Identification of the Key Residues of the Uridine Diphosphate Glycosyltransferase 91D2 and its Effect on the Accumulation of Steviol Glycosides in <i>Stevia rebaudiana</i>

Abstract

Stevia (<i>Stevia rebaudiana</i> Bertoni) possesses substantial value for its unique sweet compounds-steviol glycosides (SGs). In the metabolic glycosylation grid of SGs, SrUGT91D2 has been shown to catalyze formation of 1,2-β-d-glucoside linkages at the C₁₃- and C₁₉-positions and play a crucial role in the synthesis of SGs, including the formation of stevioside (ST), rebaudioside E (RE), and rebaudioside D (RD). However, the key residues of the SrUGT91D2 enzyme and how SrUGT91D2 affects the accumulation of SGs in <i>S. rebaudiana</i> remain unclear. In the present study, cloning and functional analysis of full-length <i>SrUGT91D2</i> gene sequences were performed in 10 different <i>S. rebaudiana</i> genotypes with divergent SG compositions. After sequence analysis, it was found that most of the sequences of this gene (more than 50%) in each genotype were consistent with the <i>UGT91D2e_No.5</i> allele, which has been reported to exert catalytic activity on 1,2-β-d-glucoside. Moreover, six variants (<i>UGT91D2e_No.5, SrUGT91D2-11-14</i>, <i>SrUGT91D2-110</i>, <i>SrUGT91D2-023, SrUGT91D2-N01</i>, and <i>SrUGT91D2-N04</i>) of this gene were obtained, and their activities were identified. Although there were some differences among these variants, the only type of mutation was partial base substitution at a very low level. In addition, the expression analysis of <i>SrUGT91D2</i> in each genotype showed that the expression level of the gene was significantly different among genotypes, and a significant positive correlation was found between the content of RD (which was closely influenced by SrUGT91D2) and the expression level of <i>SrUGT91D2</i> in each genotype (correlation coefficient = 0.91). Thus, it was indicated that <i>SrUGT91D2</i> was relatively conserved in <i>S. rebaudiana</i>, and the differential effect of <i>SrUGT91D2</i> on the accumulation of related SGs mainly derived from its expression level. Furthermore, based on homologous modeling and molecular docking analysis, T84, T144, A194, S284, E285, V286, G365, E369, R404, and G409 were predicted to be key residues in the glucosylation of SGs by SrUGT91D2. After site-mutation and enzyme assays, it was confirmed that T84, T144, R404, A194, and G409 are the key residues in the SrUGT91D2 protein, especially T144 and G409. This work provided valuable information for understanding the structure-activity relationship of the SrUGT91D2 protein and the molecular mechanism of SG accumulation in stevia.