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1.
World J Microbiol Biotechnol ; 35(7): 106, 2019 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-31267229

RESUMO

Xenorhabdus nematophila HB310 secreted the insecticidal protein toxin complex. Two chitinase genes, chi60 and chi70, were found in X. nematophila toxin complex locus. In order to clarify the function of two chitinases, chi60 and chi70 genes were cloned and expressed in Escherichia coli Transetta (DE3). As a result, we found that the Chi60 and Chi70 belonged to glycoside hydrolases (GH) family 18 with a molecular mass of 65 kDa and 78 kDa, respectively. When colloidal chitin was treated as the substrate, Chi60 and Chi70 were proved to have the highest enzymatic activity at pH 6.0 and 50 °C. Chi60 and Chi70 had obvious growth inhibition effect against the second larvae of Helicoverpa armigera with growth inhibiting rate of 81.99% and 90.51%. Chi70 had synergistic effect with the insecticidal toxicity of Bt Cry 1Ac, but the Chi60 had no synergistic effect with Bt Cry 1Ac. Chi60 and Chi70 showed antifungal activity against Alternaria brassicicola, Verticillium dahliae and Coniothyrium diplodiella. The results increased our understanding of the chitinases produced by X. nematophila and laid a foundation for further studies on the mechanism of the chitinases.


Assuntos
Antifúngicos/farmacologia , Quitinases/antagonistas & inibidores , Quitinases/genética , Quitinases/metabolismo , Xenorhabdus/metabolismo , Alternaria/efeitos dos fármacos , Animais , Ascomicetos/efeitos dos fármacos , Quitina/metabolismo , Quitinases/classificação , Clonagem Molecular , Sinergismo Farmacológico , Ensaios Enzimáticos , Estabilidade Enzimática , Escherichia coli/genética , Expressão Gênica , Glicosídeo Hidrolases/genética , Concentração de Íons de Hidrogênio , Inseticidas/metabolismo , Inseticidas/farmacologia , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Peso Molecular , Mariposas/efeitos dos fármacos , Mariposas/crescimento & desenvolvimento , Micotoxinas/genética , Micotoxinas/metabolismo , Filogenia , Domínios Proteicos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Temperatura Ambiente , Verticillium/efeitos dos fármacos , Xenorhabdus/genética
2.
Mol Immunol ; 109: 108-115, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30927621

RESUMO

Baculovirus causes liquefaction of insect cuticle to enhance the dissemination of progeny virions away from the host cadavers for increasing viral transmission rates. Antheraea pernyi nucleopolyhedrovirus (ApNPV) infects A. pernyi larvae with circular pus blotches formed in cuticle in the early stage of liquefaction. To investigate the formation mechanism of those pus blotches, the transcriptome profile changes of the cuticles between ApNPV-infected and non-infected A. pernyi larvae were analyzed using RNA-Seq. The transcriptome was de novo assembled using the Trinity platform. Comparison of gene expression levels revealed that a total of 2990 and 4427 unigenes were up- and down-regulated respectively in ApNPV-infected cuticle, of which 2620 and 1903 differentially expressed genes (DEGs) could be enriched in different GO terms and KEGG pathways. In this study, we focused on chitin metabolism related DEGs, and screened 10 genes involved in chitin synthesis and degradation with down-regulated trends, indicating that the chitin metabolism pathway was inhibited by ApNPV infection, which may promote liquefaction of A. pernyi cuticle. Besides, we also identified a large number of DEGs involved in immune related pathways via KEGG analysis, indicating that intense immune responses occurred in A. pernyi cuticle. Our research findings will serve as a basis for further researching the molecular mechanisms underlying cuticle liquefaction of A. pernyi induced by ApNPV infection.


Assuntos
Tegumento Comum/virologia , Mariposas/genética , Mariposas/virologia , Nucleopolyhedrovirus/fisiologia , Análise de Sequência de RNA , Transcriptoma/genética , Animais , Quitinases/classificação , Bases de Dados Genéticas , Perfilação da Expressão Gênica , Ontologia Genética , Anotação de Sequência Molecular , Filogenia , Reprodutibilidade dos Testes
3.
Extremophiles ; 19(6): 1055-66, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26462798

RESUMO

Chitinases are ubiquitous class of extracellular enzymes, which have gained attention in the past few years due to their wide biotechnological applications. The effectiveness of conventional insecticides is increasingly compromised by the occurrence of resistance; thus, chitinase offers a potential alternative to the use of chemical fungicides. The thermostable enzymes from thermophilic microorganisms have numerous industrial, medical, environmental and biotechnological applications due to their high stability for temperature and pH. Thermomyces lanuginosus produced a large number of chitinases, of which chitinase I and II are successfully cloned and purified recently. Molecular dynamic simulations revealed that the stability of these enzymes are maintained even at higher temperature. In this review article we have focused on chitinases from different sources, mainly fungal chitinase of T. lanuginosus and its industrial application.


