Molecular Basis for Branched Steviol Glucoside Biosynthesis

Molecular basis for branched steviol glucoside biosynthesis

Soon Goo Leea,b, Eitan Salomona,c, Oliver Yud, and Joseph M. Jeza,1

aDepartment of Biology, Washington University in St. Louis, St. Louis, MO 63130; bDepartment of Chemistry & Biochemistry, University of North Carolina Wilmington, Wilmington, NC 28403; cNational Center for Mariculture, Israel Oceanographic and Limnological Research, Eilat, 8811201, Israel; and dConagen, Inc., Bedford, MA 01730

Edited by Richard A. Dixon, University of North Texas, Denton, TX, and approved May 14, 2019 (received for review February 4, 2019) Steviol glucosides, such as stevioside and rebaudioside A, are steviol glucosides in plant tissue, which suggests this step is the natural products roughly 200-fold sweeter than sugar and are limiting reaction in the pathway (9). The UGT that modifies the used as natural, noncaloric sweeteners. Biosynthesis of rebaudio- C2′ of the C13 glucose to form steviolbioside remains to be side A, and other related stevia glucosides, involves formation of identified (6, 7). Next, UGT74G1-catalyzed glucosylation of the the steviol diterpenoid followed by a series of glycosylations carboxylate completes formation of stevioside (7). Further catalyzed by uridine diphosphate (UDP)-dependent glucosyltrans- modification by UGT76G1 at the C3′ of the C13 sugar leads to ferases. UGT76G1 from Stevia rebaudiana catalyzes the formation synthesis of rebaudioside A, which contains the branched glu- of the branched-chain glucoside that defines the stevia molecule coside (7). Variants of UGT76G1 alter levels of rebaudioside A and is critical for its high-intensity sweetness. Here, we report the in the plant (10, 11). The broad acceptor molecule activity of 3D structure of the UDP-glucosyltransferase UGT76G1, including a UGT76G1 has been explored for biocatalyst uses in industrial complex of the protein with UDP and rebaudioside A bound in the synthesis of a range of glucosylated natural products (12). Al- active site. The X-ray crystal structure and biochemical analysis of though the general sequence of modifications in the steviol site-directed mutants identifies a catalytic histidine and how the glucoside pathway has been determined, the structural basis for acceptor site of UGT76G1 achieves regioselectivity for branched- selectivity of the UGT enzymes, in particular how UGT76G1 glucoside synthesis. The active site accommodates a two-glucosyl catalyzes branched glucoside formation, remains unclear.

side chain and provides a site for addition of a third sugar molecule PLANT BIOLOGY to the C3′ position of the first C13 sugar group of stevioside. This Results and Discussion structure provides insight on the glycosylation of other naturally Overall Structure of UGT76G1. To understand the structural basis of occurring sweeteners, such as the mogrosides from monk fruit, branched steviol glucoside synthesis, the X-ray crystal structure and a possible template for engineering of steviol biosynthesis. of S. rebaudiana UGT76G1 was determined by single-wavelength anomalous dispersion phasing using selenomethionine (SeMet)- glucosyltransferase | noncaloric sweetener | plant biochemistry | stevia | substituted protein (Fig. 1B and Table 1). The SeMet-substituted X-ray crystal structure model was then used to solve 3D structures of the UGT76G1•UDP

