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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.
Adv Exp Med Biol ; 1142: 221-251, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31102249

RESUMO

Chitinases are glycosyl hydrolases that hydrolyze the ß-(1-4)-linkage of N-acetyl-D-glucosamine units present in chitin polymers. Chitinases are widely distributed enzymes and are present in a wide range of organisms including insects, plants, bacteria, fungi, and mammals. These enzymes play key roles in immunity, nutrition, pathogenicity, and arthropod molting. Humans express two chitinases, chitotriosidase 1 (CHIT1) and acid mammalian chitinase (AMCase) along with several chitinase-like proteins (CLPs). Human chitinases are reported to play a protective role against chitin-containing pathogens through their capability to degrade chitin present in the cell wall of pathogens. Now, human chitinases are gaining attention as the key players in innate immune response. Although the exact mechanism of their role in immune response is not known, studies in recent years begin to relate chitin recognition and degradation with the activation of signaling pathways involved in inflammation. The roles of both CHIT1 and AMCase in the development of various diseases have been revealed and several classes of inhibitors have been developed. However, a clear understanding could not be established due to complexities in the design of the right experiment for studying the role of human chitinase in various diseases. In this chapter, we will first outline the structural features of CHIT1 and AMcase. We will then review the progress in understanding the role of human chitinases in the development of various diseases. Finally, we will summarize the inhibitor discovery efforts targeting both CHIT1 and AMCase.


Assuntos
Quitinases/antagonistas & inibidores , Quitinases/química , Imunidade Inata , Quitina , Humanos , Inflamação , Transdução de Sinais
3.
J Agric Food Chem ; 67(13): 3575-3582, 2019 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-30865442

RESUMO

Insect chitinases play an indispensable role in shedding old cuticle during molting. Targeting chitinase inhibition is a promising pest control strategy. Of ChtI, a chitinase from the destructive insect pest Ostrinia furnacalis (Asian corn borer), has been suggested as a potential target for designing green pesticides. A 4,5,6,7-tetrahydrobenzo[ b]thiophene-3-carboxylate scaffold was previously obtained, and further derivatization generated the lead compound 1 as Of ChtI inhibitor. Here, based on the predicted binding mode of compound 1, the pocket-based lead optimization strategy was applied. A series of analogues was synthesized, and their inhibitory activities against Of ChtI were evaluated. Compound 8 with 6- tert-pentyl showed preferential inhibitory activity with a Ki value of 0.71 µM. Their structure-activity relationships suggested that the compound with larger steric hindrance at the 6-nonpolar group was essential for inhibitory activity due to its stronger interactions with surrounding amino acids. This work provides a strategy for designing potential chitinase inhibitors.


Assuntos
Quitinases/antagonistas & inibidores , Inibidores Enzimáticos/química , Proteínas de Insetos/antagonistas & inibidores , Inseticidas/química , Mariposas/enzimologia , Animais , Domínio Catalítico , Quitinases/genética , Quitinases/metabolismo , Desenho de Drogas , Inibidores Enzimáticos/farmacologia , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Inseticidas/farmacologia , Cinética , Mariposas/química , Mariposas/efeitos dos fármacos , Domínios Proteicos
4.
J Agric Food Chem ; 66(13): 3351-3357, 2018 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-29554796

RESUMO

Chitinases play a vital part in the molting phase of insect pests. Inhibiting their activities by the use of drug-like small chemical molecules is thought to be an efficient strategy in pesticide design and development. On the basis of the crystal structure of OfChtI, a chitinase indispensable for the molting of the insect pest Ostrinia furnacalis (Asian corn borer), here we report a chemical fragment and five variant compounds as inhibitors of OfChtI obtained from a library of over 200 000 chemicals by a structure-based-virtual-screening approach. The compounds were synthesized with high atom economy and tested for their OfChtI-inhibitory activities in a bioassay. Compound 3 showed preferential inhibitory activity with a Ki value of 1.5 µΜ against OfChtI. Analysis of the structure-activity relationships of the compounds provided insight into their interactions with the enzyme active site, which may inform future work in improving the potencies of their inhibitory activities.


