New substrates and activity of Phanerochaete chrysosporium Omega glutathione transferases.
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.
UMR 1136 INRA-UHP Interactions Arbres/Micro-Organismes, IFR110 Ecosystèmes Forestiers, Agroressources, Bioprocédés et Alimentation, Université de Lorraine, Faculté des Sciences et Technologies, BP 70239, 54506 Vandoeuvre-les-Nancy, France.
Edgar
E
Meux
Mélanie
M
Morel
Tiphaine
T
Lamant
Philippe
P
Gérardin
Jean-Pierre
JP
Jacquot
Stéphane
S
Dumarçay
Eric
E
Gelhaye
D020134:Catalytic Domain; D002851:Chromatography, High Pressure Liquid; D002863:Chromogenic Compounds; D003545:Cysteine / Q000737:chemistry; D029964:Fagus / Q000737:chemistry; D005452:Fluoresceins; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics; D005982:Glutathione Transferase / Q000737:chemistry* / Q000235:genetics; D055767:Immobilized Proteins / Q000737:chemistry / Q000235:genetics; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics; D007700:Kinetics; D020075:Phanerochaete / Q000737:chemistry / Q000201:enzymology*; D010936:Plant Extracts / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012685:Sepharose; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013729:Terpenes / Q000378:metabolism*; D044024:Tetralones / Q000378:metabolism*
D002863:Chromogenic Compounds; D005452:Fluoresceins; D005656:Fungal Proteins; D055767:Immobilized Proteins; D007527:Isoenzymes; D010936:Plant Extracts; D011994:Recombinant Proteins; D013729:Terpenes; D044024:Tetralones; C069306:5-chloromethylfluorescein; D012685:Sepharose; D005982:Glutathione Transferase; D003545:Cysteine