Differences in salicylic acid glucose conjugations by UGT74F1 and UGT74F2 from Arabidopsis thaliana

Abstract

Salicylic acid (SA) is a signaling molecule utilized by plants in response to various stresses. Through conjugation with small organic molecules such as glucose, an inactive form of SA is generated which can be transported into and stored in plant vacuoles. In the model organism Arabidopsis thaliana, SA glucose conjugates are formed by two homologous enzymes (UGT74F1 and UGT74F2) that transfer glucose from UDP-glucose to SA. Despite being 77% identical and with conserved active site residues, these enzymes catalyze the formation of different products: UGT74F1 forms salicylic acid glucoside (SAG), while UGT74F2 forms primarily salicylic acid glucose ester (SGE). The position of the glucose on the aglycone determines how SA is stored, further metabolized, and contributes to a defense response. We determined the crystal structures of the UGT74F2 wild-type and T15S mutant enzymes, in different substrate/product complexes. On the basis of the crystal structures and the effect on enzyme activity of mutations in the SA binding site, we propose the catalytic mechanism of SGE and SAG formation and that SA binds to the active site in two conformations, with each enzyme selecting a certain binding mode of SA. Additionally, we show that two threonines are key determinants of product specificity.

Comparative genomic and transcriptomic analyses of Family-1 UDP glycosyltransferase in three Brassica species and Arabidopsis indicates stress-responsive regulation

“…In contrast, UGT74F2 mutants have higher levels of SA and higher levels of resistance to P. syringae . Similarly, overexpression of UGT74F2 (also annotated as AtSGT1 ) results in lower levels of SA and an increased susceptibility to P. syringae 26 – 28 . According to the results of a recent study, the expression of a BSMT gene in B. oleracea appears to be involved in glycosylation rather than methyl jasmonate (MeJA) biosynthesis during Plasmodiophora brassicae infection 29 .…”

Molecular basis for branched steviol glucoside biosynthesis

“…The 3D structure of UGT76G1 shares 2.2-2.9 Å 2 root mean square deviations (rmsds) for 420-440 C α atoms with the other structurally characterized plant UGT. These include enzymes that modify terpenoids (Medicago truncatula/barrel clover UGT71G1 and Orzya sativa/rice Os79), flavonoids and isoflavonoids (Vitis vinifera/grape GT1, M. truncatula UGT85H2 and UGT78G1), chlorinated phenols (Arabidopsis thaliana/ thale cress UGT72B1), anthocyanin floral pigments (Clitoria ternatea/bluebell vine UGT78K6), salicylic acid (A. thaliana UGT74F2), and indoxyl dyes (Polygonum tinctorium/Japanese indigo GT-B) (15)(16)(17)(18)(19)(20)(21)(22)(23); however, all of these UGT transfer sugars directly to the aglycone and do not form branched natural product glucosides. The uridine ring of UDP is sandwiched between Trp338 and Gln341 with hydrogen bond interactions contributed by Val339, Pro340, and a water-mediated interaction with His260 ( Fig.…”

“…Structural and sequence comparison of UGT76G1, a branchforming glycosyltransferase, to the plant UGTs that directly glycosylate a given substrate (i.e., terpenoids, flavonoids, phenols, anthocyanins, and indoxyls; refs. [15][16][17][18][19][20][21][22][23] highlights key differences in residues forming the glc2 site (SI Appendix, Fig. S2 D-G).…”

Mechanistic differences in the uptake of salicylic acid glucose conjugates by vacuolar membrane‐enriched vesicles isolated from Arabidopsis thaliana

“…The SA UGT assays were performed as described by Edwards (1994) with the modifications described by Dean and Delaney (2008). In vitro SGE formation during SA UGT assays occurs optimally at pH 6.0 while SAG formation occurs faster at pH values of 7 or higher (Thompson et al 2017). Therefore, the pH of the extraction media was either 6.0 or 7.0 depending on the product being measured.…”