Assuntos
Quitinases/antagonistas & inibidores , Inibidores Enzimáticos/química , Proteínas de Insetos/antagonistas & inibidores , Mariposas/efeitos dos fármacos , Animais , Bioensaio , Domínio Catalítico , Quitinases/química , Avaliação Pré-Clínica de Medicamentos , Proteínas de Insetos/química , Cinética , Mariposas/enzimologia , Relação Estrutura-Atividade
5.
Int J Biol Macromol ; 106: 338-350, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28827133

RESUMO

An extracellular acido-thermostable endo-chitinase (called ChiA-Hh59) from thermophilic Hydrogenophilus hirschii strain KB-DZ44, was purified and characterized. The maximum chitinase activity recorded after 36-h of incubation at 60°C was 3000U/ml. Pure enzyme was obtained after heat and acidic treatment, precipitation by ammonium sulphate and acetone, respectively, followed by sequential column chromatographies on Sephacryl S-200 and Mono Q-Sepharose. Based on Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis, the purified enzyme is a monomer with a molecular mass of 59103.12-Da. The 22 residue NH2-terminal sequence of the enzyme showed high homology with family-18 bacterial chitinases. The optimum pH and temperature values for chitinase activity were pH 5.0 and 85°C, respectively. The pure enzyme was completely inhibited by p-chloromercuribenzoic acid (p-CMB) and N-ethylmaleimide (NEM). The obtained results suggest that ChiA-Hh59 might be an endo-chitinase. The studied chitinase exhibited high activity towards colloidal chitin, chitin azure, glycol chitin, while it did not hydrolyse chitibiose and amylose. Its Km and kcat values were 0.298mg colloidal chitin/ml and 14400s-1, respectively. Its catalytic efficiency was higher than those of chitodextrinase and ChiA-65. Additionally, Thin-layer chromatography (TLC) analysis from chitin-oligosaccharides showed that ChiA-Hh59 acted as an endo-splitting enzyme. In conclusion, this chitinase may have great potential for the enzymatic degradation of chitin.


Assuntos
Proteínas de Bactérias/química , Quitina/química , Quitinases/química , Hydrogenophilaceae/enzimologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/isolamento & purificação , Biocatálise , Quitinases/antagonistas & inibidores , Quitinases/isolamento & purificação , Inibidores Enzimáticos/química , Estabilidade Enzimática , Etilmaleimida/química , Expressão Gênica , Temperatura Alta , Concentração de Íons de Hidrogênio , Hydrogenophilaceae/química , Hydrogenophilaceae/classificação , Hidrólise , Cinética , Peso Molecular , Filogenia , Especificidade por Substrato , Ácido p-Cloromercurobenzoico/química
6.
Cell Physiol Biochem ; 42(4): 1657-1669, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28738346

RESUMO

BACKGROUND/AIMS: Pseudomonas aeruginosa (PA) is one of the major opportunistic pathogens which can cause chronic lung infection of cystic fibrosis (CF). The formation of PA biofilm promotes CF development and restricts the antimicrobial efficacies of current antibiotics. METHODS: The antimicrobial effects of azithromycin (AZM) and berberine (BER) alone and in combination were evaluated using microdilution method, checkerboard assay, time-kill test, qRT-PCR analysis and absorption method. The treatments of AZM and/or BER were further evaluated in an animal lung infection model via observing survival rate, bacterial burden and histopathology of lung, the levels of pro-/anti-inflammatory cytokines. RESULTS: AZM-BER were demonstrated to be synergistic against ten clinical PA isolates as well as the standard reference PA ATCC27853, in which PA03 was the most susceptible isolate to AZM-BER with FICI of 0.13 and chosen for subsequent experiments. The synergism of AZM-BER was further confirmed against PA03 in time-kill test and scanning electron microscope (SEM) at their concentrations showing synergism. In PA03, we found that AZM-BER could significantly attenuate productions of a series of virulence factors including alginate, LasA protease, LasB protease, pyoverdin, pyocyanin, chitinase as well as extracellular DNA, and remarkably inhibit the levels of quorum sensing (QS) molecules and the expressions of lasI, lasR, rhlI, rhlR at 1/2×MIC, 1×MIC and 2×MIC. In the infection model, the mice survival were increased markedly, the inflammations of infected lungs were improved greatly along with reduced IL-6, IL-8 and ascended IL-10 at 0.8 mg/kg of AZM combined with 3.2 mg/kg of BER. CONCLUSION: BER might be a promising synergist to enhance the antimicrobial activity of AZM in vitro and in vivo.


