Molecular basis for branched steviolglucoside biosynthesisSoon 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 CarolinaWilmington, 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, arenatural products roughly 200-fold sweeter than sugar and areused as natural, noncaloric sweeteners. Biosynthesis of rebaudio-side A, and other related stevia glucosides, involves formation ofthe steviol diterpenoid followed by a series of glycosylationscatalyzed by uridine diphosphate (UDP)-dependent glucosyltrans-ferases. UGT76G1 from Stevia rebaudiana catalyzes the formationof the branched-chain glucoside that defines the stevia moleculeand is critical for its high-intensity sweetness. Here, we report the3D structure of the UDP-glucosyltransferase UGT76G1, including acomplex of the protein with UDP and rebaudioside A bound in theactive site. The X-ray crystal structure and biochemical analysis ofsite-directed mutants identifies a catalytic histidine and how theacceptor site of UGT76G1 achieves regioselectivity for branched-glucoside synthesis. The active site accommodates a two-glucosylside chain and provides a site for addition of a third sugar moleculeto the C3′ position of the first C13 sugar group of stevioside. Thisstructure provides insight on the glycosylation of other naturallyoccurring sweeteners, such as the mogrosides from monk fruit,and a possible template for engineering of steviol biosynthesis.

glucosyltransferase | noncaloric sweetener | plant biochemistry | stevia |X-ray crystal structure

Sweeteners derived from plant natural products have signifi-cant potential as dietary supplements because they are stable

and noncaloric; can maintain good dental health by reducingsugar intake; and have possible uses by diabetic, phenylketon-uric, and obese patients (1, 2). For example, the sweetener steviais isolated from the leaves of Stevia rebaudiana (sweetleaf), aperennial herb native to Paraguay and Brazil (2, 3). The leaves ofthis plant contain a variety of ent-kaurene diterpenoid glycosidescomposed of a steviol aglycone decorated with different numbersand types of sugars attached to the C13 and C19 positions (2, 3)(Fig. 1A). The predominant steviol glucosides are stevioside (5–10% of leaf dry weight) and rebaudioside A (2–4% of leaf dryweight), which taste up to 300-fold sweeter than sucrose. Use ofthese compounds as naturally sourced noncaloric sweeteners hasexpanded globally over the last decade (1, 4). For example, rebi-ana, a commercially available sweetener, mainly contains rebau-dioside A, which reduces bitterness and aftertaste associated withother compounds isolated from the plant (4). Moreover, recentefforts to engineer steviol glucoside production in yeast aim toprovide specific types of molecules as a way to avoid variation thatresults from the use of different S. rebaudiana cultivars and growingconditions (5).The biochemical pathway for steviol glucoside biosynthesis

involves formation of the core steviol diterpenoid followed by aseries of glucosylations catalyzed by a set of uridine diphosphate(UDP)-dependent glucosyltransferases (UGT) (2, 6–12) (Fig.1A). Three UDP-glucosyltransferases in the rebaudioside Abiosynthesis pathway have been identified and shown to localizeto the cytosol (7, 8). Glycosylation of steviol by UGT85C2 beginsat the C13 hydroxyl group to yield steviolmonoside (7). Tran-script levels of UGT85C2 correlate with total accumulation of

steviol glucosides in plant tissue, which suggests this step is thelimiting reaction in the pathway (9). The UGT that modifies theC2′ of the C13 glucose to form steviolbioside remains to beidentified (6, 7). Next, UGT74G1-catalyzed glucosylation of thecarboxylate completes formation of stevioside (7). Furthermodification by UGT76G1 at the C3′ of the C13 sugar leads tosynthesis of rebaudioside A, which contains the branched glu-coside (7). Variants of UGT76G1 alter levels of rebaudioside Ain the plant (10, 11). The broad acceptor molecule activity ofUGT76G1 has been explored for biocatalyst uses in industrialsynthesis of a range of glucosylated natural products (12). Al-though the general sequence of modifications in the steviolglucoside pathway has been determined, the structural basis forselectivity of the UGT enzymes, in particular how UGT76G1catalyzes branched glucoside formation, remains unclear.

