RESUMO
A chitosanase (CvCsn46) from Chromobacterium violaceum ATCC 12472 was produced in Escherichia coli, purified, and partially characterized. When subjected to denaturing polyacrylamide gel electrophoresis, the enzyme migrated as two protein bands (38 and 36 kDa apparent molecular masses), which were both identified as CvCsn46 by mass spectrometry. The enzyme hydrolyzed colloidal chitosan, with optimum catalytic activity at 50 °C, and two optimum pH values (at pH 6.0 and pH 11.0). The chitosanolytic activity of CvCsn46 was enhanced by some ions (Ca2+, Co2+, Cu2+, Sr2+, Mn2+) and DTT, whereas Fe2+, SDS and ß-mercaptoethanol completely inhibited its activity. CvCsn46 showed a non-Michaelis-Menten kinetics, characterized by a sigmoidal velocity curve (R2 = 0.9927) and a Hill coefficient of 3.95. ESI-MS analysis revealed that the hydrolytic action of CvCsn46 on colloidal chitosan generated a mixture of low molecular mass chitooligosaccharides, containing from 2 to 7 hexose residues, as well as D-glucosamine. The chitosan oligomers generated by CvCsn46 inhibited in vitro the mycelial growth of Lasiodiplodia theobromae, significantly reducing mycelium extension and inducing hyphal morphological alterations, as observed by scanning electron microscopy. CvCsn46 was characterized as a versatile biocatalyst that produces well-defined chitooligosaccharides, which have potential to control fungi that cause important crop diseases.
Assuntos
Antifúngicos/química , Quitina/análogos & derivados , Chromobacterium/genética , Glicosídeo Hidrolases/genética , Sequência de Aminoácidos/genética , Quitina/biossíntese , Quitina/química , Quitina/genética , Quitosana/química , Chromobacterium/enzimologia , Escherichia coli/genética , Glicosídeo Hidrolases/biossíntese , Glicosídeo Hidrolases/química , Concentração de Íons de Hidrogênio , Hidrólise , Peso Molecular , OligossacarídeosRESUMO
Vicilins are 7S globulins which constitute the major seed storage proteins in leguminous species. Variant vicilins showing differential binding affinities for chitin have been implicated in the resistance and susceptibility of cowpea to the bruchid Callosobruchus maculatus. These proteins are members of the cupin superfamily, which includes a wide variety of enzymes and non-catalytic seed storage proteins. The cupin fold does not share similarity with any known chitin-biding domain. Therefore, it is poorly understood how these storage proteins bind to chitin. In this work, partial cDNA sequences encoding ß-vignin, the major component of cowpea vicilins, were obtained from developing seeds. Three-dimensional molecular models of ß-vignin showed the characteristic cupin fold and computational simulations revealed that each vicilin trimer contained 3 chitin-binding sites. Interaction models showed that chito-oligosaccharides bound to ß-vignin were stabilized mainly by hydrogen bonds, a common structural feature of typical carbohydrate-binding proteins. Furthermore, many of the residues involved in the chitin-binding sites of ß-vignin are conserved in other 7S globulins. These results support previous experimental evidences on the ability of vicilin-like proteins from cowpea and other leguminous species to bind in vitro to chitin as well as in vivo to chitinous structures of larval C. maculatus midgut.