Assuntos
Antibacterianos/farmacologia , Azitromicina/farmacologia , Berberina/farmacologia , Biofilmes/efeitos dos fármacos , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/efeitos dos fármacos , Alginatos , Animais , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Quitinases/antagonistas & inibidores , Quitinases/genética , Quitinases/metabolismo , Ciclofosfamida , Fibrose Cística/microbiologia , DNA Bacteriano/antagonistas & inibidores , DNA Bacteriano/biossíntese , Combinação de Medicamentos , Sinergismo Farmacológico , Ácido Glucurônico/antagonistas & inibidores , Ácido Glucurônico/biossíntese , Ácidos Hexurônicos/antagonistas & inibidores , Humanos , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/microbiologia , Metaloendopeptidases/antagonistas & inibidores , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Metaloproteases/antagonistas & inibidores , Metaloproteases/genética , Metaloproteases/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Neutropenia/induzido quimicamente , Neutropenia/tratamento farmacológico , Neutropenia/genética , Neutropenia/patologia , Oligopeptídeos/antagonistas & inibidores , Oligopeptídeos/biossíntese , Infecções por Pseudomonas/induzido quimicamente , Infecções por Pseudomonas/genética , Infecções por Pseudomonas/patologia , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/patogenicidade , Piocianina/antagonistas & inibidores , Piocianina/biossíntese , Fatores de Virulência/antagonistas & inibidores , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
7.
Bioorg Med Chem Lett ; 27(15): 3332-3336, 2017 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-28610983

RESUMO

In the last ten years, we identified and developed a new therapeutic class of antifungal agents, the macrocyclic amidinoureas. These compounds are active against several Candida species, including clinical isolates resistant to currently available antifungal drugs. The mode of action of these molecules is still unknown. In this work, we developed an in-silico target fishing procedure to identify a possible target for this class of compounds based on shape similarity, inverse docking procedure and consensus score rank-by-rank. Chitinase enzyme emerged as possible target. To confirm this hypothesis a novel macrocyclic derivative has been produced, specifically designed to increase the inhibition of the chitinase. Biological evaluation highlights a stronger enzymatic inhibition for the new derivative, while its antifungal activity drops probably because of pharmacokinetic issues. Collectively, our data suggest that chitinase represent at least one of the main target of macrocyclic amidinoureas.


Assuntos
Antifúngicos/farmacologia , Quitinases/antagonistas & inibidores , Desenho de Drogas , Inibidores Enzimáticos/farmacologia , Trichoderma/efeitos dos fármacos , Antifúngicos/síntese química , Antifúngicos/química , Quitinases/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , Trichoderma/enzimologia
8.
J Agric Food Chem ; 65(19): 3851-3857, 2017 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-28457127

RESUMO

Periodic chitin remodeling during insect growth and development requires a synergistic action of two glycosyl hydrolase (GH) family enzymes, GH18 chitinase and GH20 ß-N-acetylhexosaminidase (Hex). Inhibiting either or both of these enzymes is a promising strategy for pest control and management. In this study, OfChi-h (a GH18 chitinase) and OfHex1 (a GH20 Hex) from Ostrinia furnacalis were used to screen a library of microbial secondary metabolites. Phlegmacin B1 was found to be the inhibitor of both OfChi-h and OfHex1 with Ki values of 5.5 µM and 26 µM, respectively. Injection and feeding experiments demonstrated that phlegmacin B1 has insecticidal effect on O. furnacalis's larvae. Phlegmacin B1 was predicted to bind to the active pockets of both OfChi-h and OfHex1. Phlegmacin B1 also showed moderate inhibitory activities against other bacterial and insect GH18 enzymes. This work provides an example of exploiting microbial secondary metabolites as potential pest control and management agents.


Assuntos
Quitinases/antagonistas & inibidores , Inibidores Enzimáticos/química , Proteínas de Insetos/antagonistas & inibidores , Mariposas/efeitos dos fármacos , Mariposas/enzimologia , beta-N-Acetil-Hexosaminidases/antagonistas & inibidores , Animais , Domínio Catalítico , Quitina/metabolismo , Quitinases/química , Quitinases/metabolismo , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Cinética , Larva/efeitos dos fármacos , Larva/enzimologia , Larva/genética , Mariposas/química , Mariposas/genética , Metabolismo Secundário , beta-N-Acetil-Hexosaminidases/química , beta-N-Acetil-Hexosaminidases/metabolismo
9.
Microb Pathog ; 107: 62-68, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28330749