weeteners derived from plant natural products have signifi- Significance Scant potential as dietary supplements because they are stable and noncaloric; can maintain good dental health by reducing The naturally occurring noncaloric sweetener stevia is a plant sugar intake; and have possible uses by diabetic, phenylketon- natural product consisting of a core terpene structure deco- uric, and obese patients (1, 2). For example, the sweetener stevia rated with a specific pattern of glucose molecules, including a Stevia rebaudiana is isolated from the leaves of (sweetleaf), a branched three-sugar unit. Stevia and other related molecules perennial herb native to Paraguay and Brazil (2, 3). The leaves of are being explored as noncaloric dietary sweeteners because this plant contain a variety of ent-kaurene diterpenoid glycosides they can help maintain the health of diabetic, phenylketonuric, composed of a steviol aglycone decorated with different numbers and obese patients. Here, we describe the three-dimensional and types of sugars attached to the C13 and C19 positions (2, 3) structure of the plant enzyme (UGT76G1) that forms the – (Fig. 1A). The predominant steviol glucosides are stevioside (5 branched group of sugars that defines the stevia molecule and – 10% of leaf dry weight) and rebaudioside A (2 4% of leaf dry is critical for its high-intensity sweetness. Understanding how weight), which taste up to 300-fold sweeter than sucrose. Use of this enzyme forms this chemical group provides insight on how these compounds as naturally sourced noncaloric sweeteners has the stevia plant makes this sweetener and suggests how to expanded globally over the last decade (1, 4). For example, rebi- alter the protein to generate new versions of the noncaloric ana, a commercially available sweetener, mainly contains rebau- sweetener. dioside A, which reduces bitterness and aftertaste associated with other compounds isolated from the plant (4). Moreover, recent Author contributions: S.G.L., E.S., O.Y., and J.M.J. designed research; S.G.L. and E.S. per- efforts to engineer steviol glucoside production in yeast aim to formed research; O.Y. contributed new reagents/analytic tools; S.G.L., E.S., and J.M.J. provide specific types of molecules as a way to avoid variation that analyzed data; and S.G.L., E.S., O.Y., and J.M.J. wrote the paper. results from the use of different S. rebaudiana cultivars and growing Conflict of interest statement: O.Y. is a founder and employee of Conagen (New Bedford, MA). J.M.J. serves on the scientific advisory board of Conagen (New Bedford, MA). conditions (5). The biochemical pathway for steviol glucoside biosynthesis This article is a PNAS Direct Submission. involves formation of the core steviol diterpenoid followed by a Published under the PNAS license. series of glucosylations catalyzed by a set of uridine diphosphate Data deposition: Coordinates and structure factors for the UGT76G1(SeMet)•UDP com- – plex (PDB ID code 6O86), the UGT76G1•UDP complex (PDB ID code 6O87), and the (UDP)-dependent glucosyltransferases (UGT) (2, 6 12) (Fig. UGT76G1•UDP•rebaudioside A complex (PDB ID code 6O88) were deposited in the Pro- 1A). Three UDP-glucosyltransferases in the rebaudioside A tein Data Bank, https://www.rcsb.org. biosynthesis pathway have been identified and shown to localize 1To whom correspondence may be addressed. Email: [email protected]. to the cytosol (7, 8). Glycosylation of steviol by UGT85C2 begins This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. at the C13 hydroxyl group to yield steviolmonoside (7). Tran- 1073/pnas.1902104116/-/DCSupplemental. script levels of UGT85C2 correlate with total accumulation of

www.pnas.org/cgi/doi/10.1073/pnas.1902104116 PNAS Latest Articles | 1of6 Downloaded by guest on September 28, 2021 Fig. 1. Role of UGT in steviol glucoside biosynthesis and the overall structure of UGT76G1. (A) Role of UGT in the steviol biosynthesis pathway. The C13 and C19 positions of the steviol aglycone are indicated. (B) Three-dimensional structure of UGT76G1. The ribbon diagram for the UGT76G1•UDP complex shows the secondary structure with α-helices (blue) and β-strands (gold). UDP is shown as a space-filling model.

