Synthesis of fluorescently labeled UDP-GlcNAc analogues and their evaluation as chitin synthase substrates

[structure: see text] Chitin synthase (CS) polymerizes UDP-GlcNAc to form chitin (poly-beta(1,4)-GlcNAc), a key component of fungal cell wall biosynthesis. Little is known about the substrate specificity of chitin synthase or the scope of substrate modification the enzyme will tolerate. Following a previous report suggesting that 6-O-dansyl GlcNAc is biosynthetically incorporated into chitin, we became interested in developing an assay for CS activity based on incorporation of a fluorescent substrate. We describe the synthesis of two fluorescent UDP-GlcNAc analogues and their evaluation as chitin synthase substrates.     

Probing the mechanism of a fungal glycosyltransferase essential for cell wall biosynthesis. UDP-chitobiose is not a substrate for chitin synthase

Chitin synthase is responsible for the biosynthesis of chitin, an essential component of the fungal cell wall. There is a long-standing question as to whether "processive" transferases such as chitin synthase operate in the same manner as non-processive transferases. The question arises from analysis of the polysaccharide structure--in chitin, for instance, each sugar residue is rotated approximately 180 degrees relative to the preceding sugar in the chain. This requires that the enzyme account for the alternating "up/down" configuration during biosynthesis. An enzyme with a single active site, analogous to the non-processive transferases--would have to accommodate a distorted glycosidic linkage at every other synthetic step. An alternative proposal is that the enzyme might assemble the disaccharide donor, addressing the "up/down" conformational problem prior to polymer synthesis. We present compelling evidence that this latter hypothesis is incorrect.     

Elucidating the biosynthesis of chitin filaments and their configuration with specific proteins and electron microscopy

To deepen the knowledge of chitin synthesis, a yeast mutant has been used as a model. Purified chitin synthase I-containing vesicles (chitosomes) with a diameter of 85 to 120 nm are identified by electron microscopy to eject tiny fibers upon addition of UDP-N-acetylglucosamine. The filigree of extruded filaments fused gradually into a large three-dimensional network, which is degradable by a chitinase. The network is targeted and restructured by the Streptomyces chitin-binding protein CHB1, which has a very high affinity only for alpha-chitin. Within the chitosomes, filaments are found to be highly condensed within consecutive oval fibroids, which are specifically targeted by the alpha-chitin-binding protein. The presented data give new insights to the generation of chitin filaments with an antiparallel (alpha) configuration. [image: see text]     

几丁质合成酶抑制剂

几丁质合成酶是生物合成几丁质的关键物质。几丁质是昆虫表皮和真菌细胞壁的特征成分，由于存在的特殊性而成为农药、医药研发的独特靶标。几丁质合成酶抑制剂由于具有安全、高效等特点，成为农用杀虫、杀螨、杀菌剂以及医药抗真菌药物的研发热点。本文综述了天然及人工合成的几丁质合成酶抑制剂的研究进展，并对其发展趋势进行了展望。     

Design and synthesis of chitin synthase inhibitors as potent fungicides

Chitin is a structural component of fungal cell walls but is absent in vertebrates,mammals,and humans.Chitin synthase is thus an attractive molecular target for developing fungicides.Based on the structure of its donor substrate,UDP-N-acetyl-glucosamine,as well as the modelled structure of the bacterial chitin synthase NodC,we designed a novel scaffold which was then further optimized into a series of chitin synthase inhibitors.The most potent inhibitor,compound 13,exhibited high chitin synthase inhibitory activity with an IC_(50) value of 64.5 μmol/L All of the inhibitors exhibited antifungal activities against the growth of agriculturally-destructive fungi,Fusarium graminearum,Botrytis cinerea.and Colletotrichum lagenarium.This work presents a new scaffold which can be used for the development of novel fungicides.     

G-rich VEGF aptamer as a potential inhibitor of chitin trafficking signal in emerging opportunistic yeast infection

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.     

Stereoselective synthesis of novel uracil polyoxin C conjugates as substrate analogues of chitin synthase

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.     