RESUMO

In this study, a novel psychrotolerant chitinolytic bacterium Pedobacter sp. PR-M6 that displayed strong chitinolytic activity on 0.5% colloidal chitin was isolated from the soil of a decayed mushroom. Chitinase activity of PR-M6 at 25 °C (C25) after 6 days of incubation with colloidal chitin increased rapidly to a maximum level (31.3 U/mg proteins). Three chitinase isozymes (chiII, chiIII, and chiIV) from the crude enzyme at 25 °C (C25) incubation were expressed on SDS-PAGE gels at 25 °C. After purification by chitin-affinity chromatography, six chitinase isozymes (chiI, chiII, chiIII, chiIV, chiV, and chiVI) from C25-fractions were expressed on SDS-PAGE gels at 25 °C. Major bands of chitinase isozymes (chiI, chiII, and chiIII) from C4-fractions were strongly expressed on SDS-PAGE gels at 25 °C. Pedobacter sp. PR-M6 showed high inhibition rate of 60.9% and 57.5% against Rhizoctonia solani and Botrytis cinerea, respectively. These results indicated that psychrotolerant Pedobacter sp. PR-M6 could be applied widely as a microorganism agent for the biocontrol of agricultural phytopathogens at low temperatures.


Assuntos
Antifúngicos/isolamento & purificação , Quitinases/biossíntese , Quitinases/química , Quitinases/isolamento & purificação , Pedobacter/enzimologia , Agricultura , Agentes de Controle Biológico/isolamento & purificação , Botrytis/efeitos dos fármacos , Quitina/metabolismo , Quitinases/antagonistas & inibidores , Cromatografia de Afinidade/métodos , Temperatura Baixa , Eletroforese em Gel de Poliacrilamida , Ensaios Enzimáticos , Isoenzimas/química , Isoenzimas/isolamento & purificação , Micélio/efeitos dos fármacos , Micélio/crescimento & desenvolvimento , Pedobacter/classificação , Pedobacter/crescimento & desenvolvimento , Pedobacter/isolamento & purificação , Filogenia , Rhizoctonia/efeitos dos fármacos , Microbiologia do Solo
10.
J Biol Chem ; 292(6): 2080-2088, 2017 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-28053084

RESUMO

Chitinase-h (Chi-h) is of special interest among insect chitinases due to its exclusive distribution in lepidopteran insects and high sequence identity with bacterial and baculovirus homologs. Here OfChi-h, a Chi-h from Ostrinia furnacalis, was investigated. Crystal structures of both OfChi-h and its complex with chitoheptaose ((GlcN)7) reveal that OfChi-h possesses a long and asymmetric substrate binding cleft, which is a typical characteristics of a processive exo-chitinase. The structural comparison between OfChi-h and its bacterial homolog SmChiA uncovered two phenylalanine-to-tryptophan site variants in OfChi-h at subsites +2 and possibly -7. The F232W/F396W double mutant endowed SmChiA with higher hydrolytic activities toward insoluble substrates, such as insect cuticle, α-chitin, and chitin nanowhisker. An enzymatic assay demonstrated that OfChi-h outperformed OfChtI, an insect endo-chitinase, toward the insoluble substrates, but showed lower activity toward the soluble substrate ethylene glycol chitin. Furthermore, OfChi-h was found to be inhibited by N,N',N″-trimethylglucosamine-N,N',N″,N″'-tetraacetylchitotetraose (TMG-(GlcNAc)4), a substrate analog which can be degraded into TMG-(GlcNAc)1-2 Injection of TMG-(GlcNAc)4 into 5th-instar O. furnacalis larvae led to severe defects in pupation. This work provides insights into a molting-indispensable insect chitinase that is phylogenetically closer to bacterial chitinases than insect chitinases.


Assuntos
Quitinases/metabolismo , Lepidópteros/enzimologia , Animais , Catálise , Quitinases/antagonistas & inibidores , Quitinases/química , Quitinases/genética , Conformação Proteica , Proteólise , Especificidade por Substrato
11.
Biomed Res Int ; 2017: 9579736, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29457039

