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1.
Arch Biochem Biophys ; 691: 108509, 2020 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-32717225

RESUMO

Biotin protein ligase (BPL) is an essential enzyme in all kingdoms of life, making it a potential target for novel anti-infective agents. Whilst bacteria and archaea have simple BPL structures (class I and II), the homologues from certain eukaryotes such as mammals, insects and yeast (class III) have evolved a more complex structure with a large extension on the N-terminus of the protein in addition to the conserved catalytic domain. The absence of atomic resolution structures of any class III BPL hinders structural and functional analysis of these enzymes. Here, two new class III BPLs from agriculturally important moulds Botrytis cinerea and Zymoseptoria tritici were characterised alongside the homologue from the prototypical yeast Saccharomyces cerevisiae. Circular dichroism and ion mobility-mass spectrometry analysis revealed conservation of the overall tertiary and secondary structures of all three BPLs, corresponding with the high sequence similarity. Subtle structural differences were implied by the different thermal stabilities of the enzymes and their varied Michaelis constants for their interactions with ligands biotin, MgATP, and biotin-accepting substrates from different species. The three BPLs displayed different preferences for fungal versus bacterial protein substrates, providing further evidence that class III BPLs have a 'substrate validation' activity for selecting only appropriate proteins for biotinylation. Selective, potent inhibition of these three BPLs was demonstrated despite sequence and structural homology. This highlights the potential for targeting BPL for novel, selective antifungal therapies against B. cinerea, Z. tritici and other fungal species.


Assuntos
Carbono-Nitrogênio Ligases/química , Proteínas Fúngicas/química , Ascomicetos/enzimologia , Botrytis/enzimologia , Carbono-Nitrogênio Ligases/antagonistas & inibidores , Inibidores Enzimáticos/química , Proteínas Fúngicas/antagonistas & inibidores , Conformação Proteica , Estabilidade Proteica , Desdobramento de Proteína , Saccharomyces cerevisiae/enzimologia , Especificidade por Substrato
2.
Plant Cell ; 24(8): 3307-19, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22892320

RESUMO

Gibberellins (GAs) are plant hormones involved in the regulation of plant growth in response to endogenous and environmental signals. GA promotes growth by stimulating the degradation of nuclear growth-repressing DELLA proteins. In Arabidopsis thaliana, DELLAs consist of a small family of five proteins that display distinct but also overlapping functions in repressing GA responses. This study reveals that DELLA RGA-LIKE3 (RGL3) protein is essential to fully enhance the jasmonate (JA)-mediated responses. We show that JA rapidly induces RGL3 expression in a CORONATINE INSENSITIVE1 (COI1)- and JASMONATE INSENSITIVE1 (JIN1/MYC2)-dependent manner. In addition, we demonstrate that MYC2 binds directly to RGL3 promoter. Furthermore, we show that RGL3 (like the other DELLAs) interacts with JA ZIM-domain (JAZ) proteins, key repressors of JA signaling. These findings suggest that JA/MYC2-dependent accumulation of RGL3 represses JAZ activity, which in turn enhances the expression of JA-responsive genes. Accordingly, we show that induction of primary JA-responsive genes is reduced in the rgl3-5 mutant and enhanced in transgenic lines overexpressing RGL3. Hence, RGL3 positively regulates JA-mediated resistance to the necrotroph Botrytis cinerea and susceptibility to the hemibiotroph Pseudomonas syringae. We propose that JA-mediated induction of RGL3 expression is of adaptive significance and might represent a recent functional diversification of the DELLAs.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Ciclopentanos/farmacologia , Oxilipinas/farmacologia , Transdução de Sinais , Adaptação Biológica , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Botrytis/patogenicidade , Ciclopentanos/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Giberelinas/farmacologia , Imunoprecipitação , Oxilipinas/metabolismo , Doenças das Plantas/microbiologia , Plantas Geneticamente Modificadas/efeitos dos fármacos , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/microbiologia , Regiões Promotoras Genéticas , Ligação Proteica , Mapeamento de Interação de Proteínas , Pseudomonas syringae/patogenicidade , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Técnicas do Sistema de Duplo-Híbrido
3.
Plant Mol Biol ; 54(1): 137-46, 2004 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15159640

