RESUMO
GSTs represent a superfamily of multifunctional proteins which play crucial roles in detoxification processes and secondary metabolism. Instead of promoting the conjugation of glutathione to acceptor molecules as do most GSTs, members of the Lambda class (GSTLs) catalyse deglutathionylation reactions via a catalytic cysteine residue. Three GSTL genes (Pt-GSTL1, Pt-GSTL2 and Pt-GSTL3) are present in Populus trichocarpa, but two transcripts, differing in their 5' extremities, were identified for Pt-GSTL3. Transcripts for these genes were primarily found in flowers, fruits, petioles and buds, but not in leaves and roots, suggesting roles associated with secondary metabolism in these organs. The expression of GFP-fusion proteins in tobacco showed that Pt-GSTL1 is localized in plastids, whereas Pt-GSTL2 and Pt-GSTL3A and Pt-GSTL3B are found in both the cytoplasm and the nucleus. The resolution of Pt-GSTL1 and Pt-GSTL3 structures by X-ray crystallography indicated that, although these proteins adopt a canonical GST fold quite similar to that found in dimeric Omega GSTs, their non-plant counterparts, they are strictly monomeric. This might explain some differences in the enzymatic properties of both enzyme types. Finally, from competition experiments between aromatic substrates and a fluorescent probe, we determined that the recognition of glutathionylated substrates is favoured over non-glutathionylated forms.
Assuntos
Glutationa Transferase/química , Núcleo Celular/enzimologia , Cristalografia por Raios X , Citoplasma/enzimologia , Genes de Plantas , Glutationa/análogos & derivados , Glutationa/metabolismo , Glutationa Transferase/metabolismo , Cinética , Populus/enzimologia , Populus/genética , Dobramento de Proteína , Multimerização Proteica , Especificidade por SubstratoRESUMO
Glutathione S-transferases (GSTs) form a superfamily of multifunctional proteins with essential roles in cellular detoxification processes. A new fungal specific class of GST has been highlighted by genomic approaches. The biochemical and structural characterization of one isoform of this class in Phanerochaete chrysosporium revealed original properties. The three-dimensional structure showed a new dimerization mode and specific features by comparison with the canonical GST structure. An additional ß-hairpin motif in the N-terminal domain prevents the formation of the regular GST dimer and acts as a lid, which closes upon glutathione binding. Moreover, this isoform is the first described GST that contains all secondary structural elements, including helix α4' in the C-terminal domain, of the presumed common ancestor of cytosolic GSTs (i.e. glutaredoxin 2). A sulfate binding site has been identified close to the glutathione binding site and allows the binding of 8-anilino-1-naphtalene sulfonic acid. Competition experiments between 8-anilino-1-naphtalene sulfonic acid, which has fluorescent properties, and various molecules showed that this GST binds glutathionylated and sulfated compounds but also wood extractive molecules, such as vanillin, chloronitrobenzoic acid, hydroxyacetophenone, catechins, and aldehydes, in the glutathione pocket. This enzyme could thus function as a classical GST through the addition of glutathione mainly to phenethyl isothiocyanate, but alternatively and in a competitive way, it could also act as a ligandin of wood extractive compounds. These new structural and functional properties lead us to propose that this GST belongs to a new class that we name GSTFuA, for fungal specific GST class A.
Assuntos
Glutationa Transferase/química , Glutationa Transferase/metabolismo , Phanerochaete/metabolismo , Naftalenossulfonato de Anilina/farmacologia , Sítios de Ligação , Ligação Competitiva , Biotecnologia/métodos , Clonagem Molecular , Cristalografia por Raios X/métodos , Glutationa/química , Lignina , Espectrometria de Massas/métodos , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Isoformas de Proteínas , Proteínas Recombinantes/químicaRESUMO
The white rot fungus Phanerochaete chrysosporium, a saprophytic basidiomycete, possesses a large number of cytosolic glutathione transferases, eight of them showing similarity to the Omega class. PcGSTO1 (subclass I, the bacterial homologs of which were recently proposed, based on their enzymatic function, to constitute a new class of glutathione transferase named S-glutathionyl-(chloro)hydroquinone reductases) and PcGSTO3 (subclass II related to mammalian homologs) have been investigated in this study. Biochemical investigations demonstrate that both enzymes are able to catalyze deglutathionylation reactions thanks to the presence of a catalytic cysteinyl residue. This reaction leads to the formation of a disulfide bridge between the conserved cysteine and the removed glutathione from their substrate. The substrate specificity of each isoform differs. In particular PcGSTO1, in contrast to PcGSTO3, was found to catalyze deglutathionylation of S-glutathionyl-p-hydroquinone substrates. The three-dimensional structure of PcGSTO1 presented here confirms the hypothesis that it belongs not only to a new biological class but also to a new structural class that we propose to name GST xi. Indeed, it shows specific features, the most striking ones being a new dimerization mode and a catalytic site that is buried due to the presence of long loops and that contains the catalytic cysteine.