RESUMO

The carmine spider mite Tetranychus cinnabarinus is a major pest of crop and vegetable plants worldwide. Previous studies have shown that scopoletin is a promising acaricidal compound against Tetranychus cinnabarinus. However, the acaricidal mechanism of scopoletin remains unclear. In the present study, 12 full-length cDNAs of chitinase (CHIT) genes from Tetranychus cinnabarinus (designated TcCHITs) were cloned and characterized. Although TcCHITs were expressed throughout all life stages, their expression levels were significantly upregulated during the larval and nymphal stages. TcCHITs were downregulated 24 h after treatment with scopoletin and upregulated 24 h after treatment with diflubenzuron (DFB, a chitin synthesis inhibitor). Feeding double-stranded RNA effectively silenced TcCHIT transcription in Tetranychus cinnabarinus, thus increasing its susceptibility to scopoletin but reducing that to DFB. Meanwhile, TcCHIT silencing in larvae and adult resulted in an extremely low molting rate (7.3%) and high mortality rate (53.3%), respectively, compared with those in the control group. CHIT genes are closely related to arthropod survival, molting, and development in Tetranychus cinnabarinus, suggesting that acaricidal mechanisms of scopoletin and DFB may occur by inhibition and activation of CHIT gene expression, respectively. TcCHIT constitutes a possible target of scopoletin and DFB in Tetranychus cinnabarinus.


Assuntos
Acaricidas/farmacologia , Quitinases/antagonistas & inibidores , Escopoletina/farmacologia , Tetranychidae/efeitos dos fármacos , Animais , DNA Complementar/antagonistas & inibidores , Inativação Gênica , Controle de Pragas , Tetranychidae/enzimologia
12.
J Chem Inf Model ; 56(12): 2413-2420, 2016 12 27.
Artigo em Inglês | MEDLINE | ID: mdl-28024404

RESUMO

Chitinases play important roles in pathogen invasion, arthropod molting, plant defense, and human inflammation. Inhibition of the activity of a typical chitinase by small molecules is of significance in drug development and biological research. On the basis of a recent reported crystal structure of OfChtI, the insect chitinase derived from the pest Ostrinia furnacalis, we computationally identified 17 compounds from a library of over 4 million chemicals by two rounds virtual screening. Among these, three compounds from one chemical class inhibited the activity of OfChtI with single-digit-micromolar IC50 values, and one compound from another chemical class exhibited a broad inhibitory activity not only toward OfChtI but also toward bacterial, fungal, and human chitinases. A new scaffold was discovered, and a structure-inhibitory activity relationship was proposed. This work may provide a novel starting point for the development of specific or broad-spectrum chitinase inhibitors.


Assuntos
Quitinases/antagonistas & inibidores , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Inseticidas/química , Inseticidas/farmacologia , Lepidópteros/enzimologia , Animais , Bactérias/efeitos dos fármacos , Bactérias/enzimologia , Quitinases/metabolismo , Descoberta de Drogas , Fungos/efeitos dos fármacos , Fungos/enzimologia , Humanos , Lepidópteros/efeitos dos fármacos , Simulação de Acoplamento Molecular , Controle de Pragas , Relação Estrutura-Atividade
13.
Chimia (Aarau) ; 70(10): 715-720, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27779930

RESUMO

Both historically and at present, vector control is the most generally effective means of controlling malaria transmission. Insecticides are the predominant method of vector control, but the sterile insect technique (SIT) is a complementary strategy with a successful track record in both agricultural and public health sectors. Strategies of genetic and radiation-induced sterilization of Anopheles have to date been limited by logistical and/or regulatory hurdles. A safe and effective mosquito chemosterilant would therefore be of major utility to future deployment of SIT for malaria control. Here we review the prior and current use of chemosterilants in SIT, and assess the potential for future research. Recent genomic and proteomic studies reveal opportunities for specific targeting of seminal fluid proteins, and the capacity to interfere with sperm motility and storage in the female.


Assuntos
Esterilizantes Químicos/farmacologia , Insetos Vetores , Malária/prevenção & controle , Controle de Mosquitos/métodos , Animais , Aziridinas/farmacologia , Quitinases/antagonistas & inibidores , Hormônios Juvenis/farmacologia
14.
J Biochem ; 159(2): 191-200, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26330565

RESUMO

Vibrio harveyi is a bioluminescent marine bacterium that utilizes chitin as its sole source of energy. In the course of chitin degradation, the bacterium primarily secretes an endochitinase A (VhChiA) to hydrolyze chitin, generating chitooligosaccharide fragments that are readily transported into the cell and broken down to GlcNAc monomers by an exo ß-N-acetylglucosaminidase (VhGlcNAcase). Here we report that sodium salts, especially sodium azide, inhibit two classes of these chitin-degrading enzymes (VhChiA and VhGlcNAcase) with distinct modes of action. Kinetic analysis of the enzymatic hydrolysis of pNP-glycoside substrates reveals that sodium azide inhibition of VhChiA has a mixed-type mode, but that it inhibits VhGlcNAcase competitively. We propose that azide anions inhibit chitinase activity by acting as strong nucleophiles that attack Cγ of the catalytic Glu or Cß of the neighbouring Asp residues. Azide anions may bind not only to the catalytic centre, but also to the other subsites in the substrate-binding cleft of VhChiA. In contrast, azide anions may merely occupy the small-binding pocket of VhGlcNAcase, thereby blocking the accessibility of its active site by short-chain substrates.


