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1.
Biosci Biotechnol Biochem ; 79(8): 1296-304, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25818933

RESUMO

Chitinase-A (EaChiA), molecular mass 36 kDa, was purified from the vegetative stems of a horsetail (Equisetum arvense) using a series of column chromatography. The N-terminal amino acid sequence of EaChiA was similar to the lysin motif (LysM). A cDNA encoding EaChiA was cloned by rapid amplification of cDNA ends and polymerase chain reaction. It consisted of 1320 nucleotides and encoded an open reading frame of 361 amino acid residues. The deduced amino acid sequence indicated that EaChiA is composed of a N-terminal LysM domain and a C-terminal plant class IIIb chitinase catalytic domain, belonging to the glycoside hydrolase family 18, linked by proline-rich regions. EaChiA has strong chitin-binding activity, however, no antifungal activity. This is the first report of a chitinase from Equisetopsida, a class of fern plants, and the second report of a LysM-containing chitinase from a plant.


Assuntos
Sequência de Aminoácidos/genética , Quitinases/química , Quitinases/genética , Equisetum/enzimologia , Sequência de Bases , Quitinases/isolamento & purificação , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos
2.
Breed Sci ; 65(2): 145-53, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26069444

RESUMO

Sweetpotato (Ipomoea batatas L.) is an outcrossing hexaploid species with a large number of chromosomes (2n = 6x = 90). Although sweetpotato is one of the world's most important crops, genetic analysis of the species has been hindered by its genetic complexity combined with the lack of a whole genome sequence. In the present study, we constructed a genetic linkage map based on retrotransposon insertion polymorphisms using a mapping population derived from a cross between 'Purple Sweet Lord' (PSL) and '90IDN-47' cultivars. High-throughput sequencing and subsequent data analyses identified many Rtsp-1 retrotransposon insertion sites, and their allele dosages (simplex, duplex, triplex, or double-simplex) were determined based on segregation ratios in the mapping population. Using a pseudo-testcross strategy, 43 and 47 linkage groups were generated for PSL and 90IDN-47, respectively. Interestingly, most of these insertions (~90%) were present in a simplex manner, indicating their utility for linkage map construction in polyploid species. Additionally, our approach led to savings of time and labor for genotyping. Although the number of markers herein was insufficient for map-based cloning, our trial analysis exhibited the utility of retrotransposon-based markers for linkage map construction in sweetpotato.

3.
J Ind Microbiol Biotechnol ; 39(1): 55-62, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21681484

RESUMO

A heat-labile phenolic acid decarboxylase from Candida guilliermondii (an anamorph of Pichia guilliermondii) was purified to homogeneity by simple successive column chromatography within 3 days. The molecular mass was 20 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 36 kDa by gel-filtration chromatography, suggesting that the purified enzyme is a homodimer. The optimal pH and temperature were approximately 6.0 and 25°C. Characteristically, more than 50% of the optimal activity was observed at 0°C, suggesting that this enzyme is cold-adapted. The enzyme converted p-coumaric acid, ferulic acid, and caffeic acid to corresponding products with high specific activities of approximately 600, 530, and 46 U/mg, respectively. The activity was stimulated by Mg(2+) ions, whereas it was completely inhibited by Fe(2+), Ni(2+), Cu(2+), Hg(2+), 4-chloromericuribenzoate, N-bromosuccinimide, and diethyl pyrocarbonate. The enzyme was inducible and expressed inside the cells moderately by ferulic acid and p-coumaric acid and significantly by non-metabolizable 6-hydroxy-2-naphthoic acid.


