machinery regulates extracellular matrix (ECM) remodeling during cytokinesis remains poorly understood. In the budding yeast Saccharomyces cerevisiae, the primary septum (PS), a functional equivalent of animal ECM, is synthesized during cytokinesis by the chitin synthase Chs2. Here, we report that Dbf2, a conserved mitotic exit kinase, localizes to the division site after Chs2 and directly phosphorylates Chs2 on several residues, including Ser-217. Both phosphodeficient (chs2-S217A) and phosphomimic (chs2-S217D) mutations cause defects in cytokinesis, suggesting that dynamic phosphorylation-dephosphorylation of Ser-217 is critical for Chs2 function. It is striking that Chs2-S217A constricts asymmetrically with the actomyosin ring (AMR), whereas Chs2-S217D displays little or no constriction and remains highly mobile at the division site. These data suggest that Chs2 phosphorylation by Dbf2 triggers its dissociation from the AMR during the late stage of cytokinesis. Of interest, both chs2-S217A and chs2-S217D mutants are robustly suppressed by increased dosage of Cyk3, a cytokinesis protein that displays Dbf2-dependent localization and also stimulates Chs2-mediated chitin synthesis. Thus Dbf2 regulates PS formation through at least two independent pathways: direct phosphorylation and Cyk3-mediated activation of Chs2. Our study establishes a mechanism for direct cell cycle control of ECM remodeling during cytokinesis.

Chitin synthases are critical enzymes for synthesis of chitin and thus for subsequent growth and development in insects. We identified the cDNA of chitin synthase gene (CHS) in Aphis glycines, the soybean aphid, which is a serious pest of soybean. The full-length cDNA of CHS in A. glycines (AyCHS) was 5802 bp long with an open reading frame of 4704 bp that encoded for a 1567 amino acid residues protein. The predicted AyCHS protein had a molecular mass of 180.05 kDa and its amino acid sequence contained all the signature motifs (EDR, QRRRW and TWGTR) of chitin synthases. The quantitative real-time PCR (qPCR) analysis revealed that AyCHS was expressed in all major tissues (gut, fat body and integument); however, it had the highest expression in integument (~3.5 fold compared to gut). Interestingly, the expression of AyCHS in developing embryos was nearly 7 fold higher compared to adult integument, which probably is a reflection of embryonic molts in hemimetabolus insects. Expression analysis in different developmental stages of A. glycines revealed a consistent AyCHS expression in all stages. Further, through leaf dip bioassay, we tested the effect of diflubenzuron (DFB, Dimilin ®), a chitin-synthesis inhibitor, on A. glycines' survival, fecundity and body weight. When fed with soybean leaves previously dipped in 50 ppm DFB solution, A. glycines nymphs suffered significantly higher mortality compared to control. A. glycines nymphs feeding on diflubenzuron treated leaves showed a slightly enhanced expression (1.67 fold) of AyCHS compared to nymphs on untreated leaves. We discussed the potential applications of the current study to develop novel management strategies using chitin-synthesis inhibitors and using RNAi by knocking down AyCHS expression.

Chs5p is a component of the exomer, a coat complex required to transport the chitin synthase Chs3p from the trans-Golgi network to the plasma membrane. The Chs5p N-terminal region exhibits fibronectin type III (FN3) and BRCT domains. FN3 domains are present in proteins that mediate adhesion processes, whereas BRCT domains are involved in DNA repair. Several fungi--including Schizosaccharomyces pombe, which has no detectable amounts of chitin--have proteins similar to Chs5p. Here we show that the FN3 and BRCT motifs in Chs5p behave as a module that is necessary and sufficient for Chs5p localization and for cargo delivery. The N-terminal regions of S. cerevisiae Chs5p and S. pombe Cfr1p are interchangeable in terms of Golgi localization, but not in terms of exomer assembly, showing that the conserved function of this module is protein retention in this organelle and that the interaction between the exomer components is organism-specific.