Results and DiscussionOverall Structure of UGT76G1.To understand the structural basis ofbranched steviol glucoside synthesis, the X-ray crystal structureof S. rebaudianaUGT76G1 was determined by single-wavelengthanomalous dispersion phasing using selenomethionine (SeMet)-substituted protein (Fig. 1B and Table 1). The SeMet-substitutedmodel was then used to solve 3D structures of the UGT76G1•UDP

Significance

The naturally occurring noncaloric sweetener stevia is a plantnatural product consisting of a core terpene structure deco-rated with a specific pattern of glucose molecules, including abranched three-sugar unit. Stevia and other related moleculesare being explored as noncaloric dietary sweeteners becausethey can help maintain the health of diabetic, phenylketonuric,and obese patients. Here, we describe the three-dimensionalstructure of the plant enzyme (UGT76G1) that forms thebranched group of sugars that defines the stevia molecule andis critical for its high-intensity sweetness. Understanding howthis enzyme forms this chemical group provides insight on howthe stevia plant makes this sweetener and suggests how toalter the protein to generate new versions of the noncaloricsweetener.

Author contributions: S.G.L., E.S., O.Y., and J.M.J. designed research; S.G.L. and E.S. per-formed research; O.Y. contributed new reagents/analytic tools; S.G.L., E.S., and J.M.J.analyzed data; and S.G.L., E.S., O.Y., and J.M.J. wrote the paper.

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).

This article is a PNAS Direct Submission.

Published under the PNAS license.

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 theUGT76G1•UDP•rebaudioside A complex (PDB ID code 6O88) were deposited in the Pro-tein Data Bank, https://www.rcsb.org.1To whom correspondence may be addressed. Email: jjez@wustl.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1902104116/-/DCSupplemental.

Published online June 10, 2019.

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(1.75 Å resolution) and UGT76G1•UDP•rebaudioside A (1.99Å resolution) complexes by molecular replacement. The overallstructure of UGT76G1 is monomeric and adopts the GT-Bfold, which consists of an N- and C-terminal Rossmann-folddomains (13). Each structural domain of UGT76G1 containsa central β-sheet flanked by multiple α-helices (N-terminal:β1a–β1g and α1–α9; C-terminal: β2a–β2f and α10–α17) (Fig.1B and SI Appendix, Fig. S1). The C-terminal domain containsthe UDP-binding site with a cleft between the two domainsproviding the sugar acceptor-binding site (13). The amino acidsequence and 3D structure of UGT76G1 indicate that this pro-tein is a member of the carbohydrate-active enzymes (CAZy)glycosyl transferase family 1 (14), which consists of relatedenzymes that glycosylate a variety of plant natural products.The 3D structure of UGT76G1 shares 2.2–2.9 Å2 root meansquare deviations (rmsds) for 420–440 Cα atoms with theother structurally characterized plant UGT. These includeenzymes that modify terpenoids (Medicago truncatula/barrelclover UGT71G1 and Orzya sativa/rice Os79), flavonoids andisoflavonoids (Vitis vinifera/grape GT1, M. truncatula UGT85H2and UGT78G1), chlorinated phenols (Arabidopsis thaliana/thale cress UGT72B1), anthocyanin floral pigments (Clitoriaternatea/bluebell vine UGT78K6), salicylic acid (A. thalianaUGT74F2), and indoxyl dyes (Polygonum tinctorium/Japanese

indigo GT-B) (15–23); however, all of these UGT transfersugars directly to the aglycone and do not form branched naturalproduct glucosides.

Structure of the UDP Sugar Donor-Binding Site. Clear electrondensity for UDP in the C-terminal domain of UGT76G1 definesthe location of the sugar donor-binding site (Figs. 1B and 2A).The uridine ring of UDP is sandwiched between Trp338 andGln341 with hydrogen bond interactions contributed by Val339,Pro340, and a water-mediated interaction with His260 (Fig. 2B).Glu364 forms a bidentate interaction with the hydroxyl groups ofthe ribose. Direct interactions with Ser285, His356, Asn360, andSer361, along with water-mediated contacts to Ser283 andThr284, position the diphosphate group toward the cleft betweenthe N- and C-terminal domains.A structure of UGT76G1 with UDP-glucose was not obtained;