(1.75 Å resolution) and UGT76G1•UDP•rebaudioside A (1.99 indigo GT-B) (15–23); however, all of these UGT transfer Å resolution) complexes by molecular replacement. The overall sugars directly to the aglycone and do not form branched natural structure of UGT76G1 is monomeric and adopts the GT-B product glucosides. fold, which consists of an N- and C-terminal Rossmann-fold domains (13). Each structural domain of UGT76G1 contains Structure of the UDP Sugar Donor-Binding Site. Clear electron a central β-sheet flanked by multiple α-helices (N-terminal: density for UDP in the C-terminal domain of UGT76G1 defines β1a–β1g and α1–α9; C-terminal: β2a–β2f and α10–α17) (Fig. the location of the sugar donor-binding site (Figs. 1B and 2A). 1B and SI Appendix,Fig.S1). The C-terminal domain contains The uridine ring of UDP is sandwiched between Trp338 and the UDP-binding site with a cleft between the two domains Gln341 with hydrogen bond interactions contributed by Val339, providing the sugar acceptor-binding site (13). The amino acid Pro340, and a water-mediated interaction with His260 (Fig. 2B). sequence and 3D structure of UGT76G1 indicate that this pro- Glu364 forms a bidentate interaction with the hydroxyl groups of tein is a member of the carbohydrate-active enzymes (CAZy) the ribose. Direct interactions with Ser285, His356, Asn360, and glycosyl transferase family 1 (14), which consists of related Ser361, along with water-mediated contacts to Ser283 and enzymes that glycosylate a variety of plant natural products. Thr284, position the diphosphate group toward the cleft between The 3D structure of UGT76G1 shares 2.2–2.9 Å2 root mean the N- and C-terminal domains. square deviations (rmsds) for 420–440 Cα atoms with the A structure of UGT76G1 with UDP-glucose was not obtained; other structurally characterized plant UGT. These include however, Thr146, Trp359, and Asp380 bind a glycerol molecule enzymes that modify terpenoids (Medicago truncatula/barrel in proximity to the diphosphate group of UDP (Fig. 2C). The clover UGT71G1 and Orzya sativa/rice Os79), flavonoids and position of this ligand mimics how the glucose moiety of the isoflavonoids (Vitis vinifera/grape GT1, M. truncatula UGT85H2 sugar donor would interact with UGT76G1. Comparison of and UGT78G1), chlorinated phenols (Arabidopsis thaliana/ the site where glycerol binds in UGT76G1 to the structure of the thale cress UGT72B1), anthocyanin floral pigments (Clitoria Os79 UGT from rice in complex with a nonreactive UDP-glucose ternatea/bluebell vine UGT78K6), salicylic acid (A. thaliana analog, uridine-5′-diphosphate-2-deoxy-2-fluoro-α-D-glucose (U2F) UGT74F2), and indoxyl dyes (Polygonum tinctorium/Japanese (16), highlights the structural and sequence conservation of the

Table 1. Summary of crystallographic data collection and refinement statistics Data collection UGT76G1(SeMet) •UDP UGT76G1•UDP UGT76G1•UDP •rebaudioside A

Space group P3121 P3121 P3121 Cell dimensions a = b = 97.98 Å, c = 90.62 Å a = b = 98.45 Å, c = 90.67 Å a = b = 98.12 Å, c = 91.52 Å Wavelength, Å 0.979 0.979 0.979 Resolution, Å (highest shell) 38.5–1.80 (1.83–1.80) 40.0–1.75 (1.78–1.75) 42.5–1.99 (2.02–1.99) Reflections (total/unique) 320,766/47,033 304,795/48,196 317,717/35,139 Completeness (highest shell), % 99.6 (99.1) 93.5 (96.7) 99.5 (94.1) (highest shell) 13.0 (2.3) 31.8 (2.6) 25.9 (1.8)

Rsym (highest shell), % 17.4 (74.7) 4.2 (34.8) 9.32 (61.9) Figure of Merit 0.564 —— Refinement

Rcryst/Rfree,% 17.1/20.0 17.2/19.8 16.4/20.0 No. of protein atoms 3,609 3,567 3,559 No. of waters 369 267 223 No. of ligand atoms 42 42 140 rmsd, bond lengths, Å 0.007 0.007 0.007 rmsd, bond angles, ° 0.912 0.922 1.03 Avg. B-factor (Å2): protein, 28.0, 36.6, 20.2 47.2, 46.1, 31.3 40.6, 67.3 43.5 water, ligand Ramachandran plot: favored, 97.6, 2.4, 0.0 96.0, 4.0, 0.0 97.8, 2.2, 0.0 allowed, disallowed, %

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1902104116 Lee et al. Downloaded by guest on September 28, 2021 the C13 (UGT85C2) or C19 (UGT74G1) positions (Fig. 3A). For synthesis of the branched glucoside rebaudioside A, the acceptor site of UGT76G1 needs to accommodate a two-glucosyl side chain to allow for the addition of a third sugar molecule to the C3′ position of the first C13 sugar group of stevioside. To achieve this regioselectivity, UGT76G1 requires a binding site near the UDP-glucose donor site that also orients the second sugar away from the catalytic histidine. The structure of UGT76G1 in complex with UDP and rebaudioside A reveals the basis for branched steviol glucoside synthesis. Electron density for both ligands was visible in the structure with the omit map for rebaudioside A showing clear density for the three glucosyl groups extending from the steviol C13, weaker density for the steviol core, and no density for the C19 glucose (Fig. 3B). Because the solvent-exposed portion of rebaudioside A was disordered, a model of the ligand lacking the C19 sugar was used for refinement. The nucleotide is bound as observed in the UDP complex and rebaudioside A binds in a pocket of the N-terminal domain (Fig. 3C). Fig. 2. UDP binding and sugar donor-binding site of UGT76G1. (A) Electron The branched sugar portion of rebaudioside A is oriented into