Assuntos
Ascomicetos/enzimologia , Quitinases/química , Proteínas Fúngicas/química , Microbiologia Industrial/métodos , Sequência de Aminoácidos , Quitinases/classificação , Quitinases/genética , Quitinases/metabolismo , Estabilidade Enzimática , Proteínas Fúngicas/classificação , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Simulação de Dinâmica Molecular , Dados de Sequência Molecular
4.
PLoS One ; 10(6): e0129261, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26046992

RESUMO

Chitinolytic enzymes have an important physiological significance in immune and digestive systems in plants and animals, but chitinase has not been identified as having a role in the digestive system in molluscan. In our study, a novel chitinase homologue, named Ca-Chit, has been cloned and characterized as the oyster Crassostrea angulate. The 3998bp full-length cDNA of Ca-Chit consisted of 23bp 5-UTR, 3288 ORF and 688bp 3-UTR. The deduced amino acids sequence shares homologue with the chitinase of family 18. The molecular weight of the protein was predicted to be 119.389 kDa, with a pI of 6.74. The Ca-Chit protein was a modular enzyme composed of a glycosyl hydrolase family 18 domain, threonine-rich region profile and a putative membrane anchor domain. Gene expression profiles monitored by quantitative RT-PCR in different adult tissues showed that the mRNA of Ca-Chit expressed markedly higher visceral mass than any other tissues. The results of the whole mount in-situ hybridization displayed that Ca-Chit starts to express the visceral mass of D-veliger larvae and then the digestive gland forms a crystalline structure during larval development. Furthermore, the adult oysters challenged by starvation indicated that the Ca-Chit expression would be regulated by feed. All the observations made suggest that Ca-Chit plays an important role in the digestive system of the oyster, Crassostrea angulate.


Assuntos
Quitinases/metabolismo , Crassostrea/enzimologia , Sistema Digestório/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Quitinases/classificação , Quitinases/genética , Clonagem Molecular , Crassostrea/genética , Crassostrea/crescimento & desenvolvimento , DNA Complementar/química , DNA Complementar/genética , Sistema Digestório/metabolismo , Ingestão de Alimentos , Regulação da Expressão Gênica no Desenvolvimento , Hibridização In Situ , Larva/enzimologia , Larva/genética , Larva/crescimento & desenvolvimento , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Inanição
5.
PLoS One ; 10(3): e0119871, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25789746

RESUMO

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/genética
6.
Genome Biol Evol ; 7(3): 916-30, 2015 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-25716828

RESUMO

Hirsutella thompsonii (Ht) is a fungal pathogen of acarines and the primary cause of epizootics among mites. The draft genomes of two isolates of Ht (MTCC 3556: Ht3, 34.6 Mb and MTCC 6686: Ht6, 34.7 Mb) are presented and compared with the genomes of Beauveria bassiana (Bb) ARSEF 2860 and Ophiocordyceps sinensis (Os) CO18. Comparative analysis of carbohydrate active enzymes, pathogen-host interaction genes, metabolism-associated genes, and genes involved in biosynthesis of secondary metabolites in the four genomes was carried out. Reduction in gene family sizes in Ht3 and Os as compared with Ht6 and Bb is observed. Analysis of the mating type genes in Ht reveals the presence of MAT idiomorphs which is suggestive of cryptic sexual traits in Ht. We further identify and classify putative chitinases that may function as virulence factors in fungal entomopathogens due to their role in degradation of arthropod cuticle.