however, Thr146, Trp359, and Asp380 bind a glycerol moleculein proximity to the diphosphate group of UDP (Fig. 2C). Theposition of this ligand mimics how the glucose moiety of thesugar donor would interact with UGT76G1. Comparison ofthe site where glycerol binds in UGT76G1 to the structure of theOs79 UGT from rice in complex with a nonreactive UDP-glucoseanalog, uridine-5′-diphosphate-2-deoxy-2-fluoro-α-D-glucose (U2F)(16), highlights the structural and sequence conservation of the

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 andC19 positions of the steviol aglycone are indicated. (B) Three-dimensional structure of UGT76G1. The ribbon diagram for the UGT76G1•UDP complex showsthe secondary structure with α-helices (blue) and β-strands (gold). UDP is shown as a space-filling model.

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 P3121Cell 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 ÅWavelength, Å 0.979 0.979 0.979Resolution, 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) 320,766/47,033 304,795/48,196 317,717/35,139Completeness (highest shell), % 99.6 (99.1) 93.5 (96.7) 99.5 (94.1)<I/σ> (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 — —

RefinementRcryst/Rfree, % 17.1/20.0 17.2/19.8 16.4/20.0No. of protein atoms 3,609 3,567 3,559No. of waters 369 267 223No. of ligand atoms 42 42 140rmsd, bond lengths, Å 0.007 0.007 0.007rmsd, bond angles, ° 0.912 0.922 1.03Avg. B-factor (Å2): protein,

water, ligand28.0, 36.6, 20.2 47.2, 46.1, 31.3 40.6, 67.3 43.5

Ramachandran plot: favored,allowed, disallowed, %

97.6, 2.4, 0.0 96.0, 4.0, 0.0 97.8, 2.2, 0.0

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sugar donor-binding site and the position of the catalytic histidinein each active site (Fig. 2 C and D). The 3D position of glycerol inthe UGT76G1 sugar donor-binding site fills the same space as theC3–C5 portion of the U2F glucose group from the Os79 structure.The residues in Os79 (i.e., Ser142, Gln143, Trp369, Asp385, andGln386) that interact with the sugar donor are either invariant orhighly conserved in UGT76G1 (Thr146, Ser157, Trp359, Asp380,and Gln381). Likewise, the histidine (His25 in UGT76G1; His27in Os79) that serves as a general base to abstract a proton from theacceptor molecule in the SN2 mechanism and the aspartate(Asp124 in UGT76G1; Asp120 in Os79) that stabilizes the histi-dine in the transfer reaction are invariant (24).Although UGT76G1 shares ∼25% sequence identity with

UGT85C2 and UGT74G1 from the steviol glucoside bio-synthesis pathway of S. rebaudiana, key active site residues areretained across these three enzymes (SI Appendix, Fig. S1).These features include the histidine and asparate of the catalyticdyad and residues (i.e., His260, Ser283, Thr284, Ser285, Trp338,Qln341, Asn360, and Glu364 of UGT76G1) that interact withthe UDP portion of the shared sugar donor substrate (SI Ap-pendix, Fig. S1, red and blue, respectively). Similarly, residuescritical for positioning the donor glucosyl group in UGT76G1(Thr146, Ser157, Trp359, Asp380, and Gln381) are maintainedin the other UGT of this biosynthetic pathway (SI Appendix, Fig.S1, purple). A large portion of the UDP-binding and sugardonor-binding sites are part of the canonical PSPG (putativesecondary plant glycosyltransferase) sequence motif found in theplant UGT (25) (SI Appendix, Fig. S1, black box), which isconsistent with conserved sequence and structure to bind theUDP molecule. This analysis indicates that the UGTs in thesteviol pathway retain highly conserved catalytic dyads and UDP-glucose sugar donor sites, yet each enzyme displays differences inacceptor regiospecificity.