density for UDP shown as a 2Fo − Fc omit map (1.5σ). (B) Stereoview of the the interior and toward the UDP pyrophosphate. From the UDP-binding site in the UGT76G1•UDP complex. Ligand-binding interactions bottom of the binding site, the terminal glucose (glc3) fills the are shown as dotted lines. (C) Glycerol binding in the sugar donor-binding sugar donor portion of the pocket; the branching glucose (glc2) site of the UGT76G1•UDP complex. Ligand-binding interactions are shown as is positioned into a pocket away from the catalytic histidine; and dotted lines. The view is shown for comparison with D.(D) Sugar donor- the C13-linked glucose (glc1) and the steviol portion of the binding site of rice UGT Os79 (16). The orientation is the same as the glycerol molecule extend out to the solvent exposed opening of the site site from UGT76G1. The X-ray structure of the nonreactive UDP-glucose analog U2F is shown for comparison with C. (Fig. 3 C and D). In the acceptor site, Thr146, Trp359, Asp380, and Gln381 interact with glc3 and position the α1,3-linkage be- PLANT BIOLOGY tween this glucosyl unit and a glc1 in proximity to His25 (Fig. 3D). sugar donor-binding site and the position of the catalytic histidine Interactions with Met145, Ser147, Asn151, His155, and Leu379 in each active site (Fig. 2 C and D). The 3D position of glycerol in orient the α1,2-linked glc2 into a pocket away from the catalytic the UGT76G1 sugar donor-binding site fills the same space as the His25 and Asp124. The C13-linked glucose group (glc1) stacks C3–C5 portion of the U2F glucose group from the Os79 structure. with Phe22 and Ile90 and forms a water-mediated interaction with The residues in Os79 (i.e., Ser142, Gln143, Trp369, Asp385, and Gln386) that interact with the sugar donor are either invariant or highly conserved in UGT76G1 (Thr146, Ser157, Trp359, Asp380, and Gln381). Likewise, the histidine (His25 in UGT76G1; His27 in Os79) that serves as a general base to abstract a proton from the acceptor molecule in the SN2 mechanism and the aspartate (Asp124 in UGT76G1; Asp120 in Os79) that stabilizes the histidine in the transfer reaction are invariant (24). Although UGT76G1 shares ∼25% sequence identity with UGT85C2 and UGT74G1 from the steviol glucoside biosynthesis pathway of S. rebaudiana, key active site residues are retained across these three enzymes (SI Appendix, Fig. S1). These features include the histidine and asparate of the catalytic dyad and residues (i.e., His260, Ser283, Thr284, Ser285, Trp338, Qln341, Asn360, and Glu364 of UGT76G1) that interact with the UDP portion of the shared sugar donor substrate (SI Ap- pendix, Fig. S1, red and blue, respectively). Similarly, residues critical for positioning the donor glucosyl group in UGT76G1 (Thr146, Ser157, Trp359, Asp380, and Gln381) are maintained in the other UGT of this biosynthetic pathway (SI Appendix, Fig. S1, purple). A large portion of the UDP-binding and sugar Fig. 3. Structural basis for branched steviol glucoside synthesis by UGT76G1. donor-binding sites are part of the canonical PSPG (putative (A) Schematic of sugar donor and acceptor in steviol UGT. The UDP-glucose secondary plant glycosyltransferase) sequence motif found in the donor (UDP-Glc; red) and acceptor molecules (blue) are shown. Note that the plant UGT (25) (SI Appendix, Fig. S1, black box), which is oxygen in the acceptor of UGT85C2 and UGT74G1 differ. The position of the consistent with conserved sequence and structure to bind the catalytic histidine is indicated by the triangle. (B) Electron density for − σ UDP molecule. This analysis indicates that the UGTs in the rebaudioside A shown as a 2Fo Fc omit map (1.5 ). (C) Stereoview of the steviol pathway retain highly conserved catalytic dyads and UDP- molecular surface of the UGT76G1 acceptor site. Crystallographically de- glucose sugar donor sites, yet each enzyme displays differences in termined positions of UDP and rebaudioside A are shown. The surface cor- acceptor regiospecificity. responding to His25 is colored blue. The three glycosyl units (glc1, glc2, glc3) attached at the C19 position of the steviol aglycone are labeled. The fourth sugar attached at the C13 carboxylate was not modeled because of disorder. Rebaudioside A Binding and Generation of the Branched Glucoside. (D) View of the rebaudioside A binding site of UGT76G1. As in C, the glc1, As a branched sugar side chain-forming enzyme, UGT76G1 glc2, glc3, and steviol portions of the ligand are labeled. Hydrogen-bonding differs from the other two UGTs in the steviol pathway, which interactions are shown as dotted lines. The two α-helices (α3 and α8) that directly glucosylate the steviol aglycone via an oxygen at either define the steviol aglycone binding site are also labeled.

