RESUMEN
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.
Asunto(s)
Proteínas Fúngicas/química , Glutatión Transferasa/química , Phanerochaete/enzimología , Terpenos/metabolismo , Tetralonas/metabolismo , Dominio Catalítico , Cromatografía Líquida de Alta Presión , Compuestos Cromogénicos , Cisteína/química , Fagus/química , Fluoresceínas , Proteínas Fúngicas/genética , Glutatión Transferasa/genética , Proteínas Inmovilizadas/química , Proteínas Inmovilizadas/genética , Isoenzimas/química , Isoenzimas/genética , Cinética , Phanerochaete/química , Extractos Vegetales/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Sefarosa , Espectrometría de Fluorescencia , Especificidad por SustratoRESUMEN
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.
Asunto(s)
Glutatión Transferasa/química , Glutatión Transferasa/metabolismo , Phanerochaete/metabolismo , Naftalenosulfonatos de Anilina/farmacología , Sitios de Unión , Unión Competitiva , Biotecnología/métodos , Clonación Molecular , Cristalografía por Rayos X/métodos , Glutatión/química , Lignina , Espectrometría de Masas/métodos , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Isoformas de Proteínas , Proteínas Recombinantes/químicaRESUMEN
Reactive oxygen species fulfill key roles in development and signaling, but lead at high concentration to damage in macromolecules. In proteins, methionine (Met) is particularly prone to oxidative modification and can be oxidized into Met sulfoxide (MetO). MetO reduction is catalyzed by specialized enzymes, termed methionine sulfoxide reductases (MSRs), involved in senescence and protection against diseases and environmental constraints. The precise physiological functions of MSRs remain often elusive because of very poor knowledge of their substrates. In this study, affinity chromatography was used to isolate partners of Arabidopsis thaliana plastidial methionine sulfoxide reductase B1 (MSRB1). Twenty-four proteins involved in photosynthesis, translation, and protection against oxidative stress, as well as in metabolism of sugars and amino acids, were identified. Statistical analysis shows that the abundance of MSRB1 partners in chromatography affinity samples is proportional to Met content. All proteins, for which structural modeling was feasible, display surface-exposed Met and are thus potentially susceptible to oxidation. Biochemical analyses demonstrated that H(2)O(2) treatment actually converts several MSRB1-interacting proteins into MSRB substrates. In consequence, we propose that affinity chromatography constitutes an efficient tool to isolate physiological targets of MSRs.
Asunto(s)
Proteínas de Arabidopsis/aislamiento & purificación , Proteínas de Arabidopsis/metabolismo , Cromatografía de Afinidad/métodos , Metionina Sulfóxido Reductasas/metabolismo , Arabidopsis/metabolismo , Proteínas de Cloroplastos/metabolismo , Oxidación-Reducción , Factor Tu de Elongación Peptídica/metabolismo , Unión ProteicaRESUMEN
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.