This paper proposes a new method to identify communities in generally weighted complex networks and apply it to phylogenetic analysis. In this case, weights correspond to the similarity indexes among protein sequences, which can be used for network construction so that the network structure can be analyzed to recover phylogenetically useful information from its properties. The analyses discussed here are mainly based on the modular character of protein similarity networks, explored through the Newman-Girvan algorithm, with the help of the neighborhood matrix . The most relevant networks are found when the network topology changes abruptly revealing distinct modules related to the sets of organisms to which the proteins belong. Sound biological information can be retrieved by the computational routines used in the network approach, without using biological assumptions other than those incorporated by BLAST. Usually, all the main bacterial phyla and, in some cases, also some bacterial classes corresponded totally (100%) or to a great extent (>70%) to the modules. We checked for internal consistency in the obtained results, and we scored close to 84% of matches for community pertinence when comparisons between the results were performed. To illustrate how to use the network-based method, we employed data for enzymes involved in the chitin metabolic pathway that are present in more than 100 organisms from an original data set containing 1,695 organisms, downloaded from GenBank on May 19, 2007. A preliminary comparison between the outcomes of the network-based method and the results of methods based on Bayesian, distance, likelihood, and parsimony criteria suggests that the former is as reliable as these commonly used methods. We conclude that the network-based method can be used as a powerful tool for retrieving modularity information from weighted networks, which is useful for phylogenetic analysis.

Family II chitin synthases (CS), including classes IV and V enzymes, share conserved catalytic domains flanked by transmembrane regions. Here we addressed the characterization of Family II fungal CSs by heterologous expression in Saccharomyces cerevisiae. Full-length CSs from classes V or IV were not functional when expressed in S. cerevisiae and accumulated in different intracellular compartments. However, the exchange between different class IV, but not of class V, CHS domains resulted in functional proteins both in vivo and in vitro. The different domains afford the chimeric proteins distinct intracellular behaviours, ranging from endoplasmic reticulum retention to reduced endocytic turnover at the plasma membrane. These results allow a role in chitin synthesis to be assigned to all class IV enzymes, but they also highlight the involvement of the intracellular globular domain of these CSs, not only in enzymatic activity but also in the regulation of their intracellular turnover., (Copyright © 2010 Elsevier Inc. All rights reserved.)

We report the isolation and sequencing of CHS3, a gene that encodes one of several chitin synthases in Paracoccidioides brasiliensis, a medically important fungus restricted geographically to Latin America. The gene contains a single open reading frame of 3817 bp with two introns (71 and 86 bp) and encodes a 1220 amino acid polypeptide with high similarity to other fungal chitin synthases. Northern analysis reveals a high expression of CHS3 in the pathogenic yeast-like phase of the fungus and at the end of the mycelium-yeast transition. Expression of P. brasiliensis CHS3 in a Saccharomyces cerevisiae chs3 null mutant enhanced calcofluor white staining in parallel to an increase in total chitin synthase activity and chitin content in its cell wall., (Copyright (c) 2009 John Wiley & Sons, Ltd.)

Objective: To clone the chitin synthase gene PstChs II from Puccinia striiformis and to analyze its expression pattern., Methods: We isolated the cDNA and genomic DNA of PstChs II via RT-PCR and PCR,analyzed the sequences with different bioinformatic tools, characterized the gene expression pattern via real-time PCR., Results: The coding region of PstChs II (Genbank accession no: GQ329851), interrupted by 15 introns, corresponded to a 2727 bp open reading frame encoding 908 amino acids. PstChs II showed a highest 94% similarity to PgtChs II from Puccinia graminis. PstChs II had 7 transmembrance regions and several conserved chitin synthase domains and motifs such as "QXRRW", "GXGPL" and "DXD". PstChs II belonged to the class II sub-family and had a closer phylogenetic relationship to its homologs from basidiomycetes than those from ascomycetes. PstChs II expression level increased by 10-fold at the urediospore germination stage., Conclusion: PstChs II might be involved in the cell wall synthesis during the germ tube elongation process. The cloning and expression analysis of PstChs II served as a good foundation for further analyzing the role of this gene in the pathogenesis process.

The complete sequence of Paracoccidioides brasiliensis CHS5 gene, encoding a putative chitin synthase revealed a 5583nt open reading frame, interrupted by three introns of 82, 87 and 97bp (GenBank Accession No EF654132). The deduced protein contains 1861 amino acids with a predicted molecular weight of 206.9kDa. Both its large size and the presence of a N-terminal region of approx. 800 residues with a characteristic putative myosin motor-like domain, allow us to include PbrChs5 into class V fungal chitin synthases. Sequence analysis of over 4kb from the 5' UTR region in CHS5, revealed the presence of a previously reported CHS4 gene in P. brasiliensis, arranged in a head-to-head configuration with CHS5. A motif search in this shared region showed the presence of stress response elements (STREs), three binding sites for the transcription activators Rlm1p (known to be stimulated by hypo-osmotic stress) and clusters of Adr1 (related to glucose repression). A quantitative RT-PCR analysis pointed to changes in transcription levels for both genes following oxidative stress, alteration of external osmolarity and under glucose-repressible conditions, suggesting a common regulatory mechanism of transcription.