Lee et al. PNAS Latest Articles | 3of6 Downloaded by guest on September 28, 2021 Fig. S2G). Examination of the anthocyanidin glucosyltransferase UGT78K6, flavonoid glucosyltransferase GT1, and indoxyl glucosyltransferase GT-B X-ray crystal structures (SI Appendix, Fig. S2 D–F) highlight how various changes reduce the available space in the region corresponding to the glc2 site of UGT76G1. The introduction of key changes in the glc2 site of UGT76G1 are critical for the evolution of the branch chain-forming glucosyltransferase activity of this enzyme.

Biochemical Analysis of Site-Directed Mutants. To probe the con- tribution of active site residues to UGT76G1 function, 21 site- directed mutants were generated and examined for biochemical activity (Fig. 4 and Table 2). Substitution of the catalytic histidine with an alanine (H25A) eliminated enzymatic activity. The analogous histidine residue in other UDP-glucosyltransferases Fig. 4. Summary of wild-type and mutant UGT76G1 enzyme activities. A facilitates a direct displacement SN2-like mechanism. This resi- comparison of wild-type and mutant UGT76G1 catalytic efficiencies (kcat/Km) with stevioside (black) and UDP-glucose (white) is shown. Steady-state ki- due acts as a general base by abstracting a proton from the ac- netic parameters are summarized in Table 2. ceptor substrate to yield an oxyanion nucleophile that reacts with the UDP-sugar acceptor (13). Mutagenesis of residues in the rebaudioside A-binding site and Gly24. The steviol group is sandwiched between Leu126 and biochemical analysis of the mutants reveals the importance of key residues from α3 (Met88 and Ile90) and α8 (Leu200, Ile203, residues for enzymatic activity. Removal of the side chains of Asp380 Leu204, and Met207). The C19 carbonyl hydrogen bonds with the (D380A) and Gln381 (Q381A) in the glc3/sugar donor-binding site Pro84, which positions the crystallographically disordered sugar at this position toward solvent. The UGT76G1 structure reveals an active site that is generally divided between hydrophilic branched Table 2. Summary of wild-type and mutant UGT76G1 steady- glucoside-binding residues and largely apolar steviol interaction state kinetic parameters residues; however, sequence comparisons also suggest that the −1 −1 −1 Protein Varied substrate kcat,min Km, μM kcat/Km,M ·s residues forming the glc2 pocket in UGT76G1 are varied in UGT85C2 and UGT74G1 (SI Appendix,Fig.S1). WT Stevioside 33.8 ± 0.7 360 ± 23 1,560 As shown schematically in Fig. 3A, the position of the diter- UDP-Glucose 32.5 ± 1.5 943 ± 96 574 pene core of steviol in the active site needs to vary between L126I Stevioside 0.09 ± 0.01 676 ± 320 2 UGT76G1 and the other two enzymes in the stevia biosynthesis UDP-Glucose 0.06 ± 0.04 12,500 ± 10,700 0.1 pathway. UGT85C2 and UGT74G1 directly modify the terpene at M145F Stevioside 14.9 ± 1.8 3,030 ± 580 82 the C13 and C19 positions, respectively. In contrast, UGT76G1 UDP-Glucose 13.5 ± 1.2 5,940 ± 710 38 needs to bind stevioside with the terpene moiety further away from M145W Stevioside 1.0 ± 0.2 7,690 ± 2,780 2 the catalytic site. Sequence comparison indicates that residues in the UDP-Glucose 0.8 ± 0.1 17,900 ± 4,800 1 glc2- and stevia-binding regions of UGT76G1, along with the T146A Stevioside 8.9 ± 0.7 7,340 ± 1,420 20 lengths of the α3andα8 helices defining the acceptor site, vary