Assuntos
Quitinases/genética , Evolução Molecular , Genoma Fúngico , Hypocreales/genética , Beauveria/genética , Metabolismo dos Carboidratos/genética , Quitinases/química , Quitinases/classificação , Elementos de DNA Transponíveis , Genômica , Interações Hospedeiro-Patógeno/genética , Hypocreales/enzimologia , Família Multigênica , Filogenia , Estrutura Terciária de Proteína , Metabolismo Secundário/genética
7.
Exp Parasitol ; 151-152: 39-48, 2015 Apr-May.
Artigo em Inglês | MEDLINE | ID: mdl-25643862

RESUMO

The salmon louse (Lepeophtheirus salmonis spp.) is an economically important parasite on Atlantic salmon reared in aquaculture globally. Production and degradation of chitin, a major component of the exoskeleton, is the target of some pesticides (Di/Teflubenzuron) used in management of lice on farmed fish. These chemicals inhibit molting of the salmon louse leading to the death of the parasite. We found three chitinases (LsChi1, LsChi2 and LsChi4) in the salmon louse genome. Sequence analysis and phylogeny showed that they belong to the GH18 type of chitinase group and show high sequence similarity to chitinases found in other crustaceans and in insects. Expression patterns were different for all three chitinases suggesting different functions during louse development. Furthermore, the function of LsChi2 was further explored through the use of RNA interference and infection trials. Copepodids with knock down of LsChi2 transcripts were deformed and showed a highly reduced infection success.


Assuntos
Quitinases/genética , Copépodes/enzimologia , Ectoparasitoses/veterinária , Doenças dos Peixes/parasitologia , Salmo salar/parasitologia , Sequência de Aminoácidos , Animais , Quitinases/química , Quitinases/classificação , Quitinases/metabolismo , Copépodes/anatomia & histologia , Copépodes/genética , DNA Complementar/biossíntese , Ectoparasitoses/parasitologia , Feminino , Masculino , Filogenia , RNA/genética , RNA/isolamento & purificação , Interferência de RNA , RNA de Cadeia Dupla/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Alinhamento de Sequência
8.
J Biol Chem ; 290(9): 5354-66, 2015 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-25561735

RESUMO

There is emerging evidence that chitinases have additional functions beyond degrading environmental chitin, such as involvement in innate and acquired immune responses, tissue remodeling, fibrosis, and serving as virulence factors of bacterial pathogens. We have recently shown that both the human chitotriosidase and a chitinase from Salmonella enterica serovar Typhimurium hydrolyze LacNAc from Galß1-4GlcNAcß-tetramethylrhodamine (LacNAc-TMR (Galß1-4GlcNAcß(CH2)8CONH(CH2)2NHCO-TMR)), a fluorescently labeled model substrate for glycans found in mammals. In this study we have examined the binding affinities of the Salmonella chitinase by carbohydrate microarray screening and found that it binds to a range of compounds, including five that contain LacNAc structures. We have further examined the hydrolytic specificity of this enzyme and chitinases from Sodalis glossinidius and Polysphondylium pallidum, which are phylogenetically related to the Salmonella chitinase, as well as unrelated chitinases from Listeria monocytogenes using the fluorescently labeled substrate analogs LacdiNAc-TMR (GalNAcß1-4GlcNAcß-TMR), LacNAc-TMR, and LacNAcß1-6LacNAcß-TMR. We found that all chitinases examined hydrolyzed LacdiNAc from the TMR aglycone to various degrees, whereas they were less active toward LacNAc-TMR conjugates. LacdiNAc is found in the mammalian glycome and is a common motif in invertebrate glycans. This substrate specificity was evident for chitinases of different phylogenetic origins. Three of the chitinases also hydrolyzed the ß1-6 bond in LacNAcß1-6LacNAcß-TMR, an activity that is of potential importance in relation to mammalian glycans. The enzymatic affinities for these mammalian-like structures suggest additional functional roles of chitinases beyond chitin hydrolysis.


Assuntos
Proteínas de Bactérias/metabolismo , Quitinases/metabolismo , Proteínas de Insetos/metabolismo , Lactose/análogos & derivados , Salmonella typhimurium/enzimologia , Amino Açúcares/química , Amino Açúcares/metabolismo , Animais , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Sequência de Carboidratos , Quitina/química , Quitina/metabolismo , Quitinases/classificação , Quitinases/genética , Variação Genética , Humanos , Hidrólise , Proteínas de Insetos/genética , Insetos , Cinética , Lactose/química , Lactose/metabolismo , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Estrutura Molecular , Filogenia , Polissacarídeos/química , Polissacarídeos/metabolismo , Ligação Proteica , Rodaminas/química , Rodaminas/metabolismo , Salmonella typhimurium/genética , Especificidade por Substrato , Vertebrados
9.
Indian J Exp Biol ; 52(11): 1025-35, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25434097