Chitin formation depends on the activity of a family II glycosyltransferase known as chitin synthase, whose biochemical and structural properties are largely unknown. Previously, we have demonstrated that the chitin portion of the peritrophic matrix in the midgut of the tobacco hornworm, Manduca sexta, is produced by chitin synthase 2 (CHS-2), one of two isoenzymes encoded by the Chs-1 and Chs-2 genes (also named Chs-A and Chs-B), and that CHS-2 is located at the apical tips of the brush border microvilli. Here we report the purification of the chitin synthase from the Manduca midgut as monitored by its activity and immuno-reactivity with antibodies to the chitin synthase. After gel permeation chromatography, the final step of the developed purification protocol, the active enzyme eluted in a fraction corresponding to a molecular mass between 440 and 670 kDa. Native PAGE revealed a single, immuno-reactive band of about 520 kDa, thrice the molecular mass of the chitin synthase monomer. SDS-PAGE and immunoblotting indicated finally that an active, oligomeric complex of the chitin synthase was purified. In summary, the chitin synthase from the midgut of Manduca may prove to be a good model for investigating the enzymes' mode of action.

Chitin synthase (CHS) is a glucosyltransferase that converts UDP-N-acetylglucosamine into chitin, one of the main components of fungal cell wall. Class III chitin synthases act directly in the formation of the cell wall. They catalyze the conversion of the immediate precursor of chitin and are responsible for the majority of chitin synthesis in fungi. As such, they are highly specific molecular targets for drugs that can inhibit the growth and development of fungal pathogens. In this work, we have identified and characterized a chitin synthase gene of Moniliophthora perniciosa (Mopchs) by primer walking. The complete gene sequence is 3,443 bp, interrupted by 13 small introns, and comprises a cDNA with an ORF with 2,739 bp, whose terminal region was experimentally determined, encoding a protein with 913 aa that harbors all the motifs and domains typically found in class III chitin synthases. This is the first report on the characterization of a chitin synthase gene, its mature transcription product, and its putative protein in basidioma and secondary mycelium stages of M. perniciosa, a basidiomycotan fungus that causes witches' broom disease of cacao.

Chitin is one of the major cell wall components of ascomycete filamentous fungi, and chitin synthesis plays important roles in the morphogenesis of hyphae. In the Aspergillus nidulans genome, there are two genes, csmA and csmB, that encode a myosin motor-like domain (MMD) at their N-termini and a chitin synthase domain (CSD) at their C-termini. In our previous studies, we found that the MMD of CsmA was required for its functionality, and that CsmA and CsmB had certain overlapping functions essential for polarized filamentous growth. In this study, we constructed a strain that produced CsmB lacking the MMD (CSDeltaMB). This strain exhibited defects similar to those of the csmB deletion mutant. FLAG-tagged CSDeltaMB (CSDeltaMB-FLAG) did not properly localize to the hyphae. CsmB was co-immunoprecipitated with actin in vivo, whereas CSDeltaMB was not. These results suggest that the MMD of CsmB is crucial for its proper localization via interaction with actin.

Saccharomyces cerevisiae Chs2 (chitin synthase 2) synthesizes the primary septum after mitosis is completed. It is essential for proper cell separation and is expected to be highly regulated. We have expressed Chs2 and a mutant lacking the N-terminal region in Pichia pastoris in an active form at high levels. Both constructs show a pH and cation dependence similar to the wild-type enzyme, as well as increased activity after trypsin treatment. Using further biochemical analysis, we have identified two mechanisms of chitin synthase regulation. First, it is hyperactivated by a soluble yeast protease. This protease is expressed during exponential growth phase, when budding cells require Chs2 activity. Secondly, LC-MS/MS (liquid chromatography tandem MS) experiments on purified Chs2 identify 12 phosphorylation sites, all in the N-terminal domain. Four of them show the perfect sequence motif for phosphorylation by the cyclin-dependent kinase Cdk1. As we also show that phosphorylation of the N-terminal domain is important for Chs2 stability, these sites might play an important role in the cell cycle-dependent degradation of the enzyme, and thus in cell division.