甲壳素溶致液晶的研究

报道了甲壳素有溶致液晶性．对四种不同分子量的甲壳素的研究结果表明,分子量增大,临界浓度显著降低,胆甾相平均螺距减少,但液晶有序微区平均尺寸却增加．甲壳素分子量为２８４×１０６、１４７×１０６、９４×１０５和５３×１０５时,在二氯乙酸中的临界浓度（Ｗ／Ｗ）分别为０００５、００１５、００３５和００５０．根据理论分析,甲壳素可视为刚性链,与多肽相似．甲壳素的指纹状织构不同于其他溶致液晶体系,在各种分子量甲壳素的两相共存浓度区内,液晶有序存在于内含指纹的不规则微区内,而不是滴状微区内．     

含取代苯甲酰基硫脲的核苷类化合物的合成及杀菌活性研究

为了发现具有杀菌活性的新型先导化合物,基于几丁质合成酶催化作用机制,通过活性亚结构拼接方法,保留多氧霉素和尼克霉素中的活性尿苷部分,将具有良好杀菌活性的硫脲基团引入,设计合成了一系列含硫脲结构的核苷类化合物.以尿苷为原料,经5步反应制得目标物,其结构经IR,1H NMR及元素分析确证.初步生测结果表明,部分化合物对芦笋茎枯病(Phomopsisasparagi bubak)表现出明显的抑制活性,其中6m的抑制率在50μg/mL浓度下为97.2%,与相同浓度的多氧霉素B活性(100%)接近.     

Novel drug delivery system by chitin derivative

A porous chitin foam was regenerated from chitin dope in calcium chloride dihydrate saturated methanol. The porous chitin foam was shown to have cationic property, because chitin foam tended to adsorb anionic dyes through ionic binding and hydrophobic interaction. A pendant type of polymeric drug was prepared applying peptide spacer composed of phenylalanine at amino end and two step hydrolyses of polymeric drug were shown to release active drug at the final step using lysozyme and chymotrypsin in vitro.     

Quantitative in vitro biopolymerization to chitin in native chitosomal membranes supported by silica microparticles

To investigate the unknown physical mechanisms of chitin biosynthesis quantitatively, we designed a quantitative in vitro biopolymerization assay by deposition of native chitosomal membranes from Saccharomyces cerevisiae onto solid silica microparticles of a defined size (? = 3 microm). The homogeneous coating of particle surfaces with native chitosomal membranes observed by confocal microscopy agrees well with the surface coverage calculated by the phosphate analysis. The amount of the synthesized chitin polymers is determined by radioactive assays, which demonstrate that chitin synthase in particle-supported membranes retains its specific enzymatic activity. In comparison to planar substrates, particle supports of defined size (and thus surface area) enable us to amplify the signals from immobilized proteins owing to the much larger surface area and to the capability of concentrating the sample to any given sample volume. Moreover, the large density of particle supports offers unique advantages over purified chitosomes in the quick separation of particle-supported membranes and materials in bulk within 1 min. This allows for the termination of the polymerization reaction without the disruption of the whole membranes, and thus the chitin polymers released in bulk can quantitatively be extracted. The obtained results demonstrate that the native biological membranes on particle supports can be utilized as a new in vitro biopolymerization assay to study the function of transmembrane enzyme complexes.     

Chitin Extraction and Synthesis of Chitin-Based Polymer Films from Philippine Blue Swimming Crab (Portunus pelagicus) Shells

Chitin has been extracted from Philippine blue swimming crab. The extracted chitin was subjected to thermo gravimetric analysis (TGA), Fourier transform infrared (FTIR) spectroscopy and X-ray diffraction (XRD) analysis The degree of acetylation of the extracted chitin, derived from the X-ray diffraction intensity values of chitin characteristic peaks, revealed that the extracted chitin is purer than the commercially acquired high purity chitin. The extracted chitin was used to form polymer films at different formation conditions. Polymer films were also formed from commercially acquired chitin for comparison. It was shown that films prepared from the extracted chitin at different conditions have greater ultimate tensile strengths as compared to the commercially-available plastic film. Morphologies of the material surface and the fracture surface were investigated using the scanning electron microscope to identify stress concentration sites that contributed to the weakening of material under tensile loading.     

