Overexpression, purification, biochemical and structural characterization of rhamnosyltransferase UGT89C1 from Arabidopsis thaliana

Accepted Manuscript Overexpression, purification, biochemical and structural characterization of rhamnosyltransferase UGT89C1 from Arabidopsis thaliana Guangning Zong, Jie Li, Yanrong Gao, Shuang Fei, Xiao Liu, Xiaoqiang Wang, Yuequan Shen PII:

S1046-5928(18)30466-2

DOI:

https://doi.org/10.1016/j.pep.2018.12.007

Reference:

YPREP 5368

To appear in:

Protein Expression and Purification

Received Date: 26 August 2018 Revised Date:

17 December 2018

Accepted Date: 27 December 2018

Please cite this article as: G. Zong, J. Li, Y. Gao, S. Fei, X. Liu, X. Wang, Y. Shen, Overexpression, purification, biochemical and structural characterization of rhamnosyltransferase UGT89C1 from Arabidopsis thaliana, Protein Expression and Purification (2019), doi: https://doi.org/10.1016/ j.pep.2018.12.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Overexpression, purification, biochemical and structural characterization of rhamnosyltransferase UGT89C1 from

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Arabidopsis thaliana

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Xiaoqiang Wanga,b*, Yuequan Shena,c*

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Guangning Zonga,b, Jie Lia,b, Yanrong Gaoa,c, Shuang Feia,b, Xiao Liua,c,

State Key Laboratory of Medicinal Chemical Biology, b College of Pharmacy,

and c Department of Biochemistry and Molecular Biology, College of Life Sciences, Nankai University, Tianjin 300353, People’s Republic of China.

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*Correspondence e-mail: [email protected], [email protected]

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ABSTRACT The uridine diphosphate glycosyltransferase (UGT) plays the central role in

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glycosylation of small molecules by transferring sugars to various acceptors including bioactive natural products in plants. UGT89C1 from Arabidopsis thaliana is a novel UGT, a rhamnosyltransferase, specifically recognizes

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UDP-L-rhamnose as donor. To provide an insight into the sugar specificity for

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UDP-L-rhamnose and interactions between UGT89C1 and its substrates, the UGT89C1 was expressed in Escherichia coli and purified toward biochemical and structural studies. Enzyme activity assay was performed, and the recombinant UGT89C1 recognized UDP-L-rhamnose and rhamnosylated kaempferol. Crystals

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of AtUGT89C1 were obtained, they diffracted to 2.7 Å resolution and belonged to space group I41. AtUGT89C1 was also co-crystallized with UDP. Interestingly, two crystal forms were obtained in the same crystallization condition, including

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the previous I41 crystal form, and the new crystal form that diffracted to 3.0 Å

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resolution and belonged to space group P21.

Keywords: rhamnosyltransferase; rhamnosylation; UDP-L-rhamnose; X-ray

diffraction; crystallization

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ACCEPTED MANUSCRIPT 1. Introduction Glycosylation is the most common reaction in living cells and plays important roles in development, defense, and cellular homeostasis [1].

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Glycosylation reaction transfers sugar from an activated donor to different acceptors such as natural products in plants, improving their stability and solubility, enhancing their bioactivity, and facilitating their storage and

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accumulation in plant cells [2-4]. Uridine diphosphate glycosyltransferases (UGTs)

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are members of family 1 of the glycosyltransferase superfamily, which utilize UDP-activated sugars as donors and transfer the sugars to small molecules for glycosylation (http://afmb.cnrs-mrs.fr/CAZY/index.html)[5]. A large number of UGTs have been identified in genome of plants, including Arabidopsis thaliana,

consensus

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based on analysis of genomic sequences [6], and they contain a highly conserved signature

sequence

called

the

Putative

Secondary

Plant

Glycosyltransferase (PSPG) motif [7,8].

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Flavonoids and isoflavonoids are a large class of natural products, flavonoids

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are identified in all plant species, and isoflavonoids are primarily produced in legumes. (Iso)flavonoids possess antioxidant, anticancer, antibacterial and antiangiogenic activities, and they are important drug scaffolds for drug discovery

[9]. Modification of the chemical structures of flavonoids and isoflavonoids can

regulate their chemical properties and bioactivities, and glycosylation is a key modification by utilizing UGTs as synthetic biological tools in this process. So far, crystal structures of several plant UGTs have been determined, 3

ACCEPTED MANUSCRIPT including M. truncatula UGT71G1 [10], UGT85H2 [11] and UGT78G1 [12]; Vitis vinifera VvGT1 [13]; A. thaliana UGT72B1 [14], and UGT74F2 [15]; Clitoria ternatea UGT78K6 [16]; Oryza sativa Os79 [17]; and Polygonum tinctorium

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PtUGT1 [18]. However, these UGTs only recognize UDP-glucose as donor, no structure is available for UGTs which utilize other types of sugar donors such as

sugar specificity is not well understood.