Rebaudioside A Binding and Generation of the Branched Glucoside.As a branched sugar side chain-forming enzyme, UGT76G1differs from the other two UGTs in the steviol pathway, whichdirectly glucosylate the steviol aglycone via an oxygen at either

the C13 (UGT85C2) or C19 (UGT74G1) positions (Fig. 3A).For synthesis of the branched glucoside rebaudioside A, theacceptor site of UGT76G1 needs to accommodate a two-glucosylside chain to allow for the addition of a third sugar molecule tothe C3′ position of the first C13 sugar group of stevioside. Toachieve this regioselectivity, UGT76G1 requires a binding sitenear the UDP-glucose donor site that also orients the secondsugar away from the catalytic histidine.The structure of UGT76G1 in complex with UDP and

rebaudioside A reveals the basis for branched steviol glucosidesynthesis. Electron density for both ligands was visible in thestructure with the omit map for rebaudioside A showing cleardensity for the three glucosyl groups extending from the steviolC13, weaker density for the steviol core, and no density for theC19 glucose (Fig. 3B). Because the solvent-exposed portion ofrebaudioside A was disordered, a model of the ligand lacking theC19 sugar was used for refinement. The nucleotide is bound asobserved in the UDP complex and rebaudioside A binds in apocket of the N-terminal domain (Fig. 3C).The branched sugar portion of rebaudioside A is oriented into

the interior and toward the UDP pyrophosphate. From thebottom of the binding site, the terminal glucose (glc3) fills thesugar donor portion of the pocket; the branching glucose (glc2)is positioned into a pocket away from the catalytic histidine; andthe C13-linked glucose (glc1) and the steviol portion of themolecule extend out to the solvent exposed opening of the site(Fig. 3 C and D). In the acceptor site, Thr146, Trp359, Asp380,and Gln381 interact with glc3 and position the α1,3-linkage be-tween this glucosyl unit and a glc1 in proximity to His25 (Fig. 3D).Interactions with Met145, Ser147, Asn151, His155, and Leu379orient the α1,2-linked glc2 into a pocket away from the catalyticHis25 and Asp124. The C13-linked glucose group (glc1) stackswith Phe22 and Ile90 and forms a water-mediated interaction with

Fig. 2. UDP binding and sugar donor-binding site of UGT76G1. (A) Electrondensity for UDP shown as a 2Fo − Fc omit map (1.5σ). (B) Stereoview of theUDP-binding site in the UGT76G1•UDP complex. Ligand-binding interactionsare shown as dotted lines. (C) Glycerol binding in the sugar donor-bindingsite of the UGT76G1•UDP complex. Ligand-binding interactions are shown asdotted lines. The view is shown for comparison with D. (D) Sugar donor-binding site of rice UGT Os79 (16). The orientation is the same as the glycerolsite from UGT76G1. The X-ray structure of the nonreactive UDP-glucoseanalog U2F is shown for comparison with C.

Fig. 3. Structural basis for branched steviol glucoside synthesis by UGT76G1.(A) Schematic of sugar donor and acceptor in steviol UGT. The UDP-glucosedonor (UDP-Glc; red) and acceptor molecules (blue) are shown. Note that theoxygen in the acceptor of UGT85C2 and UGT74G1 differ. The position of thecatalytic histidine is indicated by the triangle. (B) Electron density forrebaudioside A shown as a 2Fo − Fc omit map (1.5σ). (C) Stereoview of themolecular surface of the UGT76G1 acceptor site. Crystallographically de-termined positions of UDP and rebaudioside A are shown. The surface cor-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 fourthsugar attached at the C13 carboxylate was not modeled because of disorder.(D) View of the rebaudioside A binding site of UGT76G1. As in C, the glc1,glc2, glc3, and steviol portions of the ligand are labeled. Hydrogen-bondinginteractions are shown as dotted lines. The two α-helices (α3 and α8) thatdefine the steviol aglycone binding site are also labeled.

