Structural and functional evolution of positively selected sites in pine glutathione S-transferase enzyme family

Abstract

Phylogenetic analyses have identified positive selection as an important driver of protein evolution, both structural and functional. However, the lack of appropriate combined functional and structural assays has generally hindered attempts to elucidate patterns of positively selected sites and their effects on enzyme activity and substrate specificity. In this study we investigated the evolutionary divergence of the glutathione S-transferase (GST) family in Pinus tabuliformis, a pine that is widely distributed from northern to central China, including cold temperate and drought-stressed regions. GSTs play important roles in plant stress tolerance and detoxification. We cloned 44 GST genes from P. tabuliformis and found that 26 of the 44 belong to the largest (Tau) class of GSTs and are differentially expressed across tissues and developmental stages. Substitution models identified five positively selected sites in the Tau GSTs. To examine the functional significance of these positively selected sites, we applied protein structural modeling and site-directed mutagenesis. We found that four of the five positively selected sites significantly affect the enzyme activity and specificity; thus their variation broadens the GST family substrate spectrum. In addition, positive selection has mainly acted on secondary substrate binding sites or sites close to (but not directly at) the primary substrate binding site; thus their variation enables the acquisition of new catalytic functions without compromising the protein primary biochemical properties. Our study sheds light on selective aspects of the functional and structural divergence of the GST family in pine and other organisms.

Keywords: Enzyme Mutation; Molecular Evolution; Plant Biochemistry; Protein Evolution; Protein Structure.

FIGURE 1.

Phylogenetic relationships of P. tabuliformis GSTs (A) and their expression patterns (B). Numbers at each node in the phylogenetic tree signify bootstrap values, and only bootstrap values >50% are shown. GST genes designated GSTU, -F, -T, -Z, and -L correspond to Tau, Phi, Theta, Zeta, and Lambda class GSTs, respectively. The gray box indicates positive detection of gene expression in the corresponding tissue under normal growth conditions.

FIGURE 2.

Phylogenetic tree of 244 Tau GSTs from S. moellendorffii (purple triangle), O. sativa (red triangle), A. thaliana (blue triangle), P. trichocarpa (green triangle), P. tabuliformis (Pta), P. taeda (Pte), G. gnemon (Gg), G. biloba (Gb), and C. rumphii (Cr). Thick branches indicate clades with strong statistical support (>50%). The protein sequences used for the tree reconstruction are provided in supplemental Table S5.

FIGURE 3.

Schematic representation of the duplication history of Tau GSTs in land plants. Gene loss events are indicated by dashed lines. The designation of each clade corresponds to the Fig. 2.

FIGURE 4.

Sequence alignment of P. tabuliformis Tau GSTs and predicted secondary structure elements. α-Helices and β strands are represented as blue cylinders and yellow arrows, respectively. Residues conserved in all P. tabuliformis Tau GSTs are shaded gray and black. The five positive selection sites predicted by the branch-site model test are colored red. Predicted G-sites and H-sites are marked with pink and blue arrows, respectively.

FIGURE 5.

Structural comparison of the P. tabuliformis Tau GSTs. A, shown is structural superposition of the 26 Tau GSTs. B, shown are predicted ligand binding cavities (blue dots) in the PtaGSTU17 model by Binding Site Analysis. C, shown are fluorescence emission spectra of ANS binding to the W171L, W171F, and W171A mutants and wild-type PtaGSTU17. In A and B, the N- and C-terminal domains are illustrated in green and red, respectively, and the linker between the two domains is shown in white. In A, positions of the positively selected sites listed in Table 1 are indicated by white arrows; the GSH binding sites are indicated by purple arrows. In B, positions of Lys-119 and Trp-171 residues of PtaGSTU17 are shown in purple.

FIGURE 6.

Enzymatic activity changes, relative to wild-type PtaGSTU17 protein, induced by mutations at the five selected sites. The boxplot shows the median (black line), interquartile range (box), and maximum and minimum scores (whiskers) for each data set. Outliers are shown as circles outside the whiskers.