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UDP-galactose, UDP-glucuronic acid, UDP-xylose and UDP-rhamnose, and the

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The current UGT structural studies revealed that the glucose moiety of sugar donor mainly interacts with the last two residues of the PSPG motif (e.g., Glu381 and Gln382 in UGT71G1) and another highly conserved tryptophan (e.g., Trp360 in UGT71G1) which may be the determinants for glucose recognition and

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specificity [10,19]. A modeling and mutagenesis study showed that Arg25 in red daisy BpUGT94B1 [20], and Arg350 in lamiales F7GAT UGT88D7 [21] are key amino acids for their specificity with UDP-glucuronic acid, and these arginines

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can interact with anionic carboxylate of the glucuronic acid moiety of

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UDP-glucuronic acid. More structural and biochemical studies on these glucuronosyltransferases and other UGTs with various sugar preferences are needed to further elucidate the complex sugar donor specificity. A. thaliana UGT89C1 was identified as a flavonol 7-O-rhamnosyltransferase

by transcriptome coexpression analysis and reverse genetics, and can recognize UDP-L-rhamnose or TDP-L-rhamnose as the sugar donor for rhamnosylation of flavonols [22,23]. In order to further reveal the detailed interactions between the 4

ACCEPTED MANUSCRIPT enzyme and its substrates, especially the new type of sugar donor UDP-L-rhamnose for understanding the sugar specificity, AtUGT89C1 was expressed and purified. Enzyme activity assays were performed. Crystals were

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obtained for both AtUGT89C1 and AtUGT89C1 with UDP. The X-ray diffraction analysis was further carried out toward structure determination by X-ray

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crystallography.

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2. Materials and methods 2.1. Cloning

The DNA sequence encoding AtUGT89C1 (residues 1-435; the numbering is according to Uniprot entry Q9LNE6) was amplified from the cDNA library of

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Arabidopsis thaliana by a sticky-end polymerase chain reaction (PCR) using the primers listed in Table 1. The PCR product was digested with BamH1 (Thermo Scientific) and EcoR1 (Thermo Scientific), and then inserted into the expression

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vector pET-32M (Novagen) with a 6xHis tag and Trx-tag, and a PreScission

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protease cleavage site (LEVLFQGP) at the N-terminus. The amplified sequence was confirmed by DNA sequencing (Genewiz, People’s Republic of China).

2.2. Overexpression and purification The recombinant plasmid containing AtUGT89C1 was transformed into E. coli BL21-CodonPlus (DE3) cells (Novagen) via heat-shock procedure. Positive transformants were selected on an LB-agar (0.5% yeast extract, 1% tryptone, 1% 5

ACCEPTED MANUSCRIPT sodium chloride, 1.5% agar) plate supplemented with 50µg/ml ampicillin (Sigma-Aldrich), and subsequently inoculated in a 5 ml LB medium (0.5% yeast extract, 1% tryptone, 1% sodium chloride) containing ampicillin and grown for 12

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h at 310 K with agitation. Then the overnight cell culture was transferred into 1 L LB medium supplemented with ampicillin, and grown at 310 K with agitation. When the OD600nm of cell culture reached about 0.6-0.8, 150 µM isopropyl

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β-D-1-thiogalactopyranoside (IPTG) was added and then further incubated at 289

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K for 16-18h. The cells were harvested by centrifugation at 5000g for 15min at 277 K and resuspended in lysis buffer (20mM Tris-HCl pH 7.5, 500mM NaCl, 10 mM imidazole). The cells were disrupted by sonication on ice with 1 s pulses and 3 s pauses for 8 min in the presence of 1.0 mg/ml lysozyme (Sigma-Aldrich), and

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then centrifuged at 18000g for 45 min. The supernatant containing soluble proteins was gently mixed with Ni2+-NTA agarose (Qiagen). After incubation for 60 min at 277 K, the mixture was transferred into a disposable column, and

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nonspecifically bound proteins were washed off from the Ni2+-NTA column with