RESUMO

After cellulose, chitin is the second most abundant organic and renewable polysaccharide in nature. This polymer is degraded by enzymes called chitinases which are a part of the glycoside hydrolase family. Chitinases have many important biophysiological functions and immense potential applications especially in control of phytopathogens, production of chito- oligosaccharides with numerous uses and in treatment and degradation of chitinous biowaste. At present many microbial sources are being explored and tapped for chitinase production which includes potential fungal cultures. With advancement in molecular biology and gene cloning techniques, research on fungal chitinases have made fast progress. The present review focuses on recent advances in fungal chitinases, containing a short introduction to types of chitinases, their fermentative production, purification and characterization and molecular cloning and expression.


Assuntos
Quitinases , Proteínas Fúngicas , Quitina/metabolismo , Quitinases/classificação , Quitinases/genética , Quitinases/isolamento & purificação , Quitinases/metabolismo , Clonagem Molecular , Fermentação , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , Fungos/enzimologia , Fungos/crescimento & desenvolvimento , Microbiologia Industrial/métodos , Micologia/métodos
10.
Int J Mol Sci ; 15(2): 2738-60, 2014 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-24552874

RESUMO

Chitinases (EC 3.2.2.14), expressed during the plant-pathogen interaction, are associated with plant defense against pathogens. In the present study, a positive correlation between chitinase activity and sugarcane smut resistance was found. ScChi (GenBank accession no. KF664180), a Class III chitinase gene, encoded a 31.37 kDa polypeptide, was cloned and identified. Subcellular localization revealed ScChi targeting to the nucleus, cytoplasm and the plasma membrane. Real-time quantitative PCR (RT-qPCR) results showed that ScChi was highly expressed in leaf and stem epidermal tissues. The ScChi transcript was both higher and maintained longer in the resistance cultivar during challenge with Sporisorium scitamineum. The ScChi also showed an obvious induction of transcription after treatment with SA (salicylic acid), H2O2, MeJA (methyl jasmonate), ABA (abscisic acid), NaCl, CuCl2, PEG (polyethylene glycol) and low temperature (4 °C). The expression levels of ScChi and six immunity associated marker genes were upregulated by the transient overexpression of ScChi. Besides, histochemical assay of Nicotiana benthamiana leaves overexpressing pCAMBIA 1301-ScChi exhibited deep DAB (3,3'-diaminobenzidinesolution) staining color and high conductivity, indicating the high level of H2O2 accumulation. These results suggest a close relationship between the expression of ScChi and plant immunity. In conclusion, the positive responses of ScChi to the biotic and abiotic stimuli reveal that this gene is a stress-related gene of sugarcane.


Assuntos
Quitinases/metabolismo , Saccharum/enzimologia , Estresse Fisiológico , Sequência de Bases , Quitinases/classificação , Quitinases/genética , Peróxido de Hidrogênio/metabolismo , Dados de Sequência Molecular , Filogenia , Folhas de Planta/metabolismo , Folhas de Planta/microbiologia , Proteínas de Plantas , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Saccharum/microbiologia , Temperatura Ambiente , Tabaco/metabolismo , Regulação para Cima , Ustilaginales/patogenicidade
11.
Mol Biol Evol ; 29(10): 2971-85, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22490823

RESUMO

Proteins produced by the large and diverse chitinase gene family are involved in the hydrolyzation of glycosidic bonds in chitin, a polymer of N-acetylglucosamines. In flowering plants, class I chitinases are important pathogenesis-related proteins, functioning in the determent of herbivory and pathogen attack by acting on insect exoskeletons and fungal cell walls. Within the carnivorous plants, two subclasses of class I chitinases have been identified to play a role in the digestion of prey. Members of these two subclasses, depending on the presence or absence of a C-terminal extension, can be secreted from specialized digestive glands found within the morphologically diverse traps that develop from carnivorous plant leaves. The degree of homology among carnivorous plant class I chitinases and the method by which these enzymes have been adapted for the carnivorous habit has yet to be elucidated. This study focuses on understanding the evolution of carnivory and chitinase genes in one of the major groups of plants that has evolved the carnivorous habit: the Caryophyllales. We recover novel class I chitinase homologs from species of genera Ancistrocladus, Dionaea, Drosera, Nepenthes, and Triphyophyllum, while also confirming the presence of two subclasses of class I chitinases based upon sequence homology and phylogenetic affinity to class I chitinases available from sequenced angiosperm genomes. We further detect residues under positive selection and reveal substitutions specific to carnivorous plant class I chitinases. These substitutions may confer functional differences as indicated by protein structure homology modeling.