Bifunctional abietadiene synthase: free diffusive transfer of the (+)-copalyl diphosphate intermediate between two distinct active sites

Abietadiene synthase (AS) catalyzes two sequential, mechanistically distinct cyclizations in the conversion of geranylgeranyl diphosphate to a mixture of abietadiene double bond isomers as the initial step of resin acid biosynthesis in grand fir (Abies grandis). The first reaction converts geranylgeranyl diphosphate to the stable bicyclic intermediate (+)-copalyl diphosphate via protonation-initiated cyclization. In the second reaction, diphosphate ester ionization-initiated cyclization generates the tricyclic perhydrophenanthrene-type backbone, and is directly coupled to a 1,2-methyl migration that generates the C13 isopropyl group characteristic of the abietane family of diterpenes. Using the transition-state analogue inhibitor 14,15-dihydro-15-azageranylgeranyl diphosphate, it was demonstrated that each reaction of abietadiene synthase is carried out at a distinct active site. Mutations in two aspartate-rich motifs specifically delete one or the other activity and the location of these motifs suggests that the two active sites reside in separate domains. These mutants effectively complement each other, suggesting that the copalyl diphosphate intermediate diffuses between the two active sites in this monomeric enzyme. Free copalyl diphosphate was detected in steady-state kinetic reactions, thus conclusively demonstrating a free diffusion transfer mechanism. In addition, both mutant enzymes enhance the activity of wild-type abietadiene synthase with geranylgeranyl diphosphate as substrate. The implications of these results for the kinetic mechanism of abietadiene synthase are discussed.     

Design,synthesis, and evaluation of 6-carboxyalkyl and 6-phosphonoxyalkyl derivatives of 7-oxo-8-ribitylaminolumazines as inhibitors of riboflavin synthase and lumazine synthase

A series of 6-carboxyalkyl and 6-phosphonoxyalkyl derivatives of 7-oxo-8-D-ribityllumazine were synthesized as inhibitors of both Escherichia coli riboflavin synthase and Bacillus subtilis lumazine synthase. The compounds were designed to bind to both the ribitylpurine binding site and the phosphate binding site of lumazine synthase. In the carboxyalkyl series, maximum activity against both enzymes was observed with the 3'-carboxypropyl compound 22. Lengthening or shortening the chain linking the carboxyl group to the lumazine by one carbon resulted in decreased activity. In the phosphonoxyalkyl series, the 3'-phosphonoxypropyl compound 33 was more potent than the 4'-phosphonoxybutyl derivative 39 against lumazine synthase, but it was less potent against riboflavin synthase. Molecular modeling suggested that the terminal carboxyl group of 6-(3'-carboxypropyl)-7-oxo-8-D-ribityllumazine (22) may bind to the side chains of Arg127 and Lys135 of the enzyme. A hypothetical molecular model was also constructed for the binding of 6-(2'-carboxyethyl)-7-oxolumazine (15) in the active site of E. coli riboflavin synthase, which demonstrated that the active site could readily accommodate two molecules of the inhibitor.     

Physicochemical characterization of α‐chitin, β‐chitin,and γ‐chitin separated from natural resources