Assuntos
Acetilglucosaminidase/antagonistas & inibidores , Azidas/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Quitinases/antagonistas & inibidores , Vibrio/enzimologia , Acetilglucosamina/metabolismo , Acetilglucosaminidase/isolamento & purificação , Ânions/farmacologia , Proteínas de Bactérias/isolamento & purificação , Catálise , Domínio Catalítico/efeitos dos fármacos , Quitina/análogos & derivados , Quitina/metabolismo , Quitinases/isolamento & purificação , Hidrólise , Cinética , Ligação Proteica
15.
Parasitol Int ; 64(6): 579-86, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26281757

RESUMO

Chitin metabolism has been shown to have a role in the development of parasitic nematodes including filarial parasites and the enzymes associated with chitin metabolism have been considered as potential vaccine and drug target. Chitinases are members of the enzyme superfamily of glycoside hydrolases, which are characterized by the ability to hydrolyze glycosidic bonds in chitin chain by either an endolytic or an exolytic mechanism. In the present study, we have demonstrated the chitinase (exochitinase and endochitinase) activity in different stages of Setaria cervi (bovine filarial parasite) and have also purified and characterized the endochitinase from microfilarial stage of the parasite. The chitinase activity has been detected in adult and microfilarial stages of S. cervi using the fluorescent substrates. The S. cervi adult stage was found to have high activity of exochitinase (28.72±0.25 nmol/min/mg) while microfilarial stage showed high activity of endochitinase (24.40±0.25 nmol/min/mg). Native polyacrylamide gel electrophoresis, followed by staining of enzyme activity with fluorescent substrates, revealed single isoenzymic form of exochitinase in adults and endochitinase in microfilariae of S. cervi. The endochitinase from S. cervi microfilariae was purified employing chitin affinity matrix and DEAE-Sephacel ion-exchange chromatography. The enzyme was purified about 55 fold with an enzyme recovery of 22.33%. The purified enzyme exhibited a doublet of protein bands on SDS-PAGE at 65-70 kDa. The closantel (chitinase inhibitor) strongly inhibited the enzyme activity of S. cervi microfilariae endochitinase with a Ki value of 4.3±0.18 µM.


Assuntos
Quitina/metabolismo , Quitinases/metabolismo , Hexosaminidases/metabolismo , Setaria (Nematoide)/enzimologia , Animais , Quitinases/antagonistas & inibidores , Eletroforese em Gel de Poliacrilamida , Hexosaminidases/antagonistas & inibidores , Microfilárias/enzimologia , Microfilárias/metabolismo , Salicilanilidas/metabolismo , Setaria (Nematoide)/crescimento & desenvolvimento , Setaria (Nematoide)/metabolismo
16.
Biotechnol Lett ; 37(10): 2083-90, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26087945

RESUMO

OBJECTIVES: The chitin synthase 1 (CHS1) gene in Phenacoccus solenopsis (PsCHS1) was evaluated as a potential target of RNA interference (RNAi) by using Potato virus X (PVX) as a vector (recombinant PVX) for expressing RNAi triggering elements in Nicotiana tabacum L. RESULTS: RT-PCR analysis confirmed the expression of PsCHS1 in N. tabacum inoculated with recombinant-PVX-PsCHS1 (treated). RT- and multiplex-PCR further showed a reduction in mRNA levels of the target gene in mealybugs feeding on treated plants. Mortality in parent adults and emerging nymphs (21 and 29%) exposed to the treated plants was significantly higher (P < 0.05) than those exposed to uninoculated (-ve control) or inoculated with non-recombinant PVX (PVX-control). The number of surviving adults and the combined number of adults and nymphs (47 and 60%) was significantly (P < 0.05) lower on the treated plants than the -ve (76%) or PVX (74%) control. The visual observations verified the physical deformities in mealybugs exposed to the treated plants. CONCLUSION: chitin synthase 1 is a potential RNAi target in P. solenopsis and the recombinant PVX can be used as a tool to evaluate candidate RNAi triggering elements in plants.