Assuntos
Candida/enzimologia , Carboxiliases/metabolismo , Ácidos Cafeicos/metabolismo , Carboxiliases/química , Carboxiliases/isolamento & purificação , Cromatografia em Gel , Ácidos Cumáricos/metabolismo , Eletroforese em Gel de Poliacrilamida , Metais/farmacologia , Peso Molecular , Naftalenos/metabolismo , Propionatos , Especificidade por Substrato
4.
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
5.
Glycobiology ; 21(5): 644-54, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21367878

RESUMO

Chitinase-A (BcChi-A) was purified from a moss, Bryum coronatum, by several steps of column chromatography. The purified BcChi-A was found to be a molecular mass of 25 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an isoelectric point of 3.5. A cDNA encoding BcChi-A was cloned by rapid amplification of cDNA ends and polymerase chain reaction. It consisted of 1012 nucleotides and encoded an open reading frame of 228 amino acid residues. The predicted mature BcChi-A consists of 205 amino acid residues and has a molecular weight of 22,654. Sequence analysis indicated that BcChi-A is glycoside hydrolase family-19 (GH19) chitinase lacking loops I, II, IV and V, and a C-terminal loop, which are present in the catalytic domain of plant class I and II chitinases. BcChi-A is a compact chitinase that has the fewest loop regions of the GH19 chitinases. Enzymatic experiments using chitooligosaccharides showed that BcChi-A has higher activity toward shorter substrates than class II enzymes. This characteristic is likely due to the loss of the loop regions that are located at the end of the substrate-binding cleft and would be involved in substrate binding of class II enzymes. This is the first report of a chitinase from mosses, nonvascular plants.


Assuntos
Antifúngicos/metabolismo , Bryopsida/enzimologia , Quitinases/metabolismo , Sequência de Aminoácidos , Antifúngicos/química , Antifúngicos/farmacologia , Domínio Catalítico , Quitinases/química , Quitinases/farmacologia , Clonagem Molecular , Ensaios Enzimáticos , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência , Trichoderma/efeitos dos fármacos
6.
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
7.
Glycobiology ; 18(5): 414-23, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18310304

RESUMO

Chitinase-A (PrChi-A), of molecular mass 42 kDa, was purified from the leaves of a fern (P. ryukyuensis) using several column chromatographies. The N-terminal amino acid sequence of PrChi-A was similar to the lysin motif (LysM). A cDNA encoding PrChi-A was cloned by rapid amplification of cDNA ends and polymerase chain reaction. It consisted of 1459 nucleotides and encoded an open-reading frame of 423-amino-acid residues. The deduced amino acid sequence indicated that PrChi-A is composed of two N-terminal LysM domains and a C-terminal catalytic domain, belonging to the group of plant class IIIb chitinases, linked by proline, serine, and threonine-rich regions. Wild-type PrChi-A had chitin-binding and antifungal activities, but a mutant without LysM domains had lost both activities. These results suggest that the LysM domains contribute significantly to the antifungal activity of PrChi-A through their binding activity to chitin in the cell wall of fungi. This is the first report of the presence in plants of a family-18 chitinase containing LysM domains.


Assuntos
Antifúngicos/química , Quitina/metabolismo , Quitinases/química , Mucoproteínas/química , Proteínas de Plantas/química , Pteris/enzimologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Antifúngicos/isolamento & purificação , Sequência de Bases , Sítios de Ligação , Domínio Catalítico , Quitinases/genética , Quitinases/metabolismo , Clonagem Molecular , DNA Complementar/metabolismo , Hidrólise , Dados de Sequência Molecular , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência
8.
Chem Biol Interact ; 220: 269-77, 2014 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-25058688

RESUMO

The erythroid differentiation-inducing effect of apigenin and its derivatives on human chronic myeloid leukemia K562 has been reported but the functional group in its structure responsible for the effect has not yet been elucidated. Here, we determined the moiety responsible for the erythroid differentiation induction effect of apigenin by using different flavonoids to represent the functional groups in its structure. In addition, we compared apigenin and apigetrin, a flavonoid similar in structure to apigenin except for the glycoside in its structure. Morphological changes as well as expressions of specific markers in K562 cells treated with apigenin were compared with those treated with apigetrin, flavone, 7-hydroxyflavone, chrysin, luteolin, or naringenin. The anti-proliferative and erythroid differentiation-inducing effect of apigenin and the five flavonoids were then investigated and their effects on the α, ß, and γ globin genes expressions were compared using real-time PCR. Results of the comparison between apigenin and apigetrin revealed that the glycoside part of apigetrin does not have a role in the induction of cell differentiation. Based on glycophorin A expression, the potency of the other flavonoids for induction of differentiation, was: apigenin>chrysin>flavone/7-hydroxyflavone>luteolin/naringenin. Results of the analysis of the relationship between the structure and function of the flavonoids suggest that the apigenin-induced K562 cell differentiation was due to the 2-3 double bond and hydroxyl groups in its structure. This is the first study that identified the specific functional group in apigenin that impact the erythroid differentiation effect in K562 cells.


