Structural and enzymatic insights into Lambda glutathione transferases from Populus trichocarpa, monomeric enzymes constituting an early divergent class specific to terrestrial plants

Abstract

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.

The still mysterious roles of cysteine-containing glutathione transferases in plants

“…This capacity usually originates from the replacement of the catalytic serine or tyrosine residues in the active site motif by a cysteinyl residue as demonstrated for mammalian, insect and fungal Omega GSTs (GSTOs) (Board et al, 2000; Kim et al, 2006; Yamamoto et al, 2009; Meux et al, 2013), for plant Lambda GSTs (GSTLs) (Dixon and Edwards, 2010b; Lallement et al, 2014) and for bacterial and fungal glutathionyl hydroquinone reductases (GHRs) (Xun et al, 2010; Meux et al, 2011). However, the physiological functions of these enzymes have rarely been elucidated.…”

“…(A) Amino acid sequence alignment of three representative members from each Cys-GST class. The sequences were structurally aligned using PROMALS3D server using as references the solved structures of PtGSTL1 [PDB code 4PQH (Lallement et al, 2014)], PtGSTL3 [PDB code 4PQI (Lallement et al, 2014)], Phanerochaete chrysosporium GHR1 [PDB code 3PPU (Meux et al, 2011)], and Macaca fascicularis mPGES-2 [PDB code 1Z9H (Yamada et al, 2005)] since there is no structure available for DHARs, GSTIs, and GSTHs. Since the structure of poplar GSTL1 has been solved, its secondary structures have been indicated as reference using ESPript 3.0 (http://espript.ibcp.fr/ESPript/ESPript/index.php), with the helices and the arrows corresponding respectively to α-helices and to β-strands.…”

“…Consistently, Arabidopsis transgenic lines expressing OsGSTL2 are more tolerant to abiotic stresses such as heavy metals, cold, drought, and salt stress (Kumar et al, 2013a,b). Recently, the expression of the three poplar GSTL genes was studied in a naturally growing Populus trichocarpa adult tree (Lallement et al, 2014). One of these genes, PtGSTL3 , generates two transcripts by alternative splicing, PtGSTL3A and PtGSTL3B , the latter being very weakly expressed.…”

“…One of these genes, PtGSTL3 , generates two transcripts by alternative splicing, PtGSTL3A and PtGSTL3B , the latter being very weakly expressed. While PtGSTL2 and PtGSTL3A seem to be constitutively expressed, all PtGSTL genes are preferentially expressed in the reproductive organs (flowers, fruits, buds) (Lallement et al, 2014). Nevertheless, poplar GSTLs have also been detected in leaves and roots (Lan et al, 2009).…”

Structural and functional characterization of tree proteins involved in redox regulation: a new frontier in forest science

“…Three GSTs of the lambda type (GSTL1 to L3) have been recently characterized until the molecular level. The proteins are monomeric and they bind glutathionylated substrates for deglutathionylation reactions (Lallement et al 2014b). Overall, this study strengthened the proposal that GSTLs are involved in the management of secondary metabolites via their redox properties.…”