We isolated α‐chitin, β‐chitin, and γ‐chitin from natural resources by a chemical method to investigate the crystalline structure of chitin. Its characteristics were identified with Fourier transform infrared (FTIR) and solid‐state cross‐polarization/magic‐angle‐spinning (CP–MAS) ¹³C NMR spectrophotometers. The average molecular weights of α‐chitin, β‐chitin, and γ‐chitin, calculated with the relative viscosity, were about 701, 612, and 524 kDa, respectively. In the FTIR spectra, α‐chitin, β‐chitin, and γ‐chitin showed a doublet, a singlet, and a semidoublet at the amide I band, respectively. The solid‐state CP–MAS ¹³C NMR spectra revealed that α‐chitin was sharply resolved around 73 and 75 ppm and that β‐chitin had a singlet around 74 ppm. For γ‐chitin, two signals appeared around 73 and 75 ppm. From the X‐ray diffraction results, α‐chitin was observed to have four crystalline reflections at 9.6, 19.6, 21.1, and 23.7 by the crystalline structure. Also, β‐chitin was observed to have two crystalline reflections at 9.1 and 20.3 by the crystalline structure. γ‐Chitin, having an antiparallel and parallel structure, was similar in its X‐ray diffraction patterns to α‐chitin. The exothermic peaks of α‐chitin, β‐chitin, and γ‐chitin appeared at 330, 230, and 310, respectively. The thermal decomposition activation energies of α‐chitin, β‐chitin, and γ‐chitin, calculated by thermogravimetric analysis, were 60.56, 58.16, and 59.26 kJ mol^?1, respectively. With the Arrhenius law, ln β was plotted against the reciprocal of the maximum decomposition temperature as a straight line; there was a large slope for large activation energies and a small slope for small activation energies. α‐Chitin with high activation energies was very temperature‐sensitive; β‐Chitin with low activation energies was relatively temperature‐insensitive. © 2004 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 42: 3423–3432, 2004     

Investigation of the catalytic mechanism of farnesyl pyrophosphate synthase by computer simulation

Farnesyl pyrophosphate synthase (FPPS) catalyses the formation of a key cellular intermediate in isoprenoid metabolic pathways, farnesyl pyrophosphate, by the sequential head-to-tail condensation of two molecules of isopentenyl diphosphate (IPP) with dimethylallyl diphosphate (DMAPP). Recently, FPPS has been shown to represent an important target for the treatment of parasitic diseases such as Chagas disease and African trypanosomiasis. Bisphosphonates, pyrophosphate analogues in which the oxygen bridge between the two phosphorus atoms has been replaced by a carbon substituted with different side chains, are able to inhibit the FPPS enzyme. Moreover, nitrogen-containing bisphosphonates have been proposed as carbocation transition state analogues of FPPS. On the basis of structural and kinetic data, different catalytic mechanisms have been proposed for FPPS. By analyzing different reaction coordinates we propose that the reaction occurs in one step through a carbocationic transition state and the subsequent transfer of a hydrogen atom from IPP to the pyrophosphate moiety of DMAPP. Moreover, we have analyzed the role of the active site amino acids on the activation barrier and the reaction mechanism. The structure of the active site is well conserved in the isoprenyl diphosphate synthase family; thus, our results are relevant for the understanding of this important class of enzymes and for the design of more potent and specific inhibitors for the treatment of parasitic diseases.     

The Nonreversibility of Adsorption-Desorption of Dieldrin and Lindane on Chitin in Seawater

Abstract

Adsorption and desorption of dieldrin and lindane on chitin were investigated in seawater batch tests as a function of chitin concentration, temperature, pH and salinity. For chitin concentrations ranging from 0.5 g l^?1 to 12.5 g l^?1, the pesticide concentrations were varied from 4 μgl^?1 to 65 μg^?1 for dieldrin and from 40 μl^?1 to 680 μg l^?1 for lindane. Both dieldrin and lindane show adsorption-desorption hysteresis at low chitin concentration. At high chitin concentrations (m > 6.25 g l^?1 for dieldrin and m > 10 gl^?1 for lindane) both pesticides exhibit reversible behaviour. However, only lindane adsorption is affected by chitin concentration. These types of behaviour remain fixed in prewashed chitin. However, an increase in the temperature and a decrease in the salinity made the process become reversible. A resistant-reversible two component model has been applied to account for these types of behaviour and provides a way to explain most of the observed effects by defining mass independent distribution coefficients.     