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washing buffer (20 mM Tris-HCl pH 7.5, 500 mM NaCl, 20 mM imidazole pH 7.5). The target protein was eluted from the column with elution buffer (20 mM Tris-HCl pH 7.5, 500 mM NaCl, 250 mM imidazole pH 7.5). The eluted protein was loaded onto a Hiload 26/60 Superdex200 size-exclusion column (GE Healthcare) for further purification and exchange into a buffer containing 20 mM Tris-HCl pH 7.5, 500 mM NaCl, 1mM DTT. The target protein was collected and digested by incubation with PreScission protease at 4℃ for 30 min, and then 6

ACCEPTED MANUSCRIPT applied to a Hiload 26/60 Superdex 200 size-exclusion column for further purification with BS200 buffer (20 mM Tris-HCl pH 7.5, 200 mM NaCl, 1mM DTT). Following the purification with the size-exclusion chromatography, the

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elution peak containing the target UGT89C1 protein was analyzed by 13% Coomassie-stained SDS-PAGE, and sample was subsequently pooled and concentrated to 10mg/ml using Amicon ultra centrifugal filter units (30kDa

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molecular-weight cutoff, Millipore). The protein concentration was measured

2.3. Assay of enzyme activity

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using the Pierce BCA protein assay kit (Thermo Scientific).

The enzyme activity of recombinant UGT89C1 was carried out in vitro using UDP-L-rhamnose as sugar donor. The glycosylation reaction was performed with

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50µg recombinant UGT89C1 in total volume of 100 µl mixing with 50 mM NaH2PO4 pH 7.6, 150 µM kaempferol, 500 µM UDP-L-rhamnose. After incubation for 30 min at 303K, the reaction was stopped by the addition of 10 µl

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of trifluoroacetic acid, and the supernatant was recovered by centrifugation at

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14000g for 10min. The resultant supernatant was immediately analyzed by HPLC system (Agilent 1260 Infinity) [22,23]. Solvent A was 0.05% trifluoracetic acid and solvent B was acetonitril. The sample (40µl) was applied to Eclipse Pl.us C18 reverse-phase column (5µm, 4.6x250mm, Alilent) and eluted with an increasing gradient of acentonitrile (0-2 min, 10%; 2-20 min, 10-70%; 20-24 min, 70%; 24-28 min, 100%; 28-30 min, 50% and 30-35 min, 50-10%) at a flow rate of 1 ml/min. The eluants were monitored at 255, 300 and 320 nm. The reaction 7

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were

identified

using

LC-QTOF-ESI/MS

(ACQUITY

UPLC

I-Class/UPCC/M-Class/SYNAPT G2-SI, Waters) in the negative ion mode. 2.4. Crystallization

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All crystals were grown by the sitting-drop vapour diffusion method by mixing 0.8µL protein solution (10mg/ml in 20 mM Tris-HCl pH 7.5, 500 mM NaCl, 1mM DTT) with 0.8µL reservoir solution in 48-well plates. Crystallization

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screening of AtUGT89C1 was initially performed at 293 K with commercially

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available kits from Hampton Research (Index, Crystal Screen, PEGRx, PEGIon, SaltRx) and Emerald Bio (Wizard 1 and Wizard 2). Small crystals appeared in 0.2M (NH4)2SO4, 0.1M sodium cacodylate pH 6.5, 30% (w/v) PEG8000 in ten days. To improve the quality of the crystals, optimization was subsequently

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performed by altering the concentration of salt from 0.05 to 0.4M, the pH of buffer from 5.0 to 8.5, and the concentration of precipitant from 20% to 40% (w/v). Additionally, we further screened crystallization conditions by using additive

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screen kits (Hampton Research) and silver bullets kits (Hampton Research). The

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best crystals were obtained with 0.2M (NH4)2SO4, 0.1M sodium cacodylate pH 6.0, 25% (w/v) PEG8000, 3% (v/v) ethylene glycol as reservoir solution. Co-crystallization of AtUGT89C1 and UDP was carried out by mixing protein solution with 5mM UDP, and two crystal forms were obtained in 0.2M ammonium acetate, 20% (w/v) PEG3350. In a search for cryoprotectants, the crystals were found to be stable in reservoir solution containing 30% (v/v) glycerol. The crystals were also picked up and washed in the crystallization solution twice prior to 8

ACCEPTED MANUSCRIPT dissolution in the gel-filtration buffer and finally analyzed in a 13% SDS-PAGE gel to confirm whether they consisted of the target protein. The detailed

2.5. X-ray diffraction data collection and processing

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information of crystallization is summarized in Table 2.