The class V chitin synthase is unique because it has a myosin motor-like domain fused to its catalytic domain. The biochemical properties of this enzyme and its function remain undefined beyond the knowledge that it is the only single chitin synthase required for sustained cell growth at elevated temperatures and, consequently, virulence. This report describes our successful efforts to isolate and purify an active and soluble form of the enzyme from the cell membranes of Wangiella by using a specific polyclonal antibody. To our knowledge, this is the first purification of a single chitin synthase of a filamentous fungus.

Chitin is an essential component of fungal cell walls, where it forms a crystalline scaffold, and chitooligosaccharides derived from it are signaling molecules recognized by the hosts of pathogenic fungi. Oomycetes are cellulosic fungus-like microorganisms which most often lack chitin in their cell walls. Here we present the first study of the cell wall of the oomycete Aphanomyces euteiches, a major parasite of legume plants. Biochemical analyses demonstrated the presence of ca. 10% N-acetyl-D-glucosamine (GlcNAc) in the cell wall. Further characterization of the GlcNAc-containing material revealed that it corresponds to noncrystalline chitosaccharides associated with glucans, rather than to chitin per se. Two putative chitin synthase (CHS) genes were identified by data mining of an A. euteiches expressed sequence tag collection and Southern blot analysis, and full-length cDNA sequences of both genes were obtained. Phylogeny analysis indicated that oomycete CHS diversification occurred before the divergence of the major oomycete lineages. Remarkably, lectin labeling showed that the Aphanomyces euteiches chitosaccharides are exposed at the cell wall surface, and study of the effect of the CHS inhibitor nikkomycin Z demonstrated that they are involved in cell wall function. These data open new perspectives for the development of antioomycete drugs and further studies of the molecular mechanisms involved in the recognition of pathogenic oomycetes by the host plants.

Stereoselective syntheses of both the natural (C5'- S) and unnatural (C5'- R) diastereoisomers of uracil polyoxin C methyl ester have been developed. The key stereocontrolled step involves nucleophilic addition of trimethylsilyl cyanide to the appropriate chiral sulfinimine derived from 2',3'-protected 5'-formyluridine and (S)-(-)-tert-butanesulfinamide or (R)-(+)-tert-butanesulfinamide, respectively. A variety of substrate mimics designed to function as inhibitors of chitin synthase have been synthesized by conjugation of the methyl ester of uracil polyoxin C (UPOC) with activated isoxazole carboxylic acids. Amide bond formation was accomplished via coupling of the amino functionality of UPOC methyl ester with a free isoxazole acid using HBTU or alternatively an isoxazole pentafluorophenyl ester. The substrate mimics incorporate features of the nucleoside-peptide antibiotics, the polyoxins and the nikkomycins, as well as features of the transition state structure expected during polymerization of the natural chitin synthase substrate uridine diphosphoryl-N-acetylglucosamine (UDP-GlcNAc), namely, a metal-binding site and glycosyl oxocarbenium ion mimic.

The chitin synthase (CHS) gene B (4781 bp) of Spodoptera exigua (SeCHSB) was cloned by reverse-transcription PCR (RT-PCR) and 3'/5' RACE from the midgut. SeCHSB contains an open reading frame of 4572 nucleotides, encoding a protein of 1523 amino acids with a predicted molecular mass of approximately 174.6 kDa. Alignment of SeCHSB with class B CHSs of other insects showed a high degree of conservation in the putative catalytic domain region. The structure of the SeCHSB gene was analyzed and was found to be the same as that of Manduca sexta CHSB (MsCHSB), including 23 exons and 22 introns but without alternative exons. Southern blot analysis revealed that SeCHSB was a single copy gene and the presence of only two chitin synthase genes in S. exigua. Further investigation indicated that SeCHSB was specifically expressed in the midgut, and its transcript existed constitutively in the midgut from the 3rd instar larval stage to prepupae and reached highest expression on the 1st day of the fifth instar larval stage. These data suggest that SeCHSB is very important in midgut formation and development. Chitin synthase gene comparisons between different classes of insects using software tools revealed some interesting aspects of the similarity and divergence of the gene in the Class Insecta.