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Gly24. The steviol group is sandwiched between Leu126 andresidues from α3 (Met88 and Ile90) and α8 (Leu200, Ile203,Leu204, and Met207). The C19 carbonyl hydrogen bonds with thePro84, which positions the crystallographically disordered sugar atthis position toward solvent. The UGT76G1 structure reveals anactive site that is generally divided between hydrophilic branchedglucoside-binding residues and largely apolar steviol interactionresidues; however, sequence comparisons also suggest that theresidues forming the glc2 pocket in UGT76G1 are varied inUGT85C2 and UGT74G1 (SI Appendix, Fig. S1).As shown schematically in Fig. 3A, the position of the diter-

pene core of steviol in the active site needs to vary betweenUGT76G1 and the other two enzymes in the stevia biosynthesispathway. UGT85C2 and UGT74G1 directly modify the terpene atthe C13 and C19 positions, respectively. In contrast, UGT76G1needs to bind stevioside with the terpene moiety further away fromthe catalytic site. Sequence comparison indicates that residues in theglc2- and stevia-binding regions of UGT76G1, along with thelengths of the α3 and α8 helices defining the acceptor site, varybetween the three UGT of the stevia pathway and that these ex-tensive changes likely contribute to different substrate preferences(SI Appendix, Fig. S1, yellow). In comparison with the residues ofthe glc2 pocket in UGT76G1, sequence comparison suggests thatlarger side chains are found in UGT85C2 and UGT74G1. For ex-ample, Met145, Ser147, and Leu379 are replaced with tryptophan,isoleucine, and tryptophan in UGT85C2 or phenylalanine, gluta-mine, and serine in UGT74G1. Each retains a histidine at position155 and has smaller side chains in place of Asn151 (a glycine inUGT85C2 and a valine in UGT74G1). Homology modeling of theother two UGT in the steviol biosynthesis pathway suggests that thevarious amino acid substitutions narrow the glc2 pocket, whichlikely occludes binding of longer side-chain steviol glucosides butallows for steviol and steviolbioside glucosylation in UGT85C2 andUGT74G1, respectively (SI Appendix, Fig. S2 A–C).Structural and sequence comparison of UGT76G1, a branch-

forming glycosyltransferase, to the plant UGTs that directlyglycosylate a given substrate (i.e., terpenoids, flavonoids, phe-nols, anthocyanins, and indoxyls; refs. 15–23) highlights key dif-ferences in residues forming the glc2 site (SI Appendix, Fig. S2D–G). For each of these enzymes, residues defining the glc3 site,in which the glucosyl group of a UDP-sugar donor binds, arehighly conserved, as expected for UGTs (SI Appendix, Fig. S2G).In contrast, each of the other plant UGT examined have multiplebulky side-chain substitutions in residues corresponding to the glc2site of UGT76G1. In particular, Leu126, Met145, and His155 ofUGT76G1 are typically replaced by phenylalanine, phenylalanine/tryptophan, and phenylalanine/tyrosine, respectively (SI Appendix,

Fig. S2G). Examination of the anthocyanidin glucosyltransferaseUGT78K6, flavonoid glucosyltransferase GT1, and indoxyl glu-cosyltransferase GT-B X-ray crystal structures (SI Appendix, Fig.S2 D–F) highlight how various changes reduce the available spacein the region corresponding to the glc2 site of UGT76G1. Theintroduction of key changes in the glc2 site of UGT76G1 arecritical for the evolution of the branch chain-forming glucosyl-transferase 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 biochemicalactivity (Fig. 4 and Table 2). Substitution of the catalytic histi-dine with an alanine (H25A) eliminated enzymatic activity. Theanalogous histidine residue in other UDP-glucosyltransferasesfacilitates a direct displacement SN2-like mechanism. This resi-due acts as a general base by abstracting a proton from the ac-ceptor substrate to yield an oxyanion nucleophile that reacts withthe UDP-sugar acceptor (13).Mutagenesis of residues in the rebaudioside A-binding site and

biochemical analysis of the mutants reveals the importance of keyresidues for enzymatic activity. Removal of the side chains of Asp380(D380A) and Gln381 (Q381A) in the glc3/sugar donor-binding site

Fig. 4. Summary of wild-type and mutant UGT76G1 enzyme activities. Acomparison of wild-type and mutant UGT76G1 catalytic efficiencies (kcat/Km)with stevioside (black) and UDP-glucose (white) is shown. Steady-state ki-netic parameters are summarized in Table 2.