Assuntos
Proteínas de Plantas/genética , Proteínas de Armazenamento de Sementes/genética , Vigna/genética , Animais , Sítios de Ligação , Quitina/química , Quitina/genética , Clonagem Molecular , Besouros/patogenicidade , DNA Complementar/genética , Resistência à Doença/genética , Doenças das Plantas/genética , Doenças das Plantas/parasitologia , Proteínas de Plantas/química , Ligação Proteica , Proteínas de Armazenamento de Sementes/química , Sementes/química , Sementes/genética , Vigna/crescimento & desenvolvimentoRESUMO
Chitinases are hydrolases that degrade chitin, a polymer of N-acetylglucosamine linked ß(1-4) present in the exoskeleton of crustaceans, insects, nematodes and fungal cell walls. A metagenome fosmid library from a wastewater-contaminated soil was functionally screened for chitinase activity leading to the isolation and identification of a chitinase gene named metachi18A. The metachi18A gene was subcloned and overexpressed in Escherichia coli BL21 and the MetaChi18A chitinase was purified by affinity chromatography as a 6xHis-tagged fusion protein. The MetaChi18A enzyme is a 92-kDa protein with a conserved active site domain of glycosyl hydrolases family 18. It hydrolyses colloidal chitin with an optimum pH of 5 and temperature of 50°C. Moreover, the enzyme retained at least 80% of its activity in the pH range from 4 to 9 and 98% at 600 mM NaCl. Thin layer chromatography analyses identified chitobiose as the main product of MetaChi18A on chitin polymers as substrate. Kinetic analysis showed inhibition of MetaChi18A activity at high concentrations of colloidal chitin and 4-methylumbelliferyl N,N'-diacetylchitobiose and sigmoid kinetics at low concentrations of colloidal chitin, indicating a possible conformational change to lead the chitin chain from the chitin-binding to the catalytic domain. The observed stability and activity of MetaChi18A over a wide range of conditions suggest that this chitinase, now characterized, may be suitable for application in the industrial processing of chitin.
Assuntos
Quitina/genética , Quitinases/genética , Biblioteca Gênica , Metagenoma/genética , Quitina/química , Quitinases/química , Cromatografia Líquida de Alta Pressão , Escherichia coli , Expressão Gênica/genética , Vetores Genéticos , Concentração de Íons de Hidrogênio , Especificidade por SubstratoRESUMO
Chitinases are hydrolases that degrade chitin, a polymer of N-acetylglucosamine linked β(1-4) present in the exoskeleton of crustaceans, insects, nematodes and fungal cell walls. A metagenome fosmid library from a wastewater-contaminated soil was functionally screened for chitinase activity leading to the isolation and identification of a chitinase gene named metachi18A. The metachi18A gene was subcloned and overexpressed in Escherichia coli BL21 and the MetaChi18A chitinase was purified by affinity chromatography as a 6xHis-tagged fusion protein. The MetaChi18A enzyme is a 92-kDa protein with a conserved active site domain of glycosyl hydrolases family 18. It hydrolyses colloidal chitin with an optimum pH of 5 and temperature of 50°C. Moreover, the enzyme retained at least 80% of its activity in the pH range from 4 to 9 and 98% at 600 mM NaCl. Thin layer chromatography analyses identified chitobiose as the main product of MetaChi18A on chitin polymers as substrate. Kinetic analysis showed inhibition of MetaChi18A activity at high concentrations of colloidal chitin and 4-methylumbelliferyl N,N′-diacetylchitobiose and sigmoid kinetics at low concentrations of colloidal chitin, indicating a possible conformational change to lead the chitin chain from the chitin-binding to the catalytic domain. The observed stability and activity of MetaChi18A over a wide range of conditions suggest that this chitinase, now characterized, may be suitable for application in the industrial processing of chitin.
Assuntos
Quitinases/genética , Quitina/genética , Metagenoma/genética , Quitinases/química , Quitina/química , Cromatografia Líquida de Alta Pressão , Escherichia coli , Expressão Gênica/genética , Biblioteca Gênica , Vetores Genéticos , Concentração de Íons de Hidrogênio , Especificidade por SubstratoRESUMO
Chitin synthases are highly important enzymes in nature, where they synthesize structural components in species belonging to different eukaryotic kingdoms, including kingdom Fungi. Unfortunately, their structure and the molecular mechanism of synthesis of their microfibrilar product remain largely unknown, probably because no fungal active chitin synthases have been isolated, possibly due to their extreme hydrophobicity. In this study we have turned to the heterologous expression of the transcript from a small chitin synthase of Rhizopus oryzae (RO3G_00942, Chs1) in Escherichia coli. The enzyme was active, but accumulated mostly in inclusion bodies. High concentrations of arginine or urea solubilized the enzyme, but their dilution led to its denaturation and precipitation. Nevertheless, use of urea permitted the purification of small amounts of the enzyme. The properties of Chs1 (Km, optimum temperature and pH, effect of GlcNAc) were abnormal, probably because it lacks the hydrophobic transmembrane regions characteristic of chitin synthases. The product of the enzyme showed that, contrasting with chitin made by membrane-bound Chs's and chitosomes, was only partially in the form of short microfibrils of low crystallinity. This approach may lead to future developments to obtain active chitin synthases that permit understanding their molecular mechanism of activity, and microfibril assembly.