Crystals were recovered in 0.3-0.4 mm nylon loops (Hampton Research) and

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immediately flash cooled in liquid nitrogen. X-ray diffraction data were collected

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on beamline BL-19U1 at Shanghai Synchrotron Radiation Facility (SSRF). Diffraction experiments were carried out at 100K and the images were recorded on a CCD Pilatus CBF. A total of 180 images were collected at a crystal-to-detector distance of 450 mm with 0.3 or 0.5 s exposure for every 1°

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oscillation frame. The Data were indexed, integrated and scaled using HKL-2000 [24]. The data-collection and processing statistics are summarized in Table 3.

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3. Results and discussion

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In order to obtain insights into the catalytic mechanism of UGT89C1 and the unique sugar specificity for UDP-L-rhamnose, we expressed, purified and crystallized

UGT89C1

for

further

biochemical

and

structural

studies.

Rhamnosyltransferase UGT89C1 was expressed in E. coli BL21-CodonPlus (DE3)

cells and purified by metal-affinity and size-exclusion chromatography, yielding approximately 0.5mg target protein per litre of LB cell culture. However, there were many proteinaceous impurities in the eluted protein sample after the 9

ACCEPTED MANUSCRIPT purification with a Ni2+-NTA column (Fig. 1a). We attempted to remove the HIS-tag of the eluted protein by digestion with PreScission protease, but the protein appeared unstable and precipitated (Fig. 1b). Therefore we changed the

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strategy and performed chromatography first, i.e., the eluted protein from Ni2+-NTA column was loaded on a size-exclusion column for exchanging the buffer and also removing proteinaceous impurities, followed by digestion with

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PreScission protease. After PreScission protease digestion, the sample was loaded

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on a size-exclusion column again for further purification, and we successfully obtained stable and pure target protein (Fig. 1c and 1d).

Enzymatic activity assays of the recombinant UGT89C1 were carried out using UDP-L-rhamnose as sugar donor and kaempferol as acceptor. The reaction

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product was separated by HPLC, the analyses revealed a new peak (retention time (tR): 14.32 min) which was well resolved from the substrate peak (tR: 17.101 min) (Fig. 2a). This peak was further analyzed by quadruple time-of-flight electrospray

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ionization mass spectrometry (QTOF-ESI/MS). The molecular mass of the

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product was consistent with 7-O-rhamnoside kaempferol (calculated mass for the molecular formula C21H19O10 [M-H]-m/z- 431.0978 and observed mass [M-H]-m/z-

431.0982) (Fig. 2d). The control sample of mixture without UGT89C1 was also analyzed by HPLC (Fig. 2b) and confirmed by ESI/MS (Fig. 2c). The data suggested the recombinant UGT89C1 can produce a single glycosylated product from kaempferol. Subsequently, we screened and optimized the crystallization condition, and 10

ACCEPTED MANUSCRIPT obtained triangular pyramidal crystals of UGT89C1 for collecting data (Fig. 3a). A diffraction data set up to 2.7Å resolution was collected (Fig. 4a), processed and scaled. The crystal belonged to space group I41, with unit-cell parameters a=80.65,

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b=80.65, c =340.19Å, α= β= γ=90.00°. A calculated Matthews coefficient VM of 2.88Å3 Da-1 [25,26] suggested that there were two molecules in each asymmetric unit with the solvent content of 57.29%. The data-processing statistics are given in

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Table 3.

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For the co-crystallization of UGT89C1 and UDP, two crystal forms were obtained in the same crystallization drop including one similar to that of apo-enzyme described above, and a new crystal form (Fig. 3b). A diffraction data set up to 3.0Å resolution was collected for the new crystal form of UGT89C1

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co-crystallized with UDP (Fig. 4b), and the crystal belonged to space group P21 with unit-cell parameters a = 92.97, b = 84.14, c = 129.15Å, β = 108.90°. A calculated Matthews coefficient VM of 2.49Å3 Da-1 [25,26] suggested that there

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were four molecules in each asymmetric unit with the solvent content of 50.57%

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(Table 3). It is possible that the new P21 crystal form may contain the UDP, and the I41 form which is the same as that of apo-enzyme may not contain the UDP although it was obtained in the experiment with 5mM UDP. The further structural study will help to confirm this hypothesis. The sequence-alignment analysis indicated that the structure of UGT71G1 (PDB: 2acv) may be the best template for a molecular replacement study of AtUGT89C1. They share 27% sequence identity. A molecular-replacement (MR) 11

ACCEPTED MANUSCRIPT study was performed using a program phaser in PHENIX [27], and a solution with a translation-function Z-score (TFZ) of 8.0 and a convincing log-likelihood (LLG) gain of 38 was identified. Then, refinement was performed using phenix-refine in

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PHENIX [28], resulted in a model with Rwork and Rfree as 44.09% and 55.20%, respectively. However, the model rebuilding and further refinement are challenging. Preparation of a selenomethionine derivative of UGT89C1 and

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further crystallization optimization are in progress.

