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.
Highlights
Salicylic acid (SA) is a plant hormone involved in regulating plant stress responses including local and systemic pathogen responses; UV-C stress, osmotic stress, high light stress, heat, heavy metals, drought, and chilling[1,2,3,4,5,6]
One example of this conjugation is the formation of SA: glucose conjugates, of which two forms are possible: either an SA-glucoside (SAG) – where glucose is conjugated to the hydroxyl group, or an SA glucose ester (SGE) – with glucose attached to the carboxylate group[8,9,10,11,12,13,14] (SAG and SGE structures, Fig. 1a)
UGT74F1 and UGT74F2 are the enzymes responsible for the in vivo glucosylation of SA in A. thaliana. Homologs of these proteins are present in many other plant species, including fruiting trees, citrus trees and the Brassica genus, suggesting that these enzymes and their products work together in plant immune responses
Summary
Salicylic acid (SA) is a plant hormone involved in regulating plant stress responses including local and systemic pathogen responses; UV-C stress, osmotic stress, high light stress, heat, heavy metals, drought, and chilling[1,2,3,4,5,6]. Because of its reactivity and lipophilicity, small hydrophilic molecules are often added to SA to aid in transport and storage One example of this conjugation is the formation of SA: glucose conjugates, of which two forms are possible: either an SA-glucoside (SAG) – where glucose is conjugated to the hydroxyl group, or an SA glucose ester (SGE) – with glucose attached to the carboxylate group[8,9,10,11,12,13,14] (SAG and SGE structures, Fig. 1a). In Arabidopsis thaliana, SA glucosylation in vivo is performed by two UDP-glucosyltransferase (UGT) enzymes: UGT74F1 and UGT74F215,16 Deletion of these two enzymes at the gene level results in different phenotypes in response to bacterial infection. In vivo evidence indicates that UGT74F2 produces anthranilate glucose ester[19]
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