RESUMO

Nicotiana tabacum Togt encodes a scopoletin glucosyltransferase (UDPglucose:scopoletin O -beta-D-glucosyltrans- ferase, EC 2.4.1.128) known to act in vitro on many different substrates including the 6-methoxy-7-hydroxy- coumarin scopoletin. This phenolic compound accumulates in vast amounts, essentially in its glucosylated form scopolin, in tobacco during the hypersensitive response (HR) to tobacco mosaic virus (TMV). To identify the physiological role of this pathogen-inducible UDP-Glc glucosyltransferase (UGT), we generated TOGT over-expressing transgenic plants. Although no endogenous scopoletin or scopolin could be detected before infection, the accumulation of both the aglycone and the glucoside was found to be 2-fold higher in transgenic plants after inoculation with TMV than in wild-type plants. Scopoletin UGT activity in plants over-expressing Togt was significantly higher during the HR than in control plants. This up-regulated activity was associated with a strong increase of the bright blue fluorescence surrounding the HR-necrotic lesions under UV light, which is known to correlate with scopoletin and scopolin abundance. Necrosis appeared sooner in transgenic plants and lesions developed faster, suggesting an accelerated HR. Unexpectedly, the viral content in each lesion was not significantly different in transgenic and in wild-type plants. These results are discussed in relation to the role of TOGT as the major UDP-Glc: scopoletin glucosyltransferase and to the importance of scopoletin accumulation during the HR.


Assuntos
Glucosiltransferases/genética , Nicotiana/genética , Doenças das Plantas/genética , Vírus do Mosaico do Tabaco/crescimento & desenvolvimento , Cumarínicos/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Glucosídeos/metabolismo , Glucosiltransferases/metabolismo , Imunidade Inata/genética , Doenças das Plantas/virologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Folhas de Planta/virologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Escopoletina/metabolismo , Fatores de Tempo , Nicotiana/metabolismo , Nicotiana/virologia , Vírus do Mosaico do Tabaco/metabolismo
4.
Plant Cell ; 14(5): 1093-107, 2002 May.
Artigo em Inglês | MEDLINE | ID: mdl-12034899

RESUMO

Plant UDP-Glc:phenylpropanoid glucosyltransferases (UGTs) catalyze the transfer of Glc from UDP-Glc to numerous substrates and regulate the activity of compounds that play important roles in plant defense against pathogens. We previously characterized two tobacco salicylic acid- and pathogen-inducible UGTs (TOGTs) that act very efficiently on the hydroxycoumarin scopoletin and on hydroxycinnamic acids. To identify the physiological roles of these UGTs in plant defense, we generated TOGT-depleted tobacco plants by antisense expression. After inoculation with Tobacco mosaic virus (TMV), TOGT-inhibited plants exhibited a significant decrease in the glucoside form of scopoletin (scopolin) and a decrease in scopoletin UGT activity. Unexpectedly, free scopoletin levels also were reduced in TOGT antisense lines. Scopolin and scopoletin reduction in TOGT-depleted lines resulted in a strong decrease of the blue fluorescence in cells surrounding TMV lesions and was associated with weakened resistance to infection with TMV. Consistent with the proposed role of scopoletin as a reactive oxygen intermediate (ROI) scavenger, TMV also triggered a more sustained ROI accumulation in TOGT-downregulated lines. Our results demonstrate the involvement of TOGT in scopoletin glucosylation in planta and provide evidence of the crucial role of a UGT in plant defense responses. We propose that TOGT-mediated glucosylation is required for scopoletin accumulation in cells surrounding TMV lesions, where this compound could both exert a direct antiviral effect and participate in ROI buffering.


Assuntos
Glucosiltransferases/genética , Nicotiana/enzimologia , Doenças das Plantas/virologia , Ácido Clorogênico/metabolismo , Ácido Clorogênico/farmacologia , Cumarínicos/metabolismo , Cumarínicos/farmacologia , Regulação para Baixo , Indução Enzimática/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Glucosídeos/metabolismo , Glucosídeos/farmacologia , Glucosiltransferases/biossíntese , Peróxido de Hidrogênio/metabolismo , Imunidade Inata/genética , Dados de Sequência Molecular , Mutação , Estresse Oxidativo , Fenilalanina Amônia-Liase/biossíntese , Fenilalanina Amônia-Liase/genética , Doenças das Plantas/genética , Folhas de Planta/virologia , Plantas Geneticamente Modificadas , Ácido Salicílico/metabolismo , Ácido Salicílico/farmacologia , Escopoletina/metabolismo , Escopoletina/farmacologia , Transdução de Sinais , Nicotiana/genética , Nicotiana/virologia , Vírus do Mosaico do Tabaco/efeitos dos fármacos , Vírus do Mosaico do Tabaco/crescimento & desenvolvimento
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