UDP-Glucose 5.3 ± 0.2 8,770 ± 674 10 between the three UGT of the stevia pathway and that these ex- S147A Stevioside 1.4 ± 0.1 2,570 ± 558 9 tensive changes likely contribute to different substrate preferences UDP-Glucose 0.8 ± 0.1 3,750 ± 355 4 (SI Appendix,Fig.S1, yellow). In comparison with the residues of S147T Stevioside 0.6 ± 0.1 927 ± 163 11 the glc2 pocket in UGT76G1, sequence comparison suggests that UDP-Glucose 0.7 ± 0.1 477 ± 49 24 larger side chains are found in UGT85C2 and UGT74G1. For ex- S147N Stevioside 0.6 ± 0.1 880 ± 162 11 ample, Met145, Ser147, and Leu379 are replaced with tryptophan, UDP-Glucose 0.7 ± 0.1 2,160 ± 320 5 isoleucine, and tryptophan in UGT85C2 or phenylalanine, gluta- N151A Stevioside 9.2 ± 0.4 1,600 ± 125 96 mine, and serine in UGT74G1. Each retains a histidine at position UDP-Glucose 8.6 ± 0.5 1,870 ± 190 76 155 and has smaller side chains in place of Asn151 (a glycine in N151Q Stevioside 27.8 ± 0.4 1,210 ± 34 390 UGT85C2 and a valine in UGT74G1). Homology modeling of the UDP-Glucose 105 ± 18 21,200 ± 4,090 83 other two UGT in the steviol biosynthesis pathway suggests that the H155A Stevioside 11.4 ± 0.8 424 ± 69 448 various amino acid substitutions narrow the glc2 pocket, which UDP-Glucose 12.8 ± 0.1 577 ± 16 370 ± ± likely occludes binding of longer side-chain steviol glucosides but H155R Stevioside 3.4 0.1 1,040 72 54 ± ± allows for steviol and steviolbioside glucosylation in UGT85C2 and UDP-Glucose 4.3 0.3 3,290 290 22 ± ± UGT74G1, respectively (SI Appendix,Fig.S2A–C). H155W Stevioside 7.8 0.4 2,090 180 62 ± ± Structural and sequence comparison of UGT76G1, a branch- UDP-Glucose 6.1 0.1 3,220 96 32 ± ± forming glycosyltransferase, to the plant UGTs that directly L200I Stevioside 43.0 19 1,750 1,120 410 ± ± glycosylate a given substrate (i.e., terpenoids, flavonoids, phe- UDP-Glucose 30.7 2.0 2,270 240 225 ± ± nols, anthocyanins, and indoxyls; refs. 15–23) highlights key dif- L204I Stevioside 27.1 2.7 762 150 593 ± ± ferences in residues forming the glc2 site (SI Appendix, Fig. S2 UDP-Glucose 24.7 1.2 1,090 113 378 ± ± D–G). For each of these enzymes, residues defining the glc3 site, M207F Stevioside 22.9 2.4 885 172 431 ± ± in which the glucosyl group of a UDP-sugar donor binds, are UDP-Glucose 19.2 0.5 1,050 56 305 ± ± highly conserved, as expected for UGTs (SI Appendix, Fig. S2G). M207W Stevioside 34.7 18.9 4,710 3090 123 ± ± In contrast, each of the other plant UGT examined have multiple UDP-Glucose 68.6 22.4 15,600 5,700 73 L379I Stevioside 42.6 ± 3.5 1,120 ± 160 634 bulky side-chain substitutions in residues corresponding to the glc2 ± ± site of UGT76G1. In particular, Leu126, Met145, and His155 of UDP-Glucose 33.2 0.7 1,420 54 390 UGT76G1 are typically replaced by phenylalanine, phenylalanine/ Assays were performed as described in Methods. Average values ± SD tryptophan, and phenylalanine/tyrosine, respectively (SI Appendix, (n = 3) are shown.