Catalytic flexibility of rice glycosyltransferase OsUGT91C1 for the production of palatable steviol glycosides

Abstract

Steviol glycosides are the intensely sweet components of extracts from Stevia rebaudiana. These molecules comprise an invariant steviol aglycone decorated with variable glycans and could widely serve as a low-calorie sweetener. However, the most desirable steviol glycosides Reb D and Reb M, devoid of unpleasant aftertaste, are naturally produced only in trace amounts due to low levels of specific β (1-2) glucosylation in Stevia. Here, we report the biochemical and structural characterization of OsUGT91C1, a glycosyltransferase from Oryza sativa, which is efficient at catalyzing β (1-2) glucosylation. The enzyme's ability to bind steviol glycoside substrate in three modes underlies its flexibility to catalyze β (1-2) glucosylation in two distinct orientations as well as β (1-6) glucosylation. Guided by the structural insights, we engineer this enzyme to enhance the desirable β (1-2) glucosylation, eliminate β (1-6) glucosylation, and obtain a promising catalyst for the industrial production of naturally rare but palatable steviol glycosides.

Fig. 1. Steviol glucosides related to OsUGT91C1 and the catalysis of the UGTs in synthesizing steviol glucosides.

a Chemical structure of steviol, Reb M, and cartoon representations of the steviol glucoside species. The glycan units are represented by individually colored hexagons with glucose 1-R1 and 1-R2 in cyan, glucose 2-1-R1 and 2-1-R2 in yellow, glucose 3-1-R1 and 3-1-R2 in green, and glucose 6-1-R1 in pink. The anomeric hydroxyl is marked by a black dot. The glycosidic bonds marked in red can be formed by OsUGT91C1. Where known, the sweetness potency of the steviol glycoside sweetener to sucrose is indicated by the number in the bracket. Reb D and Reb M have desirable taste properties. b Reactions catalyzed by UGTs in the steviol glycoside biosynthesis pathway. There is no obligate order of the first glucose additions by UGT85C2 (to the R1 end) and UGT74G1 (to the R2 end). The subsequent addition of glucose, the subject of this study, is boxed in blue. UGT91D2 adds glucose 2-1-R1 but shows only trace catalytic activity for the addition of glucose 2-1-R2 (denoted as a dashed arrow). OsUGT91C1, studied here, efficiently adds both glucose 2-1-R1 and glucose 2-1-R2 (as red arrows). UGT76G1 has been previously studied, which adds both glucose 3-1-R1 and glucose 3-1-R2.

Fig. 2. β (1–2) sugar transfers by OsUGT91C1 at both the R1 and R2 ends of Rubu.

a LC-MS was used to monitor the reaction of Rubu with OsUGT91C1 and UDP-glucose. The HPLC traces represent the 18 h control reaction without the enzyme (black); the incubations for 10 min, 20 min, and 30 min with the enzyme at 0.4 mg ml⁻¹ (blue); the incubations were repeated with the enzyme (5x) at 2.0 mg ml^-1 (green). The HPLC traces in pink report the incubation with the enzyme at 0.05 mg ml^-1 for 2 h and 18 h, repeated with 0.25 mg ml⁻¹ enzyme (5x). The authentic standards ST and Reb E are shown in the red dashed box. b–d Mass spectra of the three new peaks in LC-MS are consistent with the assignment of ST (b), Rub-X (c), and Reb E (d). The main negative derived ions of the products ST, Rub-X, and Reb E are labeled in MS analyses. The negative parent ions [ST-H]⁻, [(Rub-X)-H]⁻, and [Reb E-H]⁻ with m/z at 803, 803, and 965 were explicitly isolated and then characterized by MS/MS, where the more labile ester bond breaks first in MS/MS, leading to the first mass loss of ester-linked glycan at the R2 end from the parent ion. The size of the mass loss of the abundant fragment ions of ST, Rub-X, and Reb E indicates the number of ester-linked glucose units at the R2 end. The insert denotes where the ester bond breaks first during MS/MS fragmentation with a red dash line. e Cartoon of the reactions catalyzed by OsUGT91C1 on Rubu.