Assuntos
Apigenina/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células Eritroides/efeitos dos fármacos , Antineoplásicos/química , Antineoplásicos/farmacologia , Apigenina/química , Células Eritroides/citologia , Humanos , Células K562 , Estrutura Molecular , Reação em Cadeia da Polimerase
9.
Cytotechnology ; 65(6): 899-907, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23138267

RESUMO

α-Linolenic acid (ALA), a major fatty acid in flaxseed oil, has multiple functionalities such as anti-cardiovascular and anti-hypertensive activities. In this study, we investigated the effects of ALA on lipid metabolism and studied the possible mechanisms of its action in differentiated 3T3-L1 adipocytes using DNA microarray analysis. From a total of 34,325 genes in the DNA chip, 87 genes were down-regulated and 185 genes were up-regulated at least twofold in differentiated 3T3-L1 adipocyte cells treated with 300 µM ALA for a week, 5-12 days after induction of cell differentiation, compared to ALA-untreated 3T3-L1 adipocytes (control). From the Reactome analysis results, eight lipid metabolism-related genes involved in cholesterol and triacylglycerol biosynthesis pathway and lipid transport were significantly down-regulated by ALA treatment. Furthermore, ALA significantly decreased the mRNA expressions of sterol regulatory element binding protein (SREBP)-2, SREBP-1a, SREBP-1c and fatty acid synthase (FAS) in 3T3-L1 adipocyte cells. On the other hand, the average levels of the gene expressions of carnitine palmitoyltransferase 1a (CPT-1a) and leptin in 300 µM ALA treatment were increased by 1.7- and 2.9-fold, respectively, followed by an increase in the intracellular ATP content. These results show that ALA is likely to inhibit cholesterol and fatty acid biosynthesis pathway by suppressing the expression of transcriptional factor SREBPs. Furthermore, ALA promotes fatty acid oxidation in 3T3-L1 adipocytes, thereby increasing its health benefits.

10.
J Biol Chem ; 283(8): 5178-87, 2008 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-18083709

RESUMO

The LysM domain probably binds peptidoglycans, but how it does so has yet to be described. For this report, we measured the thermal stabilities of recombinant LysM domains derived from Pteris ryukyuensis chitinase-A (PrChi-A) and monitored their binding to N-acetylglucosamine oligomers ((GlcNAc)n) using differential scanning calorimetry, isothermal titration calorimetry, and NMR spectroscopy. We thereby characterized certain of the domains' functional and structural features. We observed that the domains are very resistant to thermal denaturation and that this resistance depends on the presence of disulfide bonds. We also show that the stoichiometry of (GlcNAc)n/LysM domain binding is 1:1. (GlcNAc)5 titration experiments, monitored by NMR spectroscopy, allowed us to identify the domain residues that are critical for (GlcNAc)5 binding. The binding site is a shallow groove formed by the N-terminal part of helix 1, the loop between strand 1 and helix 1, the C-terminal part of helix 2, and the loop between helix 2 and strand 2. Furthermore, mutagenesis experiments reiterate the critical involvement of Tyr72 in (GlcNAc)n/LysM domain binding. Ours is the first report describing the physical structure of a LysM oligosaccharide-binding site based on experimental data.


Assuntos
Acetilglucosamina/química , Quitinases/química , Oligossacarídeos/química , Proteínas de Plantas/química , Pteridium/enzimologia , Acetilglucosamina/metabolismo , Substituição de Aminoácidos , Quitinases/genética , Quitinases/metabolismo , Dissulfetos/química , Dissulfetos/metabolismo , Ressonância Magnética Nuclear Biomolecular , Oligossacarídeos/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ligação Proteica/fisiologia , Desnaturação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína/fisiologia , Pteridium/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade
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