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1902104116 Lee et al. Downloaded by guest on September 28, 2021 ∼ resulted in inactive enzyme. Based on their position in the conserved was transformed into E. coli BL21 (DE3) cells, which were grown to A600 0.8 − PSPG sequence motif (SI Appendix,Fig.S1), loss of either side chain at 37 °C in M9 minimal media supplemented with SeMet containing 50 μg·mL 1 kanamycin (33). Protein expression was induced by addition of isopropyl 1- removes key interactions with the sugar being transferred from β donor to acceptor and likely results in compromised substrate thio- -D-galactopyranoside (IPTG; 1 mM final) with cells growth continued overnight (16 °C). Cells were pelleted by centrifugation (10,000 × g)and binding, which prevents efficient catalysis. Likewise, alteration resuspended in lysis buffer [50 mM Tris, pH 8.0, 500 mM NaCl, 20 mM imid- of Thr146, which is situated between the glc3/sugar donor- azole, 1 mM β-mercaptoethanol, 10% (vol/vol) glycerol, and 1% (vol/vol) binding site and the glc2 site, either lost activity (T146N) or Tween-20]. After sonication of the resuspended cells, debris was removed by decreased catalytic efficiency by 80-fold (T146A). Mutations of centrifugation (30,000 × g).ThelysatewaspassedoveraNi2+-nitriloacetic acid residues in the glc2 site resulted in a range of effects. Substi- (Qiagen) column equilibrated with wash buffer (lysis buffer minus Tween-20). tutions of Ser147 yielded mutants with ∼170-fold reductions in Bound his-tagged protein was eluted with elution buffer (wash buffer with catalytic efficiency (S147A, S147T, and S147N). Modest 3- to 250 mM imidazole). The eluant was further purified by size-exclusion chro- 15-fold changes in k /K were observed with the N151A, matography using a Superdex-200 26/60 HiLoad FPLC column equilibrated cat m with 50 mM Tris, pH 8.0, 25 mM NaCl, 1 mM Tris(2-carboxyethyl)phosphine. N151Q, H155A, and L379I mutants. Mutations that introduce Peak fractions were collected and concentrated to ∼10 mg·mL−1 using cen- larger side chains in to the glc2 site at His155 (H155R and trifugal concentrators (Amicon). Bradford assay with BSA as the standard was H155W) led to ∼25-fold less-efficient variants. Change of used to determine protein concentration. For storage at −80 °C, purified Met145 to either a phenylalanine (M145F) or tryptophan protein was flash-frozen in liquid nitrogen. Expression of wild-type and mu- (M145W) resulted in 20- and 750-fold reductions in kcat/Km.In tant UGT76G1 proteins used similar protocols, except that Terrific broth the apolar steviol-binding cleft, a subtle change of Leu126 to replaced the minimal media. isoleucine (L126I) leads to a 750-fold decrease in catalytic efficiency. Other point mutants, such as L200I, L204I, and Protein Crystallization and Structure Determination. Purified UGT76G1 was − M207F, displayed modest threefold changes with the M207W concentrated to 10 mg·mL 1 and crystallized using the hanging-drop vapor- mutant having a 13-fold effect. Overall, biochemical analysis of diffusion method with a 2-μL drop (1:1 concentrated protein and crystalli- the UGT76G1 mutants confirms the conserved function of the zation solution). Diffraction quality crystals of both SeMet-substituted and native protein were obtained at 4 °C with 15% (wt/vol) PEG 4000, 20% 2- catalytic histidine and identifies critical residues in the glc2- propanol (vol/vol), 100 mM sodium citrate tribasic dihydrate buffer (pH 5.6), and steviol-binding sites. and either 5 mM UDP or 5 mM UDP and 5 mM rebaudioside A. To generate the UGT76G1•UDP•rebaudioside A complex, 5 mM UDP and 5 mM rebau- Conclusion. This first structure of a branched steviol glucoside- dioside A [in 10% (vol/vol) DMSO] were added during protein concentration.