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A detailed structural analysis will help us to understand the molecular mechanism of rhamnosylation mediated by UGT89C1 and the sugar donor

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specificity for UDP-L-rhamnose.

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Figures

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Fig. 1. Purification of UGT89C1 and SDS-PAGE analysis. (a) SDS-PAGE analysis

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of recombinant UGT89C1 protein purification with Ni2+-NTA column. Lane 1, lysate of recombinant protein before loaded on Ni2+-NTA column; Lane 2, the flow through sample after binding with Ni2+-NTA column; Lane 3-4, the samples from

washing with buffer of low concentration imidazole; Lane 5-8, the eluent with buffer of high concentration imidazole. (b) SDS-PAGE analysis of the UGT89C1 protein His/Trx-tag cleavage by PreScission protease. Lane M, molecular-mass marker (labelled in kDa); Lane 1, the sample after purification with Ni2+-NTA 13

ACCEPTED MANUSCRIPT agarose and before cleavage of the His/Trx-tag; Lane 2, the soluble portion of sample after cleavage of the His/Trx-tag; Lane 3, the precipitated portion of sample after cleavage of the His/Trx-tag. (c) Size-exclusion FPLC chromatogram of the

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metal-affinity-purified and tag-cleaved UGT89C1. (d) SDS-PAGE analysis of protein samples purified by size-exclusion FPLC chromatography. Lane L, protein sample before loaded onto the gel-filtration column; lanes 1-6, fraction samples

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lane M, molecular-mass marker (labelled in kDa).

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corresponding to the major elution peak in the size-exclusion FPLC chromatogram;

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Fig. 2. HPLC analyses of the reaction products of UGT89C1 enzyme assay. (a) Elution profiles of reaction mixture of kaempferol with UGT89C1 at the UV

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absorbance 350 nm. (b) Elution profiles of reaction mixture of kaempferol with no UGT89C1 as control. (c) Mass spectrograms of kamepferol and (d) its product. The

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kamepferol and its product were further confirmed by high resolution mass analyses at negative ion, respectively.

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Fig. 3. Characterization of the crystals of UGT89C1. (a) Crystals of UGT89C1 obtained from 0.2 M (NH4)2SO4, 0.1 M sodium cacodylate, pH 6.0; 25% (w/v) PEG8000, and 3% ethylene glycol; (b) Crystals of UGT89C1 co-crystallized with

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UDP in 0.2M ammonia acetate, 20% (w/v) PEG3350 (labelled with red box); (c) SDS-PAGE analysis

of the dissolved crystals of the UGT89C1 and

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UGT89C1-UDP. The gel was stained with Coomassie Brilliant Blue R-250. Lane M, molecular-mass marker (labelled in kDa); lane 1, dissolved crystals of the

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UGT89C1; lane 2, dissolved crystals of the UGT89C1 co-crystallized with UDP.

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Fig. 4. Representative diffraction images obtained from a crystal of the UGT89C1 (a)

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and UGT89C1 co-crystallized with UDP (b).

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Table 1. Macromolecule-production information Protein

UGT89C1

Source organism

Arabidopsis thaliana

DNA source

A. thaliana cDNA ATCCTCGGATCCATGACAACAACAACAACGAAGAAGC

**

Reverse primers

AGCATTGAATTCTTACAAACACATCTCTGCAACGAG

Cloning vector

pET-32M (containing N-terminal 6xHis tag, Trx tag and a PreScission

Forward primers

protease cleavage site)

