Identification of a residue responsible for UDP-sugar donor selectivity of a dihydroxybenzoic acid glycosyltransferase from Arabidopsis natural accessions

UDP-glycosyltransferase (UGT) plays a major role in the diversity and reactivity of plant specialized metabolites by catalyzing the transfer of the sugar moiety from activated UDP-sugars to various acceptors. Arabidopsis UGT89A2 was previously identified from a genome-wide association study as a key factor that affects the differential accumulation of dihydroxybenzoic acid (DHBA) glycosides in distinct Arabidopsis natural accessions, including Col-0 and C24. The in vitro enzyme assays indicate that these distinct metabolic phenotypes reflect the divergence of UGT89A2 enzyme properties in the Col-0 and C24 accessions. UGT89A2 from Col-0 is highly selective toward UDP-xylose as the sugar donor, and the isoform from C24 can utilize both UDP-glucose and UDP-xylose but with a higher affinity to the glucose donor. The sequences of the two isozymes only differ at six amino acid residues. Examination of these amino acid residues in more natural accessions revealed a strong correlation between the amino acid polymorphism at position 153 and the DHBA glycoside accumulation pattern. Site-directed mutagenesis that swapped residue 153 between UGT89A2 from Col-0 and C24 reversed the UDP-sugar preferences, indicating that residue 153 plays an important role in determining sugar donor specificity of UGT89A2. This study provides insight into the key amino acid changes that confer sugar donor selectivity on UGTs, and demonstrates the usefulness of natural variation in understanding the structure-function relationship of enzymes involved in specialized metabolism.