Assuntos
Carnivoridade , Caryophyllaceae/enzimologia , Caryophyllaceae/genética , Quitinases/genética , Evolução Molecular , Substituição de Aminoácidos/genética , Quitinases/classificação , Genoma de Planta/genética , Cadeias de Markov , Modelos Genéticos , Filogenia , Seleção Genética , Homologia de Sequência do Ácido Nucleico , Especificidade por Substrato
12.
Biochemistry ; 50(25): 5693-703, 2011 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-21615077

RESUMO

Enzymatic features that determine transglycosylating activity have been investigated through site-directed mutagenesis studies on two family 18 chitinases, ChiA and ChiB from Serratia marcescens, with inherently little transglycosylation activity. The activity was monitored for the natural substrate (GlcNAc)(4) using mass spectrometry and HPLC. Mutation of the middle Asp in the diagnostic DxDxE motif, which interacts with the catalytic Glu during the catalytic cycle, yielded the strongly transglycosylating mutants ChiA-D313N and ChiB-D142N, respectively. Mutation of the same Asp(313/142) to Ala or the mutation of Asp(311/140) to either Asn or Ala had no or much smaller effects on transglycosylating activity. Mutation of Phe(396) in the +2 subsite of ChiA-D313N to Trp led to a severalfold increase in transglycosylation rate while replacement of aromatic residues with Ala in the aglycon (sugar acceptor-binding) subsites of ChiA-D313N and ChiB-D142N led to a clear reduction in transglycosylating activity. Taken together, these results show that the transglycosylation properties of family 18 chitinases may be manipulated by mutations that affect the configuration of the catalytic machinery and the affinity for sugar acceptors. The hypertransglycosylating mutant ChiA-D313N-F396W may find applications for synthetic purposes.


Assuntos
Proteínas de Bactérias/genética , Quitinases/síntese química , Quitinases/genética , Mutagênese Sítio-Dirigida , Asparagina/genética , Ácido Aspártico/genética , Proteínas de Bactérias/síntese química , Domínio Catalítico/genética , Quitinases/classificação , Estabilidade Enzimática/genética , Glicosilação , Hidrólise , Família Multigênica/genética , Oligossacarídeos/química , Oligossacarídeos/genética , Mutação Puntual , Serratia marcescens/enzimologia , Serratia marcescens/genética , Especificidade por Substrato/genética
13.
Glycobiology ; 21(1): 122-33, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20843785

RESUMO

Fungi have a plethora of chitinases, which can be phylogenetically divided into three subgroups (A, B and C). Subgroup C (sgC) chitinases are especially interesting due to their multiple carbohydrate-binding modules, but they have not been investigated in detail yet. In this study, we analyzed sgC chitinases in the mycoparasites Trichoderma atroviride and Trichoderma virens. The expression of sgC chitinase genes in T. atroviride was induced during mycoparasitism of the fungal prey Botrytis cinerea, but not Rhizoctonia solani and correspondingly only by fungal cell walls of the former. Interestingly, only few sgC chitinase genes were inducible by chitin, suggesting that non-chitinous cell wall components can act as inducers. In contrast, the transcriptional profile of the most abundantly expressed sgC chitinase gene tac6 indicated a role of the protein in hyphal network formation. This shows that sgC chitinases have diverse functions and are not only involved in the mycoparasitic attack. However, sequence analysis and 3D modelling revealed that TAC6 and also its ortholog in T. virens have potentially detrimental deletions in the substrate-binding site and are thus probably not catalytically active enzymes. Genomic analysis showed that the genes neighboring sgC chitinases often encode proteins that are solely composed of multiple LysM modules, which were induced by similar stimuli as their neighboring sgC chitinase genes. This study provides first insights into fungal sgC chitinases and their associated LysM proteins.


