Hydrophobic recognition allows the glycosyltransferase UGT76G1 to catalyze its substrate in two orientations

Abstract

Diets high in sugar are recognized as a serious health problem, and there is a drive to reduce their consumption. Steviol glycosides are natural zero-calorie sweeteners, but the most desirable ones are biosynthesized with low yields. UGT76G1 catalyzes the β (1-3) addition of glucose to steviol glycosides, which gives them the preferred taste. UGT76G1 is able to transfer glucose to multiple steviol substrates yet remains highly specific in the glycosidic linkage it creates. Here, we report multiple complex structures of the enzyme combined with biochemical data, which reveal that the enzyme utilizes hydrophobic interactions for substrate recognition. The lack of a strict three-dimensional recognition arrangement, typical of hydrogen bonds, permits two different orientations for β (1-3) sugar addition. The use of hydrophobic recognition is unusual in a regio- and stereo-specific catalysis. Harnessing such non-specific hydrophobic interactions could have wide applications in the synthesis of complex glycoconjugates.

Fig. 1

The steviol glucosides related to UGT76G1 and the reactions in the biosynthesis pathway of steviol glucoside. a The chemical structure of Reb M and cartoon representations of the typical steviol glucosides Reb M, ST, Reb A, Reb D, sophorose, STB, Reb E, and Rubu. In the cartoon representation, the glycone units are represented by individually colored hexagons with the letters A, B, and C for clarity and the position of the 1-hydroxyl of glycone is marked by a black dot. All β (1–3) glycolic bonds formed by UGT76G1 are marked in red. b The reactions in the steviol glucoside biosynthesis pathway of Stevia rebaudiana Bertoni. The reaction and the glycosidic bonds involving UGT76G1 are labeled in red

Fig. 2

The steviol products of the sugar transfers by UGT76G1. a–d HPLC traces of the reactions of STB, Reb A, ST, Reb E were analyzed, respectively, by HPLC. The five HPLC traces from top to bottom represent the following reaction conditions: no enzyme for 18 h (black), 0.03 mg ml⁻¹ enzyme (1×) for 2 h (blue), 0.03 mg ml⁻¹ enzyme (1×) for 18 h (purple), 0.15 mg ml⁻¹ enzyme (5×) for 2 h (green) and 0.15 mg ml⁻¹ enzyme (5×) for 18 h (red). The product is identified by the authentic standard. The yields of the products are related to the enzyme concentration and the reaction duration. The reactions catalyzed by UGT76G1 are shown in the box

Fig. 3

Biochemical assays of two sugar transfers to Rubu by UGT76G1. a HPLC traces of the reactions of Rubu. The five HPLC traces from top to bottom represent the same reaction conditions as Fig. 2. The first product of Rubu is arbitrarily assigned as RX, and the second one is assigned as RY. The yields of the products are related to the enzyme concentration and the reaction duration. The reactions catalyzed by UGT76G1 are shown in the box. b Direct MS of two sugar transfers of Rubu by 0.15 mg ml⁻¹ UGT76G1 (5×) for 2 and 18 h. The two main negative ions derived from the products RX and RY are labeled and show the same characteristics in terms of the relative contents of the products as a function of the reaction duration as shown by HPLC (Fig. 3a). c MS/MS of the authentic Rubu standard and the collected HPLC peaks of the products RX and RY. The negative ions [Rubu–H]⁻, [RX–H]⁻, and [RY–H]⁻ with m/z at 641.3, 803.4 and 965.4, respectively, were specifically isolated as the parent ions and characterized by MS/MS. The most labile ester bond breaks first, which was consistently indicated by the abundant fragment ions of Rubu, RX, and RY and was used to identify the positions of the added sugars transferred by UGT76G1. The inlet suggests where the ester bond breaks first during MS/MS fragmentation

Fig. 4

The reactions of steviol monoglucosides by UGT76G1. Direct MS of the single sugar transfer at R1 (a) and R2 position (b) of two self-made steviol monoglucosides. The reaction of the steviol monoglucoside was catalyzed by 0.03 mg ml⁻¹ UGT76G1 (1×) for 18 h. The reaction schemes to synthesize steviol monoglucosides and the single sugar transfer catalyzed by UGT76G1 are shown. The main negative ions derived from the products are labeled and related to the chemical compounds in the reaction schemes. The reaction conditions for these two self-made steviol glucosides are identical, and the relative contents of the substrate (M) and the product (P) show the preferred reaction at the R1 position

Fig. 5

The structure of UGT76G1 and analysis of the active site. a The overall structure of UGT76G1. The structure is shown in cartoon representation. The β-sheet of the N-terminus is shown in cyan and that of the C-terminus in pink. The bound UDP is shown in stick-ball mode with carbons colored yellow, oxygen red and nitrogen blue. Two molecules of the steviol compound Reb A are shown in stick representation with oxygen colored red, carbons of the nonreactive Reb A colored white, carbons of the reactive Reb A colored differently, carbons of steviol aglycone and the glucose A in cyan, the glucose B in light yellow and the incoming glucose C in pale green. b The binding pocket of UDP in the presence of Reb A with atoms colored as shown in Fig. 5a. The hydrogen bonds are shown by dashed lines. c The binding pocket of the reactive Reb A. d Comparison of the ternary complex structures with Rubu and Reb A. Two ternary complex structures are shown in cartoon representation with the proteins in light blue (Rubu) and white (Reb A). The residues associated with Rubu are shown in stick representation with carbons colored light blue, oxygen red and nitrogen blue. Two residues, Asn 196 and Trp 197, in the structure of Reb A are shown in stick representation with carbons colored white, which highlight the flexible loop between Ser 195 and Lys 201. Rubu and a glycerol molecule (GOL) in the active site are shown in stick with carbons colored gold, while Reb A is shown in a transparent stick representation after overlapping their ternary complex structures. The glycerol molecule occupies a similar position of the incoming glucose C of Reb A. e The S_N2 mechanism of UGT76G1 catalysis. His 25, with the aid of Asp 124, specifically deprotonates the 3-hydroxyl of the glucose A and makes it a nucleophile to attack the sugar donor UDPG. The transition state of the S_N2 mechanism is shown in the brackets

Fig. 6

The two binding modes of the steviol substrates for the sugar transfers. a The normal binding mode for the R1 reaction. The binding mode for the R1 reaction is represented by the ternary complex structure with Rubu, in which the positions of Rubu, GOL, UDP and the catalytic residue His 25 are shown in the cross-section of the binding pocket. b The flipped binding model for the R2 reaction. The flipped binding mode for R2 reaction is modeled in the ternary complex structure with Reb A, in which the flipped steviol glucoside fragment (with carbons colored light purple) stretches the 3-hydroxyl of the glucose AR2 toward the catalytic site without any obvious clash in the binding pocket. c The cartoon representation of the normal and flipped binding modes. The catalytic residue His 25 and its reactivation region are represented by a red star and red dots, respectively, toward which the 3-hydroxyl group of either the R1 or R2 position would be presented

Fig. 7

Replacement of the steviol aglycone ring of the substrate by another hydrophobic group. a Direct MS of the reaction of 4-nitrophenyl β-D-glucopyranoside by UGT76G1. The nitrophenyl ring shows a similar role to give the reactivity to the glycone unit. The reaction of 4-nitrophenyl β-d-glucopyranoside was catalyzed by 0.25 mg ml⁻¹ UGT76G1 for 12 h. The two main negative ions derived from the substrate (M) and product (P) are labeled. b The slow reaction scheme and the binding mode of 4-nitrophenyl β-D-glucopyranoside by UGT76G1