Fig. 3. Structure of OsUGT91C1 and the binding modes to recognize substrate.

a Overall structure of OsUGT91C1. The structure is shown in cartoon representation with the N-terminus domain colored cyan and the C-terminus white. The bound UDP is shown in stick-ball mode with carbon colored orange, oxygen red, and nitrogen blue. The steviol compound derived from Reb E is shown in stick with carbons of the steviol aglycone colored white, while carbons of the glycan are colored differently, carbons of glucose 1-R2 in cyan, glucose 1-R1 in dark cyan, glucose 2-1-R1 in yellow, and glucose 6-1-R1 in pink. b Binding of UDP mainly involves the C-terminal domain with the hydrogen bonds shown by dashed lines. A cluster of water molecules, shown as red balls, fills the space most likely occupied by the glucose of UDP-glucose. c, d Binding of the steviol compounds Reb E (c) and ST (d) with the R2 end at the active site and the R1 end at the “out” site. e Binding of the steviol compound STB with the R1 end at the active site and the R2 end at the “out” site. The catalytic His27 is marked with a red asterisk in c–e. f Comparison of two binding modes using ST (the R2 end at the active site, denoted as R2 “in”) and STB (the R1 end at the active site, denoted as R1 “in”).

Fig. 4. β (1–6) sugar transfer by OsUGT91C1 at the R1 end of steviol substrates STB and Reb E.

a, d LC-MS was used to monitor the reaction progress of STB (a) or Reb E (d) with OsUGT91C1. The HPLC traces represent the 18 h control reaction without the enzyme (black); the incubations for 2 h and 18 h with the enzyme at 0.15 mg ml⁻¹; repeated with the enzyme (5x) at 0.75 mg ml⁻¹ (blue). The yields of the products are related to the enzyme concentration and the reaction duration, consistent with enzymatic-catalyzed reaction. b, e Mass spectra of the new peak in LC-MS are consistent with a single glucose mass addition at the R1 end of the molecule. The products are arbitrarily named STB-X (b) and Reb E-X (e). The main negative derived ions are labeled in MS analyses. The negative parent ions [(STB-X)-H]⁻ with m/z at 803 and [(Reb E-X)-H]⁻ with m/z at 1127 were explicitly isolated and characterized by MS/MS. The insert notes where the ester bond breaks first during MS/MS fragmentation with a red dash line. c, f Reaction schemes catalyzed by OsUGT91C1 on substrate STB (c) and Reb E (f).

Fig. 5. Origins of OsUGT91C1 promiscuity.

a OsUGT91C1 binds Reb E with the R2 end at the active site is ready to add glucose 2-1-R2 to glucose 1-R2. The protein is represented in the surface, and the loose-fitting substrate tunnel of OsUGT91C1 is delimited by white dashed lines. The catalytic residue His27 is colored yellow (marked with a red asterisk), and the residues Phe208 and Glu283 are colored salmon on the surface. Phe379 cannot be seen as a result of the cutaway surface presentation, and this residue is shown in Fig. 3c–e. The positions of the R1 and R2 ends, glucose units, the reactive 2-hydroxyl, and 6-hydroxyl are labeled with a circle shadow for clarity. b OsUGT91C1 is able to bind the aglycone in the opposite orientation (the transition is shown in the red dashed box) so that the R1 end is now at the active site positioned for the addition of glucose 2-1-R1 to glucose 1-R1. The figure uses the same design as a. c After the addition of glucose 2-1-R1, glucose 1-R1 has flipped by 180° from that seen in b (the transition is shown in the red dashed box). As a result, glucose 2-1-R1 is held in a new pocket, freeing up the enzyme to bind fresh UDP-glucose and positioning the 6-OH of glucose 1-R1 for catalysis. The figure uses the same design as a. d Structurally related β (1–3) glycosyltransferase UGT76G1 (Fig. 1b) binds the aglycone in a different location. As a result, 3-OH, not 2-OH, is positioned for catalysis. The figure follows the design of a with color differences. The orientation of UGT76G1 here matches that of OsUGT91C1 in a–c. In UGT76G1, His25 serves as the catalytic base and is colored yellow (marked with a red asterisk) on the surface.