Assuntos
Quitina/metabolismo , Quitinases/química , Quitinases/classificação , Trichoderma/enzimologia , Sítios de Ligação , Quitinases/genética , Genes Fúngicos , Genoma Fúngico , Genômica , Filogenia , Alinhamento de Sequência , Trichoderma/metabolismo
14.
J Phys Chem B ; 114(18): 6144-9, 2010 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-20397673

RESUMO

Binding of allosamidin to the three family 18 chitinases of Serratia marcescens has been studied using isothermal titration calorimetry (ITC). Interestingly, the thermodynamic signatures of allosamidin binding were different for all three chitinases. At pH 6.0, chitinase A (ChiA) binds allosamidin with a K(d) value of 0.17 +/- 0.06 microM where the main part of the driving force is due to enthalpic change (DeltaH(r) degrees = -6.2 +/- 0.2 kcal/mol) and less to entropic change (-TDeltaS(r) degrees = -3.2 kcal/mol). A large part of DeltaH is due to allosamidin stacking with Trp(167) in the -3 subsite. Binding of allosamidin to both chitinase B (ChiB) (K(d) = 0.16 +/- 0.04 microM) and chitinase C (ChiC) (K(d) = 2.0 +/- 0.2 microM) is driven by entropy (DeltaH(r) degrees = 3.8 +/- 0.2 kcal/mol and -TDeltaS(r) degrees = -13.2 kcal/mol for ChiB and DeltaH(r) degrees = -0.6 +/- 0.1 and -TDeltaS(r) degrees = -7.3 kcal/mol for ChiC). For ChiC, the entropic term is dominated by changes in solvation entropy (DeltaS(conf) = 1 cal/K.mol and DeltaS(solv) = 31 cal/K.mol), while, for ChiB, changes in conformational entropy dominate (DeltaS(conf) = 37 cal/K x mol and DeltaS(solv) = 15 cal/K x mol). Corresponding values for ChiA are DeltaS(conf) = 4 cal/K x mol and DeltaS(solv) = 15 cal/K x mol. These remarkable differences in binding parameters reflect the different architectures of the catalytic centers in these enzymes that are adapted to different types of actions: ChiA and ChiB are processive enzymes that move in opposite directions, meaning that allosamidin binds in to "product" subsites in ChiB, while it binds to polymer-binding subsites in ChiA. The values for ChiC are compatible with this enzyme being a nonprocessive endochitinase with a much more open and solvated substrate-binding-site cleft.


Assuntos
Acetilglucosamina/análogos & derivados , Quitina/metabolismo , Quitinases/fisiologia , Serratia marcescens/enzimologia , Trissacarídeos/farmacologia , Acetilglucosamina/metabolismo , Acetilglucosamina/farmacologia , Domínio Catalítico , Quitinases/classificação , Quitinases/efeitos dos fármacos , Cristalografia por Raios X , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Estrutura Terciária de Proteína , Termodinâmica , Trissacarídeos/metabolismo
15.
Artigo em Inglês | MEDLINE | ID: mdl-20197105

RESUMO

Chitinases are essential enzymes for crustaceans and animal alike for their molting and digestion of foods containing chitin. From the Penaeus monodon EST database, cDNA contigs and singletons for three chitinases, namely PmChi1, 2 and 3, were identified. The complete sequences for the mature PmChi1, 3 and partial PmChi2 were amplified and cloned. The reading frames of PmChi1 and 3 encoded mature proteins of 644 and 468 amino acids with calculated molecular masses of 72.4 and 51.9kDa, respectively. The amino acid sequence comparison among the penaeid chitinases revealed homology around 90%. Therefore, they were grouped together along with those of other crustaceans and insects into three groups separated from those of mammals. PmChi1, 2 and 3 were expressed mainly in hepatopancreas, gill and hepatopancreas, respectively, though small amounts were expressed in other tissues. After molting, only the expression of PmChi2 was down-regulated, while the expression of PmChi1 and 3 was relatively unchanged. The results suggested that the PmChi2 was likely involved in molting while the others might function in the digestion of chitinous foods. The recombinant PmChi1 (rPmChi1) over-produced from Escherichia coli had its optimal pH 5 but it was most stable at neutral pH. Interestingly, the optimal temperature was relatively high at 55 degrees C. Nevertheless, it was stable at lower temperature below 40 degrees C. The rPmChi1 preferentially hydrolyzed the more soluble substrates like partially N-acetylated chitin (PNAC) and colloidal chitin from shrimp shell as compared to the beta-chitin from squid pen.