producing enzyme (i.e., UGT76G1) provides insight on how the Individual crystals were flash-frozen in liquid nitrogen with the mother li- PLANT BIOLOGY enzyme accommodates large sugar side chains and differs from quor containing 25% glycerol as a cryoprotectant. Diffraction data (100 K) the other glycosyltransferases in the pathway. Importantly, the was collected at the Argonne National Laboratory Advanced Photon Source branched-chain glycosylation pattern built from the C19 position 19-ID beamline. HKL3000 (34) was used to index, integrate, and scale dif- of rebaudioside A leads to rebaudiosides D and M, which are fraction data. The structure of SeMet-substituted UGT76G1 was determined by single-wavelength anomalous diffraction (SAD) phasing. SHELX (35) was more commercially interesting steviosides because they deliver used to determine SeMet positions and to estimate initial phases from the high-intensity sweetness with less off-taste than rebaudioside A peak wavelength dataset. Refinement of SeMet positions and parameters (26). Similar glycosylation patterns are found in the biosynthesis was performed with MLPHARE (36). Solvent flattening using density modi- of other molecules, such as the mogrosides from monk fruit fication implemented with ARP/wARP (37) was employed to build an initial (Siraitia grosvenorii), which are being explored as stevia alter- model. Subsequent iterative rounds of manual model building and refine- nates (27). Knowledge of the active site architecture also pro- ment, which included translation-libration-screen parameter refinement, vides a template for enzyme engineering that may lead to the used COOT (38) and PHENIX (39), respectively. The structures of UGT76G1 in development of variants with altered regiospecificity and/or complex with either UDP or UDP and rebaudioside A were solved by mo- substrate glycosylation patterns that can be combined with varied lecular replacement in PHASER (40) using the SeMet structure as a search – model with refinement and building performed as above. The final model donor substrates either in vitro or in vivo (28 31). Moreover, the of the SeMet-substituted UGT76G1 includes residues Arg12-Pro169 and amino sequence of the glc2 site of UGT76G1, which allows for Arg174-Leu458, one UDP molecule, one glycerol molecule, and 369 waters. formation of a branched-sugar modified product (i.e., rebau- The UGT76G1•UDP complex includes the same residues and ligands, but with dioside A), may provide a useful “signature motif” to bio- 267 waters. The UGT76G1•UDP•rebaudioside A complex includes the same informatically distinguish branched chain-forming UGT from residues, one UDP molecule, one rebaudioside A molecule (modeled with- those that directly glucosylate various substrates when assessing out the C19 sugar), and 223 waters. Data collection and refinement statistics are summarized in Table 1. Coordinates and structure factors for the potential metabolic function. Such structure-guided efforts offer • • the potential for altered pathways of steviol production to gen- UGT76G1(SeMet) UDP complex (PDB ID code: 6O86), the UGT76G1 UDP complex (PDB ID code: 6O87), and the UGT76G1•UDP•rebaudioside A com- erate tailored variants of this noncaloric sweetener. plex (PDB ID code: 6O88) were deposited in the Protein Data Bank. Methods Enzyme Assays. UDP-glycosyltransferase activity was monitored spectropho- Chemicals, Codon Optimized Gene Synthesis, and Site-Directed Mutagenesis. tometrically (A ) using a coupled assay system (41). Standard reaction All reagents used were purchased from Sigma-Aldrich unless noted other- 340 conditions were 50 mM Hepes, pH 7.5, 200 μM NADH, 500 μM phosphoenol wise. An Escherichia coli codon-optimized version of the gene encoding pyruvate, 10 mM MgCl , two units of pyruvate kinase, and six units of lactate UGT76G1 was generated for protein expression. The original sequence 2 dehydrogenase in a 0.1-mL reaction at 25 °C. Reactions were initiated by (SwissProt Q6VAB4; ref. 7) was optimized and synthesized by GenScript. The addition of protein with changes in absorbance measured on a Tecan 96- resulting gene was inserted into pET-28a to yield the pET-28a-UGT76G1 for well plate reader. Steady-state kinetic parameters were determined by ini- expression of an N-terminally His -tagged fusion protein. Site-directed mu- 6 tial velocity experiments with either varied stevioside concentrations and tants of UGT76G1 (H25A, L126I, M145F, M145W, T146A, T146N, S147A, 5 mM UDP-glucose or varied UDP-glucose and 2 mM stevioside. Data were fit S147T, S147N, N151A, N151Q, H155A, H155R, H155W, L200I, L204I, M207F, to the Michaelis–Menton equation, v = kcat[S]/(Km + [S]), using Kaleidagraph. M207W, L379I, D380A, and Q381A) were generated using QuikChange PCR mutagenesis with the pET-28a-UGT76G1 vector as template and appropriate oligonucleotides. ACKNOWLEDGMENTS. E.S. was supported by US-Israel Vaadia Binational Agricultural Research Development Fund Postdoctoral Fellowship FI-504-14. Portions of this research were carried out at the Argonne National Protein Expression and Purification. The general protein expression and pu- Laboratory Structural Biology Center of the Advanced Photon Source, a rification protocol for UGT76G1 uses a combination of affinity and size- national user facility operated by the University of Chicago by Department exclusion chromatographies based on a previously published protocol (32). of Energy Office of Biological and Environmental Research Grant DE-AC02- For production of SeMet-substituted protein, the pET-28a-UGT76G1 construct 06CH11357.

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