Assuntos
Quitinases/metabolismo , Hemípteros/enzimologia , Hemípteros/fisiologia , Potexvirus/crescimento & desenvolvimento , Interferência de RNA , Tabaco/parasitologia , Tabaco/virologia , Animais , Quitinases/antagonistas & inibidores , Quitinases/genética , Reação em Cadeia da Polimerase Multiplex , Ninfa/fisiologia , Plantas , Potexvirus/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sobrevida
17.
J Med Chem ; 58(12): 4984-97, 2015 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-26030312

RESUMO

Argifin, a 17-membered pentapeptide, inhibits chitinase. As argifin has properties that render it unsuitable as a drug development candidate, we devised a mechanism to create the structural component of argifin that bestows the chitinase inhibition and introduce it into a 14-membered macrolide scaffold. Here we describe (1) the designed macrolide, which exhibits ∼200-fold more potent chitinase inhibition than argifin, (2) the binding modes of the macrolide with Serratia marcescens chitinase B, and (3) the computed analysis explaining the reason for derivatives displaying increased inhibition compared to argifin, the macrolide aglycone displaying inhibition in a nanomolar range. This promises a class of chitinase inhibitors with novel skeletons, providing innovative insight for drug design and the use of macrolides as adaptable, flexible templates for use in drug discovery research and development.


Assuntos
Quitinases/antagonistas & inibidores , Macrolídeos/química , Macrolídeos/farmacologia , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Serratia marcescens/enzimologia , Bactérias/efeitos dos fármacos , Bactérias/enzimologia , Infecções Bacterianas/tratamento farmacológico , Infecções Bacterianas/microbiologia , Quitinases/metabolismo , Cristalografia por Raios X , Desenho de Drogas , Inibidores Enzimáticos/farmacologia , Humanos , Modelos Moleculares , Infecções por Serratia/tratamento farmacológico , Infecções por Serratia/microbiologia , Serratia marcescens/efeitos dos fármacos , Relação Estrutura-Atividade
18.
Planta ; 242(4): 895-907, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25998529

RESUMO

MAIN CONCLUSION: We first solved the crystal structure of class III catalytic domain of a chitinase from fern (PrChiA-cat), and found a structural difference between PrChiA-cat and hevamine. PrChiA-cat was found to have reduced affinities to chitin oligosaccharides and allosamidin. Plant class III chitinases are subdivided into enzymes with three disulfide bonds and those without disulfide bonds. We here referred to the former enzymes as class IIIa chitinases and the latter as class IIIb chitinases. In this study, we solved the crystal structure of the class IIIb catalytic domain of a chitinase from the fern Pteris ryukyuensis (PrChiA-cat), and compared it with that of hevamine, a class IIIa chitinase from Hevea brasiliensis. PrChiA-cat was found to adopt an (α/ß)8 fold typical of GH18 chitinases in a similar manner to that of hevamine. However, PrChiA-cat also had two large loops that extruded from the catalytic site, and the corresponding loops in hevamine were markedly smaller than those of PrChiA-cat. An HPLC analysis of the enzymatic products revealed that the mode of action of PrChiA-cat toward chitin oligosaccharides, (GlcNAc) n (n = 4-6), differed from those of hevamine and the other class IIIa chitinases. The binding affinities of (GlcNAc)3 and (GlcNAc)4 toward the inactive mutant of PrChiA-cat were determined by isothermal titration calorimetry, and were markedly lower than those toward other members of the GH18 family. The affinity and the inhibitory activity of allosamidin toward PrChiA-cat were also lower than those toward the GH18 chitinases investigated to date. Several hydrogen bonds found in the crystal structure of hevamine-allosamidin complex were missing in the modeled structure of PrChiA-cat-allosamidin complex. The structural findings for PrChiA-cat successfully interpreted the functional data presented.


Assuntos
Quitinases/metabolismo , Dissulfetos/química , Pteris/enzimologia , Sequência de Aminoácidos , Calorimetria , Quitinases/antagonistas & inibidores , Quitinases/química , Cromatografia Líquida de Alta Pressão , Cristalografia por Raios X , Inibidores Enzimáticos/farmacologia , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Homologia de Sequência de Aminoácidos
19.
J Biol Chem ; 290(18): 11678-91, 2015 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-25767120