Assuntos
Quitinases/metabolismo , Penaeidae/enzimologia , Sequência de Aminoácidos , Animais , Quitinases/classificação , Quitinases/genética , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Filogenia , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Temperatura Ambiente , Distribuição Tecidual
16.
J Invertebr Pathol ; 104(2): 75-82, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20153333

RESUMO

Serratia marcescens GEI strain was isolated from the gut of the workers of Chinese honey bee Apis cerana and evaluated in the laboratory for the control of Varroa destructor, a parasite of western honey bee A. mellifera. The supernatant and the collected proteins by ammonium sulfate from the bacterial cultures showed a strong miticidal effect on the female mites, with 100% mite mortality in 5days. Heat (100 degrees C for 10min) and proteinase K treatment of the collected proteins destroyed the miticidal activity. The improved miticial activity of this bacterial strain on chitin medium indicated the involvement of chitinases. The expressed chitinases ChiA, ChiB and ChiC1 from S. marcescens GEI by recombinant Escherichia coli showed pathogenicity against the mites in the laboratory. These chitinases were active in a broad pH range (5-9) and the optimum temperatures were between 60 and 75 degrees C. Synergistic effects of ChiA and ChiB on the miticidal activity against V. destructor were observed. The workers of both honey bee species were not sensitive to the spraying and feeding chitinases. These results provided alternative control strategies for Varroa mites, by formulating chitinase agents and by constructing transgenetic honey bees.


Assuntos
Proteínas de Bactérias/isolamento & purificação , Abelhas/parasitologia , Quitinases/isolamento & purificação , Infestações por Ácaros/veterinária , Controle Biológico de Vetores , Serratia marcescens/enzimologia , Acaricidas , Análise de Variância , Animais , Proteínas de Bactérias/classificação , Criação de Abelhas , Abelhas/microbiologia , Quitinases/classificação , Feminino , Isoenzimas , Infestações por Ácaros/prevenção & controle , Especificidade da Espécie , Varroidae
17.
Biochim Biophys Acta ; 1804(4): 668-75, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19879383

RESUMO

Class V chitinase from cycad, Cycas revoluta, (CrChi-A) is the first plant chitinase that has been found to possess transglycosylation activity. To identify the structural determinants that bring about transglycosylation activity, we mutated two aromatic residues, Phe166 and Trp197, which are likely located in the acceptor binding site, and the mutated enzymes (F166A, W197A) were characterized. When the time-courses of the enzymatic reaction toward chitin oligosaccharides were monitored by HPLC, the specific activity was decreased to about 5-10% of that of the wild type and the amounts of transglycosylation products were significantly reduced by the individual mutations. From comparison between the reaction time-courses obtained by HPLC and real-time ESI-MS, we found that the transglycosylation reaction takes place under the conditions used for HPLC but not under the ESI-MS conditions. The higher substrate concentration (5 mM) used for the HPLC determination is likely to bring about chitinase-catalyzed transglycosylation. Kinetic analysis of the time-courses obtained by HPLC indicated that the sugar residue affinity of +1 subsite was strongly reduced in both mutated enzymes, as compared with that of the wild type. The IC(50) value for the inhibitor allosamidin determined by real-time ESI-MS was not significantly affected by the individual mutations, indicating that the state of the allosamidin binding site (from -3 to -1 subsites) was not changed in the mutated enzymes. We concluded that the aromatic side chains of Phe166 and Trp197 in CrChi-A participate in the transglycosylation acceptor binding, thus controlling the transglycosylation activity of the enzyme.


Assuntos
Quitinases/genética , Quitinases/metabolismo , Cycas/enzimologia , Cycas/genética , Acetilglucosamina/análogos & derivados , Acetilglucosamina/farmacologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Sequência de Bases , Domínio Catalítico/genética , Quitinases/química , Quitinases/classificação , Cromatografia Líquida de Alta Pressão , Primers do DNA/genética , DNA de Plantas/genética , Inibidores Enzimáticos/farmacologia , Glicosilação , Cinética , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização por Electrospray , Especificidade por Substrato , Trissacarídeos/farmacologia
18.
Cell Mol Life Sci ; 67(2): 201-16, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19816755

RESUMO

Insect chitinases belong to family 18 glycosylhydrolases that hydrolyze chitin by an endo-type of cleavage while retaining the anomeric beta-(1-->4) configuration of products. There are multiple genes encoding chitinases and chitinase-like proteins in all insect species studied using bioinformatics searches. These chitinases differ in size, domain organization, physical, chemical and enzymatic properties, and in patterns of their expression during development. There are also differences in tissue specificity of expression. Based on a phylogenetic analysis, insect chitinases and chitinase-like proteins have been classified into several different groups. Results of RNA interference experiments demonstrate that at least some of these chitinases belonging to different groups serve non-redundant functions and are essential for insect survival, molting or development. Chitinases have been utilized for biological control of insect pests on transgenic plants either alone or in combination with other insecticidal proteins. Specific chitinases may prove to be useful as biocontrol agents and/or as vaccines.


