Fungal glutathione transferases
Fungi have a strong effect on plant health. On one hand, symbionts (such as mycorrhizae) can amplify yield of plant feedstock, on the other hand pathogens can have dramatic negative consequences on crops. The recent release of several fungi genome sequences allows understanding of the mechanisms of their interaction with plants and identifying the enzymes involved, for example, in the wood degradation process (Eastwood et al. 2011). Our analysis is focused on glutathione transferases (GSTs) which constitute a complex and widespread superfamily classified as enzymes of secondary metabolism. This project concerns the characterization of all GSTs found in the wood-degrading basidiomycete Phanerochaete chrysosporium by identifying their substrate specificities and by studying their structure-function relationships. Saprophytic fungi are of great interest for biotechnological applications such as wood preservation and resistance to fungicides. Our phylogenetic analysis of fungal GSTs revealed at least five classes of fungal GSTs : Omega, Ure2p, GTT1, GTT2 and GSTFuA, the last four being specific to fungi. We recently published the biochemical and the crystallographic studies of the PcGSTO1 which belongs to a new biological class and a new structural class, named Xi and also PcGSTFuA1,2 and 3 which are fungal specific enzymes with explicit ligandin properties.
Mathieu et al., (2013). Diversification of Fungal Specific Class A Glutathione Transferases in Saprotrophic Fungi. Plos One 8.
Glutathione transferases (GSTs) form a superfamily of multifunctional proteins with essential roles in cellular detoxification processes. The distribution of the fungal specific class A GST was investigated among saprotrophic fungi revealing a recent diversification within this class. Biochemical characterization of eight isoforms belonging to this class from Phanerochaete chrysosporium and Coprinus cinereus demonstrated functional divergence among wood decomposing fungi. The three-dimensional structure of three isoforms belonging to P. chrysosporium exhibited structural differences explaining the functional diversity of these enzymes. Competition experiments between fluorescent probes, and various molecules, showed that these GSTs display ligandin property, the L-site overlapping the glutathione binding pocket in all studied enzymes. These proteins are indeed able to bind various wood extractives molecules. By combining the genomic data with structural and biochemical properties we propose that this class of GST has evolved in response to environmental constraints induced by wood chemistry.
Mathieu et al., (2012). Characterization of a Phanerochaete chrysosporium Glutathione Transferase Reveals a Novel Structural and Functional Class with Ligandin Properties. Journal of Biological Chemistry 287, 39001-39011.
Glutathione S-transferases (GSTs) form a superfamily of multifunctional proteins with essential roles in cellular detoxification processes. Anew 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 beta-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 alpha 4′ in the C-terminal domain, of the pre-sumed 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.
Meux et al. (2011). Glutathione Transferases of Phanerochaete chrysosporium : S-Glutathionyl-p-hydroquinone reductase belongs to a new structural class Journal of Biological Chemistry 286, 9162-9173.
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.
Recent studies revealed that glutaredoxins (Grxs) constitute a multi-faceted protein family, involved not only in oxidative stress response but also in other essential functions such as iron-sulphur assembly and haem biosynthesis. These discoveries also led to propose a new classification extended to six classes (Couturier et al., Cell Mol Life Sci. 2009, 66, 2539-57). All Grxs structures reported so far are in fact class I or class II Grxs, with a Cxx[C/S] active site. One part of our project is to elucidate the structure of Grxs belonging to the unexplored classes III to VI. The class III Grxs are characterised by a CCxx active site and are found in higher plants only. The Grxs of class IV, V or VI contain one Grx domain fused with one or two domains of unknown function. Another aspect of our project is to study the structure of complexes between Grxs and their biological partners, in order to gain better insight into their roles in particular in plant iron homeostasis.
Couturier et al. (2011). Arabidopsis Chloroplastic Glutaredoxin C5 as a Model to Explore Molecular Determinants for Iron-Sulfur Cluster Binding into Glutaredoxins. Journal of Biological Chemistry 286, 27515-27527.
Unlike thioredoxins, glutaredoxins are involved in iron-sulfur cluster assembly and in reduction of specific disulfides i.e. protein-glutathione adducts, and thus they are also important redox regulators of chloroplast metabolism. Using GFP fusion, AtGrxC5 isoform, present exclusively in Brassicaceae, is shown to be localized in chloroplasts. A comparison of the biochemical, structural and spectroscopic properties of Arabidopsis GrxC5 (WCSYC active site) with poplar GrxS12 (WCSYS active site), a chloroplastic paralog, indicates that, contrary to the solely apomonomeric GrxS12 isoform, AtGrxC5 exists as two forms when expressed in Escherichia coli. The monomeric apoprotein possesses deglutathionylation activity mediating the recycling of plastidial methionine sulfoxide reductase B1 and peroxiredoxin IIE, whereas the dimeric holoprotein incorporates a [2Fe-2S] cluster. Site-directed mutagenesis experiments and resolution of the X-ray crystal structure of AtGrxC5 in its holoform revealed that, although not involved in its ligation, the presence of the second active site cysteine (Cys32) is important for cluster formation and stability. In addition, thiol titrations, fluorescence measurements and mass spectrometry analyses show that, despite the presence of a dithiol active site, AtGrxC5 does not form any inter- or intramolecular disulfide bond and that its activity relies on a monothiol mechanism.