Identification of cellulose synthase(s) in higher plants: sequence analysis of processive β-glycosyltransferases with the common motif ’D, D, D35Q(R,Q)XRW‘

More than ten β-glycosyltransferases are now recognized that have limited similarity to the amino acid sequence of cellulose synthase from Acetobacter xylinum. Using hydrophobic cluster analysis (HCA), we recently identified two domains and putative catalytic residues in the processive β-glycosyltransferases. In this study, we have found expressed sequence tags (ESTs) from higher plants (Arabidopsis thaliana, Brassica campestris, and Oryza sativa) that exhibit a limited sequence similarity to the A. xylinum cellulose synthase. These ESTs contain some of the conserved residues identified in the processive β-glycosyltransferases. Complete sequencing of an EST clone (T88271) from A. thaliana led to the identification of all the conserved residues in the derived truncated polypeptide which appears to be part of a putative cellulose synthase. Sequence comparison of proteins with known function and several unidentified proteins have the ‘D, D, D35Q(R,Q)XRW’ motif which is considered a strong predictor for β-glycosyltransferasesthat includes, among other proteins, cellulose and chitin synthases. The first two conserved aspartic acid residues in this motif were analysed by site-directed mutagenesis, and their replacement by another amino acid led to a loss of cellulose synthase activity in A. xylinum, suggesting that they are essential for enzyme activity. A correlation between the second residue (R or Q) in the Q(R,Q)XRW sequence and the synthesis of along glucan chain (polysaccharide) or a short glucan chain(oligosaccharide) suggests that this residue may be involved in the degree of processivity This revised version was published online in August 2006 with corrections to the Cover Date.     

Molecular characterization of selected dermatophytes and their identification by electrophoretic mutation scanning

Dermatophytes are fungi that can be contagious and cause infections in the keratinized skin of mammals, including humans. The etiological diagnosis of dermatophytosis relies on a combination of in vitro‐culture and microscopic methods. Effective molecular tools could overcome the limitations of conventional methods of identification. In the present study, following phenetic identification as M. canis, M. fulvum, M. gypseum, T. mentagrophytes and T. terrestre, we genetically characterized key dermatophytes, employing the sequences of the first and second internal transcribed spacers of nuclear ribosomal DNA as well as part of the chitin synthase‐1 gene, and assessed the utility of these DNA regions (based on levels of nucleotide variation within and among species/taxa) as markers for the classification of species and genotypes. Employing partial chitin synthase‐1 gene as the marker, we also established a PCR‐coupled SSCP approach as a diagnostic/analytical mutation‐scanning tool. This tool should facilitate fundamental investigations of the ecology, epidemiology and population genetics of dermatophytes and, importantly, should assist in allowing a more rapid diagnosis of dermatophytoses in humans and other animals, thus overcoming the significant delays in targeted chemotherapy following diagnosis using conventional methods. (Nucleotide sequence data reported in this paper are available in the EMBL, GenBank and DDJB datadases under accession numbers FJ897707–FJ897713 (ITS‐1), FJ897714–FJ897720 (ITS‐2) and FJ897700–FJ897706 (pchs‐1)).     

Understanding the different activities of highly promiscuous MbtI by computational methods

Salicylate synthase from Mycobacterium tuberculosis, MbtI, is a highly promiscuous Mg(2+) dependent enzyme with up to four distinct activities detected in vitro: isochorismate synthase (IS), isochorismate pyruvate lyase (IPL), salicylate synthase (SS) and chorismate mutase (CM). In this paper, Molecular Dynamic (MD) simulations employing hybrid quantum mechanics/molecular mechanics (QM/MM) potentials have been carried out to get a detailed knowledge of the IS and the IPL activities at the molecular level. According to our simulations, the architecture of the MbtI active site allows catalyzing the two reactions: the isochorismate formation, by means of a stepwise mechanism, and the salicylate production from isochorismate, that appears to be pericyclic in nature. Findings also explain the role of the magnesium cation and the pH dependence activity experimentally observed in MbtI. Mg(2+) would be polarizing and pre-organizing the substrate and active site, as well as shifting the pK(a) values of key active site residues.