Assuntos
Quitina Sintase/biossíntese , Quitina/biossíntese , Rhizopus/enzimologia , Arginina/química , Quitina/genética , Quitina Sintase/genética , Escherichia coli/genética , Regulação Fúngica da Expressão Gênica , Rhizopus/genética , Ureia/químicaRESUMO
Mo-CBP3 is a chitin-binding protein from M. oleifera seeds that inhibits the germination and mycelial growth of phytopathogenic fungi. This protein is highly thermostable and resistant to pH changes, and therefore may be useful in the development of new antifungal drugs. However, the relationship of MoCBP3 with the known families of carbohydrate-binding domains has not been established. In the present study, full-length cDNAs encoding 4 isoforms of Mo-CBP3 (Mo-CBP3-1, Mo-CBP3-2, Mo-CBP3-3 and Mo-CBP3-4) were cloned from developing seeds. The polypeptides encoded by the Mo-CBP3 cDNAs were predicted to contain 160 (Mo-CBP3-3) and 163 amino acid residues (Mo-CBP3-1, Mo-CBP3-2 and Mo-CBP3-4) with a signal peptide of 20-residues at the N-terminal region. A comparative analysis of the deduced amino acid sequences revealed that Mo-CBP3 is a typical member of the 2S albumin family, as shown by the presence of an eight-cysteine motif, which is a characteristic feature of the prolamin superfamily. Furthermore, mass spectrometry analysis demonstrated that Mo-CBP3 is a mixture of isoforms that correspond to different mRNA products. The identification of Mo-CBP3 as a genuine member of the 2S albumin family reinforces the hypothesis that these seed storage proteins are involved in plant defense. Moreover, the chitin-binding ability of Mo-CBP3 reveals a novel functionality for a typical 2S albumin.
Assuntos
Albuminas 2S de Plantas/genética , Proteínas de Transporte/genética , Quitinases/genética , Moringa oleifera/genética , Proteínas de Plantas/genética , Albuminas 2S de Plantas/metabolismo , Sequência de Aminoácidos , Proteínas de Transporte/metabolismo , Quitina/genética , Quitina/metabolismo , Quitinases/classificação , Sementes/química , Sementes/genéticaRESUMO
The human genome encodes a gene for an enzymatically active chitinase (CHIT1) located in a single copy on Chromosome 1, which is highly expressed by activated macrophages and in other cells of the innate immune response. Several dysfunctional mutations are known in CHIT1, including a 24-bp duplication in Exon 10 causing catalytic deficiency. This duplication is a common variant conserved in many human populations, except in West and South Africans. Thus it has been proposed that human migration out of Africa and the consequent reduction of exposure to chitin from environmental factors may have enabled the conservation of dysfunctional mutations in human chitinases. Our data obtained from 85 indigenous Amerindians from Peru, representative of populations characterized by high prevalence of chitin-bearing enteroparasites and intense entomophagy, reveal a very high frequency of the 24-bp duplication (47.06%), and of other single nucleotide polymorphisms which are known to partially affect enzymatic activity (G102S: 42.7% and A442G/V: 25.5%). Our finding is in line with a founder effect, but appears to confute our previous hypothesis of a protective role against parasite infection and sustains the discussion on the redundancy of chitinolytic function.