Table 2. Summary of wild-type and mutant UGT76G1 steady-state kinetic parameters

Protein Varied substrate kcat, min−1 Km, μM kcat/Km, M−1·s−1

WT Stevioside 33.8 ± 0.7 360 ± 23 1,560UDP-Glucose 32.5 ± 1.5 943 ± 96 574

L126I Stevioside 0.09 ± 0.01 676 ± 320 2UDP-Glucose 0.06 ± 0.04 12,500 ± 10,700 0.1

M145F Stevioside 14.9 ± 1.8 3,030 ± 580 82UDP-Glucose 13.5 ± 1.2 5,940 ± 710 38

M145W Stevioside 1.0 ± 0.2 7,690 ± 2,780 2UDP-Glucose 0.8 ± 0.1 17,900 ± 4,800 1

T146A Stevioside 8.9 ± 0.7 7,340 ± 1,420 20UDP-Glucose 5.3 ± 0.2 8,770 ± 674 10

S147A Stevioside 1.4 ± 0.1 2,570 ± 558 9UDP-Glucose 0.8 ± 0.1 3,750 ± 355 4

S147T Stevioside 0.6 ± 0.1 927 ± 163 11UDP-Glucose 0.7 ± 0.1 477 ± 49 24

S147N Stevioside 0.6 ± 0.1 880 ± 162 11UDP-Glucose 0.7 ± 0.1 2,160 ± 320 5

N151A Stevioside 9.2 ± 0.4 1,600 ± 125 96UDP-Glucose 8.6 ± 0.5 1,870 ± 190 76

N151Q Stevioside 27.8 ± 0.4 1,210 ± 34 390UDP-Glucose 105 ± 18 21,200 ± 4,090 83

H155A Stevioside 11.4 ± 0.8 424 ± 69 448UDP-Glucose 12.8 ± 0.1 577 ± 16 370

H155R Stevioside 3.4 ± 0.1 1,040 ± 72 54UDP-Glucose 4.3 ± 0.3 3,290 ± 290 22

H155W Stevioside 7.8 ± 0.4 2,090 ± 180 62UDP-Glucose 6.1 ± 0.1 3,220 ± 96 32

L200I Stevioside 43.0 ± 19 1,750 ± 1,120 410UDP-Glucose 30.7 ± 2.0 2,270 ± 240 225

L204I Stevioside 27.1 ± 2.7 762 ± 150 593UDP-Glucose 24.7 ± 1.2 1,090 ± 113 378

M207F Stevioside 22.9 ± 2.4 885 ± 172 431UDP-Glucose 19.2 ± 0.5 1,050 ± 56 305

M207W Stevioside 34.7 ± 18.9 4,710 ± 3090 123UDP-Glucose 68.6 ± 22.4 15,600 ± 5,700 73

L379I Stevioside 42.6 ± 3.5 1,120 ± 160 634UDP-Glucose 33.2 ± 0.7 1,420 ± 54 390

Assays were performed as described in Methods. Average values ± SD(n = 3) are shown.

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resulted in inactive enzyme. Based on their position in the conservedPSPG sequence motif (SI Appendix, Fig. S1), loss of either side chainremoves key interactions with the sugar being transferred fromdonor to acceptor and likely results in compromised substratebinding, which prevents efficient catalysis. Likewise, alterationof Thr146, which is situated between the glc3/sugar donor-binding site and the glc2 site, either lost activity (T146N) ordecreased catalytic efficiency by 80-fold (T146A). Mutations ofresidues in the glc2 site resulted in a range of effects. Substi-tutions of Ser147 yielded mutants with ∼170-fold reductions incatalytic efficiency (S147A, S147T, and S147N). Modest 3- to15-fold changes in kcat/Km were observed with the N151A,N151Q, H155A, and L379I mutants. Mutations that introducelarger side chains in to the glc2 site at His155 (H155R andH155W) led to ∼25-fold less-efficient variants. Change ofMet145 to either a phenylalanine (M145F) or tryptophan(M145W) resulted in 20- and 750-fold reductions in kcat/Km. Inthe apolar steviol-binding cleft, a subtle change of Leu126 toisoleucine (L126I) leads to a 750-fold decrease in catalytic ef-ficiency. Other point mutants, such as L200I, L204I, andM207F, displayed modest threefold changes with the M207Wmutant having a 13-fold effect. Overall, biochemical analysis ofthe UGT76G1 mutants confirms the conserved function of thecatalytic histidine and identifies critical residues in the glc2-and steviol-binding sites.

