Sequencing Analysis and Enzyme Activity Assay of SrUGT76G1 Revealed the Mechanism Toward on/off Production of Rebaudioside-A in Stevia Plants
Abstract Stevia plants is known for its ability to synthesize steviol glycosides (SGs), a natural sweetener blend. Stevioside (STEV) and Rebaudioside-A (Reb-A) are the main SGs. However, Reb-A is more palatable than STEV and shows reduced bitter aftertaste. SrUGT76G1 catalyzes the conversion of STEV to Reb-A, improving their organoleptic properties. The better understanding of the structure/activity of SrUGT76G1 would allow shedding light up on on/off production of Reb-A in stevia plants. Thus, we analyzed the STEV and Reb-A content in stevia leaves of plants from Brazil and Spain and did not find detectable levels of Reb-A in Brazilian samples (off production). For this reason, we used a sequencing tool to study at the genetic and structural level the SrUGT76G1 gene. Changes in key amino acid residues in Brazilian samples were found, such as Leu204Phe, Thr284Leu and Leu126Ile. Leu204Phe mutants can narrow substrate-binding pocket to favor flavonoids recognition and decrease SGs synthesis, while Thr284 is considered key for 1,3-glucosylation of SGs, including Reb-A. These punctual mutations may partly explain the lack of functionality of UGT76G1 enzyme and off production of Reb-A in stevia plants from Brazil. Following this trend, Brazilian samples exhibited a T-to-A substitution, resulting in premature stop codon. As expected, the relative expression of SrUGT76G1 gene showed a higher level in Spanish samples than in Brazilian ones. Collectively, the results presented here show the structure-activity interplay of SrUGT76G1 enzyme and provide new insights on structural features and its role toward Reb-A synthesis.