Discovery of specific catalytic activity toward IAA/FA by LaSABATHs based on genome-wide phylogenetic and enzymatic analysis of SABATH gene family from Larix kaempferi.

The SABATH methyltransferases catalyze methylation of small-molecule metabolites, which participate in plant growth, development and defense response. Given lack of genome-wide studies on gymnosperms SABATH family, the formation and functional differentiation mechanism of the Larix kaempferi SABATH gene family was systematically and exhaustively explored by analyzing gene sequence characteristics, phylogenetic relationship, expression pattern, and enzyme activities. Phylogenetic analysis showed that 247 SABATH genes from 14 land plants were divided into 4 clades, and lineage-specific gene duplication events were important factors that contributed to the evolution of the SABATH gene family in gymnosperms and angiosperms. Substrate specificity analysis of 18 Larix SABATH proteins showed that LaSABATHs could catalyze O-methylation of indole-3-acetic acid (IAA) and farnesic acid (FA), N-methylation of theobromine, and S-methylation of thiobenzoic acid. Furthermore, only LaSABATH2 and LaSABATH29 could catalyze O-methylation of FA, and only LaSABATH30 could catalyze O-methylation of IAA. Homology modeling and molecular docking studies showed the hydrogen bond formed between the His188 of LaSABATH30 and IAA and the noticeable hydrophobic IAA-binding pocket may be helpful for IAA methylation. In this study, identification of proteins with significant specific catalytic activity toward FA and IAA provided high-quality candidate genes for forest genetics and breeding.

Non-synonymous substitution of evolutionarily conserved residue in Tau class glutathione transferases alters structural and catalytic features.

Plant-specific tau glutathione transferases (GSTs) are basically involved in catalysing γ-glutathione (GSH)-dependent conjugation reactions with pesticides and herbicides, which play an important role in the detoxification of pollutants. Given the lack of systematic biochemical and structural information on tau GSTs, the study of their mediated defence mechanisms against toxic compounds has been greatly hindered. Here, we reveal the importance of the Ile residue closely interacting with GSH for the structural stability and catalytic function of GST. Evolutionary conservation analysis indicated that the crucial G-site Ile55 in the SbGSTU6 was converted to Thr53 of SbGSTU7. The comparative biochemical data on SbGSTU6, SbGSTU7 and their mutants showed that the substitution of Ile by Thr caused significant decrease in the affinity and catalytic efficiency of the GSTs. The unfavourable structural flexibility and pKa distribution of the active cavity residues were also demonstrated. Crystallography studies and molecular dynamics simulations showed that the conversion resulted in the hydrogen bond recombination with GSH and conformational rearrangement of GST active cavity, in which the Ile residue was more conducive to the formation of enzyme substrate complexes. The extensive biochemical and structural data not only reveal the critical role of the conserved G-site Ile residue in catalysing GSH-conjugate reactions but also provide valuable resources for the development of GST engineering in analytical and agricultural biotechnology.

Biochemical Functions of Glutathione S-Transferase Family of Salix babylonica.

Glutathione S-transferases (GSTs) are ubiquitous enzymes that are encoded by a large gene family, and they contribute to the detoxification of endogenous or xenobiotic compounds and oxidative stress metabolism in plants. Although the GSTs gene family has been reported in many land plants, our knowledge of the evolution and function of the willow GSTs is still limited. In this study, 22 full-length GST genes were cloned from Salix babylonica and divided into three classes based on the conserved domain analysis, phylogenetic tree and gene structure: tau, phi and DHAR. The tissue-specific expression patterns were substantially different among the tau and phi GSTs. The Salix GST proteins showed functional divergences in the substrate specificities, substrate activities and kinetic characteristics. The site-directed mutagenesis studies revealed that a single amino acid mutation (Ile/Val53→Thr53) resulted in the lowest activity of SbGSTU7 among the Salix GSTs. These results suggest that non-synonymous substitution of an amino acid at the putative glutathione-binding site may play an important role in the divergence of enzymatic functions of Salix GST family.