Conclusion. This first structure of a branched steviol glucoside-producing enzyme (i.e., UGT76G1) provides insight on how theenzyme accommodates large sugar side chains and differs fromthe other glycosyltransferases in the pathway. Importantly, thebranched-chain glycosylation pattern built from the C19 positionof rebaudioside A leads to rebaudiosides D and M, which aremore commercially interesting steviosides because they deliverhigh-intensity sweetness with less off-taste than rebaudioside A(26). Similar glycosylation patterns are found in the biosynthesisof other molecules, such as the mogrosides from monk fruit(Siraitia grosvenorii), which are being explored as stevia alter-nates (27). Knowledge of the active site architecture also pro-vides a template for enzyme engineering that may lead to thedevelopment of variants with altered regiospecificity and/orsubstrate glycosylation patterns that can be combined with varieddonor substrates either in vitro or in vivo (28–31). Moreover, theamino sequence of the glc2 site of UGT76G1, which allows forformation of a branched-sugar modified product (i.e., rebau-dioside A), may provide a useful “signature motif” to bio-informatically distinguish branched chain-forming UGT fromthose that directly glucosylate various substrates when assessingpotential metabolic function. Such structure-guided efforts offerthe potential for altered pathways of steviol production to gen-erate tailored variants of this noncaloric sweetener.

MethodsChemicals, Codon Optimized Gene Synthesis, and Site-Directed Mutagenesis.All reagents used were purchased from Sigma-Aldrich unless noted other-wise. An Escherichia coli codon-optimized version of the gene encodingUGT76G1 was generated for protein expression. The original sequence(SwissProt Q6VAB4; ref. 7) was optimized and synthesized by GenScript. Theresulting gene was inserted into pET-28a to yield the pET-28a-UGT76G1 forexpression of an N-terminally His6-tagged fusion protein. Site-directed mu-tants of UGT76G1 (H25A, L126I, M145F, M145W, T146A, T146N, S147A,S147T, S147N, N151A, N151Q, H155A, H155R, H155W, L200I, L204I, M207F,M207W, L379I, D380A, and Q381A) were generated using QuikChange PCRmutagenesis with the pET-28a-UGT76G1 vector as template and appropriateoligonucleotides.

Protein Expression and Purification. The general protein expression and pu-rification protocol for UGT76G1 uses a combination of affinity and size-exclusion chromatographies based on a previously published protocol (32).For production of SeMet-substituted protein, the pET-28a-UGT76G1 construct

was transformed into E. coli BL21 (DE3) cells, which were grown to A600 ∼ 0.8at 37 °C in M9minimal media supplementedwith SeMet containing 50 μg·mL−1

kanamycin (33). Protein expression was induced by addition of isopropyl 1-thio-β-D-galactopyranoside (IPTG; 1 mM final) with cells growth continuedovernight (16 °C). Cells were pelleted by centrifugation (10,000 × g) andresuspended in lysis buffer [50 mM Tris, pH 8.0, 500 mM NaCl, 20 mM imid-azole, 1 mM β-mercaptoethanol, 10% (vol/vol) glycerol, and 1% (vol/vol)Tween-20]. After sonication of the resuspended cells, debris was removed bycentrifugation (30,000 × g). The lysate was passed over a Ni2+-nitriloacetic acid(Qiagen) column equilibrated with wash buffer (lysis buffer minus Tween-20).Bound his-tagged protein was eluted with elution buffer (wash buffer with250 mM imidazole). The eluant was further purified by size-exclusion chro-matography using a Superdex-200 26/60 HiLoad FPLC column equilibratedwith 50 mM Tris, pH 8.0, 25 mM NaCl, 1 mM Tris(2-carboxyethyl)phosphine.Peak fractions were collected and concentrated to ∼10 mg·mL−1 using cen-trifugal concentrators (Amicon). Bradford assay with BSA as the standard wasused to determine protein concentration. For storage at −80 °C, purifiedprotein was flash-frozen in liquid nitrogen. Expression of wild-type and mu-tant UGT76G1 proteins used similar protocols, except that Terrific brothreplaced the minimal media.