Assuntos
Basidiomycota/enzimologia , Proteínas Fúngicas/química , Glutationa Transferase/química , Multimerização Proteica , Dissulfetos/química , Proteínas Fúngicas/classificação , Glutationa Transferase/classificação , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Especificidade por SubstratoRESUMO
Glutathionyl-hydroquinone reductases (GHRs) catalyze the deglutathionylation of quinones via a catalytic cysteine. The two GHR genes in the Populus trichocarpa genome, Pt-GHR1 and Pt-GHR2, are primarily expressed in reproductive organs. Both proteins are localized in plastids. More specifically, Pt-GHR2 localizes in nucleoids. At the structural level, Pt-GHR1 adopts a typical GHR fold, with a dimerization interface comparable to that of the bacterial and fungal GHR counterparts. Pt-GHR1 catalyzes the deglutathionylation of both reduced and oxidized glutathionylated quinones, but the enzyme is more catalytically efficient with the reduced forms.
Assuntos
Proteínas de Cloroplastos/metabolismo , Oxirredutases/metabolismo , Populus/enzimologia , Dobramento de Proteína , Multimerização Proteica/fisiologia , Domínio Catalítico , Proteínas de Cloroplastos/química , Proteínas de Cloroplastos/genética , Oxirredução , Oxirredutases/química , Oxirredutases/genética , Populus/genéticaRESUMO
Omega glutathione transferases (GSTO) constitute a family of proteins with variable distribution throughout living organisms. It is notably expanded in several fungi and particularly in the wood-degrading fungus Phanerochaete chrysosporium, raising questions concerning the function(s) and potential redundancy of these enzymes. Within the fungal families, GSTOs have been poorly studied and their functions remain rather sketchy. In this study, we have used fluorescent compounds as activity reporters to identify putative ligands. Experiments using 5-chloromethylfluorescein diacetate as a tool combined with mass analyses showed that GSTOs are able to cleave ester bonds. Using this property, we developed a specific activity-based profiling method for identifying ligands of PcGSTO3 and PcGSTO4. The results suggest that GSTOs could be involved in the catabolism of toxic compounds like tetralone derivatives. Biochemical investigations demonstrated that these enzymes are able to catalyze deglutathionylation reactions thanks to the presence of a catalytic cysteine residue. To access the physiological function of these enzymes and notably during the wood interaction, recombinant proteins have been immobilized on CNBr Sepharose and challenged with beech wood extracts. Coupled with GC-MS experiments this ligand fishing method allowed to identify terpenes as potential substrates of Omega GST suggesting a physiological role during the wood-fungus interactions.
Assuntos
Proteínas Fúngicas/química , Glutationa Transferase/química , Phanerochaete/enzimologia , Terpenos/metabolismo , Tetralonas/metabolismo , Domínio Catalítico , Cromatografia Líquida de Alta Pressão , Compostos Cromogênicos , Cisteína/química , Fagus/química , Fluoresceínas , Proteínas Fúngicas/genética , Glutationa Transferase/genética , Proteínas Imobilizadas/química , Proteínas Imobilizadas/genética , Isoenzimas/química , Isoenzimas/genética , Cinética , Phanerochaete/química , Extratos Vegetais/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Sefarose , Espectrometria de Fluorescência , Especificidade por SubstratoRESUMO
Fungal degradation of wood is mainly restricted to basidiomycetes, these organisms having developed complex oxidative and hydrolytic enzymatic systems. Besides these systems, wood-decaying fungi possess intracellular networks allowing them to deal with the myriad of potential toxic compounds resulting at least in part from wood degradation but also more generally from recalcitrant organic matter degradation. The members of the detoxification pathways constitute the xenome. Generally, they belong to multigenic families such as the cytochrome P450 monooxygenases and the glutathione transferases. Taking advantage of the recent release of numerous genomes of basidiomycetes, we show here that these multigenic families are extended and functionally related in wood-decaying fungi. Furthermore, we postulate that these rapidly evolving multigenic families could reflect the adaptation of these fungi to the diversity of their substrate and provide keys to understand their ecology. This is of particular importance for white biotechnology, this xenome being a putative target for improving degradation properties of these fungi in biomass valorization purposes.
Assuntos
Adaptação Fisiológica/genética , Basidiomycota/enzimologia , Sistema Enzimático do Citocromo P-450/metabolismo , Glutationa Transferase/metabolismo , Redes e Vias Metabólicas/genética , Madeira/química , Madeira/microbiologia , Basidiomycota/genética , Basidiomycota/metabolismo , Basidiomycota/fisiologia , Biodegradação Ambiental , Sistema Enzimático do Citocromo P-450/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genoma Fúngico , Glutationa Transferase/genética , Madeira/metabolismo , Xenobióticos/metabolismoRESUMO
SpLigG is one of the three glutathione transferases (GSTs) involved in the process of lignin breakdown in the soil bacterium Sphingobium sp. SYK-6. Sequence comparisons showed that SpLigG and several proteobacteria homologues form an independent cluster within cysteine-containing GSTs. The relationship between SpLigG and other GSTs was investigated. The X-ray structure and biochemical properties of SpLigG indicate that this enzyme belongs to the omega class of glutathione transferases. However, the hydrophilic substrate binding site of SpLigG, together with its known ability to stereoselectively deglutathionylate the physiological substrate α-glutathionyl-ß-hydroxypropiovanillone, argues for broadening the definition of the omega class.