RESUMO

Processive enzymes are major components of the efficient enzyme systems that are responsible for the degradation of the recalcitrant polysaccharides cellulose and chitin. Despite intensive research, there is no consensus on which step is rate-limiting for these enzymes. Here, we performed a comparative study of two well characterized enzymes, the cellobiohydrolase Cel7A from Hypocrea jecorina and the chitinase ChiA from Serratia marcescens. Both enzymes were inhibited by their disaccharide product, namely chitobiose for ChiA and cellobiose for Cel7A. The products behaved as noncompetitive inhibitors according to studies using the (14)C-labeled crystalline polymeric substrates (14)C chitin nanowhiskers and (14)C-labeled bacterial microcrystalline cellulose for ChiA and Cel7A, respectively. The resulting observed Ki (obs) values were 0.45 ± 0.08 mm for ChiA and 0.17 ± 0.02 mm for Cel7A. However, in contrast to ChiA, the Ki (obs) of Cel7A was an order of magnitude higher than the true Ki value governed by the thermodynamic stability of the enzyme-inhibitor complex. Theoretical analysis of product inhibition suggested that the inhibition strength and pattern can be accounted for by assuming different rate-limiting steps for ChiA and Cel7A. Measuring the population of enzymes whose active site was occupied by a polymer chain revealed that Cel7A was bound predominantly via its active site. Conversely, the active-site-mediated binding of ChiA was slow, and most ChiA exhibited a free active site, even when the substrate concentration was saturating for the activity. Collectively, our data suggest that complexation with the polymer chain is rate-limiting for ChiA, whereas Cel7A is limited by dissociation.


Assuntos
Celulose 1,4-beta-Celobiosidase/antagonistas & inibidores , Celulose 1,4-beta-Celobiosidase/metabolismo , Quitinases/antagonistas & inibidores , Quitinases/metabolismo , Polissacarídeos/metabolismo , Animais , Domínio Catalítico , Celulose/metabolismo , Celulose 1,4-beta-Celobiosidase/química , Quitina/química , Quitina/metabolismo , Quitinases/química , Dissacarídeos/metabolismo , Dissacarídeos/farmacologia , Hidrólise , Hypocrea/enzimologia , Cinética , Peso Molecular , Nanoestruturas , Polissacarídeos/farmacologia , Ligação Proteica , Serratia marcescens/enzimologia
20.
Plant J ; 82(1): 54-66, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25652217

RESUMO

A class V (glycoside hydrolase family 18) chitinase from the cycad Cycas revoluta (CrChiA) is a plant chitinase that has been reported to possess efficient transglycosylation (TG) activity. We solved the crystal structure of CrChiA, and compared it with those of class V chitinases from Nicotiana tabacum (NtChiV) and Arabidopsis thaliana (AtChiC), which do not efficiently catalyze the TG reaction. All three chitinases had a similar (α/ß)8 barrel fold with an (α + ß) insertion domain. In the acceptor binding site (+1, +2 and +3) of CrChiA, the Trp168 side chain was found to stack face-to-face with the +3 sugar. However, this interaction was not found in the identical regions of NtChiV and AtChiC. In the DxDxE motif, which is essential for catalysis, the carboxyl group of the middle Asp (Asp117) was always oriented toward the catalytic acid Glu119 in CrChiA, whereas the corresponding Asp in NtChiV and AtChiC was oriented toward the first Asp. These structural features of CrChiA appear to be responsible for the efficient TG activity. When binding of the inhibitor allosamidin was evaluated using isothermal titration calorimetry, the changes in binding free energy of the three chitinases were found to be similar to each other, i.e. between -9.5 and -9.8 kcal mol(-1) . However, solvation and conformational entropy changes in CrChiA were markedly different from those in NtChiV and AtChiC, but similar to those of chitinase A from Serratia marcescens (SmChiA), which also exhibits significant TG activity. These results provide insight into the molecular mechanism underlying the TG reaction and the molecular evolution from bacterial chitinases to plant class V chitinases.


Assuntos
Acetilglucosamina/análogos & derivados , Quitinases/química , Cycas/enzimologia , Inibidores Enzimáticos/metabolismo , Trissacarídeos/metabolismo , Acetilglucosamina/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Arabidopsis/enzimologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Quitina/metabolismo , Quitinases/antagonistas & inibidores , Quitinases/genética , Cristalografia por Raios X , Evolução Molecular , Glicosilação , Dados de Sequência Molecular , Proteínas de Plantas/antagonistas & inibidores , Proteínas de Plantas/química , Proteínas de Plantas/genética , Alinhamento de Sequência , Serratia/enzimologia , Temperatura Ambiente , Tabaco/enzimologia
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