Protein Crystallization and Structure Determination. Purified UGT76G1 wasconcentrated to 10 mg·mL−1 and crystallized using the hanging-drop vapor-diffusion method with a 2-μL drop (1:1 concentrated protein and crystalli-zation solution). Diffraction quality crystals of both SeMet-substituted andnative protein were obtained at 4 °C with 15% (wt/vol) PEG 4000, 20% 2-propanol (vol/vol), 100 mM sodium citrate tribasic dihydrate buffer (pH 5.6),and either 5 mM UDP or 5 mM UDP and 5 mM rebaudioside A. To generatethe UGT76G1•UDP•rebaudioside A complex, 5 mM UDP and 5 mM rebau-dioside A [in 10% (vol/vol) DMSO] were added during protein concentration.Individual crystals were flash-frozen in liquid nitrogen with the mother li-quor containing 25% glycerol as a cryoprotectant. Diffraction data (100 K)was collected at the Argonne National Laboratory Advanced Photon Source19-ID beamline. HKL3000 (34) was used to index, integrate, and scale dif-fraction data. The structure of SeMet-substituted UGT76G1 was determinedby single-wavelength anomalous diffraction (SAD) phasing. SHELX (35) wasused to determine SeMet positions and to estimate initial phases from thepeak wavelength dataset. Refinement of SeMet positions and parameterswas performed with MLPHARE (36). Solvent flattening using density modi-fication implemented with ARP/wARP (37) was employed to build an initialmodel. Subsequent iterative rounds of manual model building and refine-ment, which included translation-libration-screen parameter refinement,used COOT (38) and PHENIX (39), respectively. The structures of UGT76G1 incomplex with either UDP or UDP and rebaudioside A were solved by mo-lecular replacement in PHASER (40) using the SeMet structure as a searchmodel with refinement and building performed as above. The final modelof the SeMet-substituted UGT76G1 includes residues Arg12-Pro169 andArg174-Leu458, one UDP molecule, one glycerol molecule, and 369 waters.The UGT76G1•UDP complex includes the same residues and ligands, but with267 waters. The UGT76G1•UDP•rebaudioside A complex includes the sameresidues, one UDP molecule, one rebaudioside A molecule (modeled with-out the C19 sugar), and 223 waters. Data collection and refinement statis-tics are summarized in Table 1. Coordinates and structure factors for theUGT76G1(SeMet)•UDP complex (PDB ID code: 6O86), the UGT76G1•UDPcomplex (PDB ID code: 6O87), and the UGT76G1•UDP•rebaudioside A com-plex (PDB ID code: 6O88) were deposited in the Protein Data Bank.

Enzyme Assays. UDP-glycosyltransferase activity was monitored spectropho-tometrically (A340) using a coupled assay system (41). Standard reactionconditions were 50 mM Hepes, pH 7.5, 200 μM NADH, 500 μM phosphoenolpyruvate, 10 mMMgCl2, two units of pyruvate kinase, and six units of lactatedehydrogenase in a 0.1-mL reaction at 25 °C. Reactions were initiated byaddition of protein with changes in absorbance measured on a Tecan 96-well plate reader. Steady-state kinetic parameters were determined by ini-tial velocity experiments with either varied stevioside concentrations and5 mM UDP-glucose or varied UDP-glucose and 2 mM stevioside. Data were fitto the Michaelis–Menton equation, v = kcat[S]/(Km + [S]), using Kaleidagraph.

ACKNOWLEDGMENTS. E.S. was supported by US-Israel Vaadia BinationalAgricultural Research Development Fund Postdoctoral Fellowship FI-504-14.Portions of this research were carried out at the Argonne NationalLaboratory Structural Biology Center of the Advanced Photon Source, anational user facility operated by the University of Chicago by Departmentof Energy Office of Biological and Environmental Research Grant DE-AC02-06CH11357.

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