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*

E. coli BL21 CodonPlus (DE3)

Complete amino-acid sequence of

MSDKIIHLTDDSFDTDVLKADGAILVD

the construct produced***

FWAEWCGPCKMIAPILDEIADEYQGK

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Expression host

LTVAKLNIDQNPGTAPKYGIRGIPTLLL

FKNGEVAATKVGALSKGQLKEFLDAN

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LAGSGSGHMHHHHHHSSGLEVLFQGP

GSMTTTTTKKPHVLVIPFPQSGHMVPH LDLTHQILLRGATVTVLVTPKNSSYLDA LRSLHSPEHFKTLILPFPSHPCIPSGVESL QQLPLEAIVHMFDALSRLHDPLVDFLSR QPPSDLPDAILGSSFLSPWINKVADAFSI KSISFLPINAHSISVMWAQEDRSFFNDL

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ETATTESYGLVINSFYDLEPEFVETVKT RFLNHHRIWTVGPLLPFKAGVDRGGQ SSIPPAKVSAWLDSCPEDNSVVYVGFG SQIRLTAEQTAALAAALEKSSVRFIWAV RDAAKKVNSSDNSVEEDVIPAGFEERV

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KEKGLVIRGWAPQTMILEHRAVGSYLT HLGWGSVLEGMVGGVMLLAWPMQA DHFFNTTLIVDKLRAAVRVGENRDSVP

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DSDKLARILAESAREDLPERVTLMKLR EKAMEAIKEGGSSYKNLDELVAEMCL†

* The BamH1 site is underlined.

** The EcoR1 site is underlined.

*** Blue colours denote the amino-acid sequences of N-terminal tag will be cleaved before

crystallization.

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ACCEPTED MANUSCRIPT Table 2. Crystallization information of UGT89C1 UGT89C1

UGT89C1+UDP

Method

Sitting-drop vapour diffusion

Sitting-drop vapour diffusion

Plate type

48-well plate

48-well plate

20℃

20℃

Protein concentration (mg ml )

10.0

10.0

Buffer composition of protein

20 mM Tris-HCl pH 7.5, 200 mM

20 mM Tris-HCl pH 7.5, 200 mM

solution

NaCl, 1mM DTT

NaCl, 1mM DTT

Composition of reservoir solution

0.2 M (NH4)2SO4, 0.1M sodium

0.2M ammonia acetate, 20% (w/v)

cacodylate pH 6.0, 25% (w/v)

PEG3350

Temperature -1

PEG8000 1.6µL(1:1 ratio of protein solution and reservoir solution) 100µL

Table 3. Data collection and processing

and reservoir solution)

100µL

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Volume of reservoir

1.6µL(1:1 ratio of protein solution

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Volume and ratio of drop

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Crystal

UGT89C1

Diffraction source

BL-19U1, SSRF

BL-19U1, SSRF

Wavelength(Å)

0.9775

0.97853

Temperature(K)

100

Detector

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Crystal

UGT89C1+UDP

100

CCD Pilatus CBF

CCD Pilatus CBF

Crystal-to-detector distance (mm)

450

450

Rotation range per image(°)

1.0

1.0

180

180

0.3

0.5

I41

P21

a =80.65, b=80.65, c =340.19

a =92.97, b=84.14, c =129.15

α= β=γ=90.00

α=γ=90.00, β=108.90

Total rotation range(°) Space group

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Exposure time per image (s)

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Unit-cell parameters(Å, °) Resolution range(Å)

50.00-2.7

50.0-3.0

No. of unique reflections

16058

39311

Completeness(%)

99.9(99.5)

98.7(97.4)

Multiplicity

6.8(5.7)

3.4(3.4)

˂ I/σ (I)˃

16.5(1.9)

14.3(2.0)

11.9(57.1)

8.3(61.6)

Rmeas† (%)

† Rmeas = ∑hkl{N(hkl)/[N(hkl)-1] ∑i│Ii(hkl) - │/∑hkl∑iIi (hkl), where N(hkl) is the multiplicity, Ii(hkl) 1/2

is the intensity of the ith measurement of reflection hklandis the average value over multiple measurements.

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Acknowledgements We thank the staff of beamline BL19U1 at the Shanghai Synchrotron Radiation Facility for excellent technical assistance during the data collection. This work was

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Technology Commission (15JCZDJC65500).

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supported by the Fund for Natural Sciences of Tianjin Municipal Science and

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References

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Highlights •

Expression in Escherichia coli and purification of the recombinant UGT89C1

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have been established • The recombinant UGT89C1 specifically recognized UDP-L-rhamnose and rhamnosylated kaempferol

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• UGT89C1 was crystallized without and with UDP, and two crystal forms were

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obtained in the same co-crystallization experiment with UDP .

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• The X-ray crystallographic analysis of UGT89C1 was performed and discussed

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