Assuntos
Quitinases , Controle de Insetos , Insetos/enzimologia , Sequência de Aminoácidos , Animais , Quitinases/química , Quitinases/classificação , Quitinases/genética , Sequência Conservada , Regulação Enzimológica da Expressão Gênica , Dados de Sequência Molecular , Praguicidas/metabolismo , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Estrutura Terciária de Proteína , Especificidade por Substrato
19.
Glycobiology ; 19(12): 1452-61, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19696236

RESUMO

Chitinase-A (CrChi-A) was purified from leaf rachises of Cycas revoluta by several steps of column chromatography. It was found to be a glycoprotein with a molecular mass of 40 kDa and an isoelectric point of 5.6. CrChi-A produced mainly (GlcNAc)(3) from the substrate (GlcNAc)(6) through a retaining mechanism. More interestingly, CrChi-A exhibited transglycosylation activity, which has not been observed in plant chitinases investigated so far. A cDNA encoding CrChi-A was cloned by rapid amplification of cDNA ends and polymerase chain reaction procedures. It consisted of 1399 nucleotides and encoded an open reading frame of 387-amino-acid residues. Sequence analysis indicated that CrChi-A belongs to the group of plant class V chitinases. From peptide mapping and mass spectrometry of the native and recombinant enzyme, we found that an N-terminal signal peptide and a C-terminal extension were removed from the precursor (M1-A387) to produce a mature N-glycosylated protein (Q24-G370). This is the first report on a plant chitinase with transglycosylation activity and posttranslational modification of a plant class V chitinase.


Assuntos
Quitinases/genética , Quitinases/metabolismo , Cycas , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Quitinases/química , Quitinases/classificação , Quitinases/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , Cycas/química , Cycas/genética , Cycas/metabolismo , DNA Complementar/isolamento & purificação , Genes de Plantas , Dados de Sequência Molecular , Mapeamento de Peptídeos , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
20.
Plant Physiol Biochem ; 47(5): 426-34, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19246207

RESUMO

In plants, various chitinases have been identified and categorized into several groups based on the analysis of their sequences and domains. We have isolated SafchiA, a novel class of chitinase from saffron (Crocus sativus L.). The cDNA encoding SafchiA is mainly expressed in roots and corms, and its expression is induced by elicitor treatment, methyl jasmonate, wounding, and by the fungi Fusarium oxysporum, Beauveria and Phoma sp., suggesting a defence role of the protein. Furthermore, in vitro assays with the recombinant native protein showed chitinolytic, and antifungal activity. The deduced protein shares high similarity with chitinases belonging to family 19 of glycosyl-hydrolases, although some changes in the enzyme active site are present. To explore the properties of SafchiA we have expressed recombinant SafchiA in Escherichia coli and generated four different mutants affected in residues involved in the catalytic activity. One glutamic acid essential for family 19 chitinases activity is not present in C. sativus chitinase suggesting that only one acidic residue is necessary for the enzyme activity, in a similar manner as family 18 glycosyl-hydrolases.


Assuntos
Quitinases/metabolismo , Crocus/enzimologia , Proteínas de Plantas/metabolismo , Estruturas Vegetais/enzimologia , Sequência de Aminoácidos , Antifúngicos/farmacologia , Ascomicetos/efeitos dos fármacos , Ascomicetos/fisiologia , Beauveria/efeitos dos fármacos , Beauveria/fisiologia , Catálise , Domínio Catalítico/genética , Quitinases/classificação , Quitinases/genética , Quitinases/isolamento & purificação , Clonagem Molecular , Crocus/genética , Crocus/microbiologia , DNA Complementar/química , DNA Complementar/genética , Fusarium/efeitos dos fármacos , Fusarium/fisiologia , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Interações Hospedeiro-Patógeno , Dados de Sequência Molecular , Mutação , Filogenia , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Proteínas de Plantas/isolamento & purificação , Estruturas Vegetais/genética , Estruturas Vegetais/microbiologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Estresse Mecânico
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