Assuntos
Quitina/química , Hexosaminidases/genética , Imunidade Inata/genética , Animais , Quitina/genética , Dieta , Hexosaminidases/deficiência , Humanos , Indígenas Sul-Americanos , Macrófagos/metabolismo , Macrófagos/parasitologia , Mutação , Parasitos/química , Parasitos/metabolismo , Peru , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Paracoccidioides brasiliensis is a thermodimorphic fungus associated with paracoccidioidomycosis (PCM), the most common systemic mycosis in Latin America. The infection is initiated by inhalation of environmentally dispersed conidia produced by the saprophytic phase of the fungus. In the lungs, P. brasiliensis assumes the parasitic yeast form and must cope with the adverse conditions imposed by cells of the host immune system, which includes a harsh environment, highly concentrated in reactive oxygen species (ROS). In this work, we used the ROS-generating agent paraquat to experimentally simulate oxidative stress conditions in order to evaluate the stress-induced modulation of gene expression in cultured P. brasiliensis yeast cells, using a microarray hybridization approach. The large-scale evaluation inherent to microarray-based analyses identified 2070 genes differentially transcribed in response to paraquat exposure, allowing an integrated visualization of the major metabolic changes that constitute the systemic defense mechanism used by the fungus to overcome the deleterious effects of ROS. These include overexpression of detoxifying agents, as well as of molecular scavengers and genes involved in maintenance of the intracellular redox potential. Particularly noteworthy was to verify that the oxidative stress resistance mechanism of P. brasiliensis also involves coordinated overexpression of a series of genes responsible for chitin-biosynthesis, suggesting that this pathway may constitute a specific regulon. Further analyses aiming at confirming and understanding the mechanisms that control such regulon may provide interesting new targets for chemotherapeutic approaches against P. brasiliensis and other pathogenic fungi.
Assuntos
Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Paracoccidioides/genética , Paracoccidioides/metabolismo , Paraquat/farmacologia , Quitina/biossíntese , Quitina/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expressão Gênica , Perfilação da Expressão Gênica , Herbicidas/farmacologia , Análise em Microsséries , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/genética , Paracoccidioides/imunologia , Paracoccidioidomicose/tratamento farmacológico , Paracoccidioidomicose/genética , Paracoccidioidomicose/microbiologia , Espécies Reativas de OxigênioRESUMO
Metarhizium anisopliae is a filamentous fungus used in the biological control of arthropods and produces several chitinases in order to break the host cuticle chitin fibers. Chitinase function during fungal cell development and/or infection processes is also an important aspect when analyzing the life cycle of entomopathogens. The expression profile analysis of the endochitinase chi2 gene acquired by RT-PCR experiments indicated the presence of two different transcripts, suggesting the occurrence of alternative splicing in the chi2 gene. The presence of two transcripts, characterized by the removal or retention of the second 72 bp intron, was further confirmed by DNA sequencing, Northern blot and qRT-PCR. Furthermore, we detected the synthesis of two different proteins from the transcripts by two-dimensional Western blot and mass spectrometry analyses. This is the first reported occurrence of alternative splicing in M. anisopliae.
Assuntos
Quitinases/genética , Metarhizium/genética , Processamento Alternativo , Sequência de Bases , Western Blotting , Quitina/genética , Quitina/metabolismo , Quitinases/biossíntese , Quitinases/metabolismo , Genes , Metarhizium/metabolismo , Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Paracoccidioides brasiliensis is an amenable model to study the molecular and biochemical events that lead to morphological transition in fungi, because temperature seems to be the only factor regulating this process. It is the causative agent of paracoccidioidomycosis, a systemic mycosis that affects humans and that is geographically confined to Latin America, where it constitutes one of the most prevalent deep mycoses. With the help of molecular tools, events leading to the morphological transition have been traced to genes that control cell wall glucan and chitin syntheses, and other metabolic processes such as production of heat shock proteins and ornithine decarboxylase activity. Molecular diagnosis and epidemiology of paracoccidioidomycosis are also the focus of intensive research, with several primers being proposed as specific probes for clinical and field uses. Although P. brasiliensis is refractory to cytogenetic analysis, electrophoretic methods have allowed an approximation of its genomic organization and ploidy. Finally, the recognition of P. brasiliensis as an anamorph in the phylum Ascomycota, order Onygenales, family Onygenaceae, has been accomplished by means of molecular tools. This phylogenetic placement has revised the taxonomic position of this fungus, which was traditionally included within now-abandoned higher anamorph taxa, the phylum Deuteromycota and the class Hyphomycetes.