Method Article

Structural Biology and Analytical Chemistry Approaches for Characterizing C-Glycoside Metabolic Enzymes in Human Gut Microbiota

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DOI:

10.3791/67629

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May 23rd, 2025

 ,  ,  ,  , 
1The School of Life Science, Beijing University of Chinese Medicine

* These authors contributed equally

In This Article

Summary

Recent studies highlight the roles of human gut microbiota enzymes in metabolizing C-glycosides. Here, we present a comprehensive methodology integrating analytical chemistry techniques (LC-MS/MS and NMR) with structural biology approaches to systematically characterize the structural and enzymatic reaction chains of these C-glycoside-metabolizing enzymes.

Abstract

The gut microbiota has emerged as a key modulator of host health, particularly because of its capacity to metabolize dietary glycosides. While C-glycosides demonstrate superior chemical stability and resistance to enzymatic cleavage compared to O-glycosides, specialized gut bacteria can selectively metabolize them, improving their bioavailability. Since the identification of microbial C-glycosidase enzymes, the precise reaction mechanisms remain incompletely understood, and standardized methodologies for studying these processes are still not well-established. To address this gap, we introduced an integrated multidisciplinary approach combining structural biology, enzymology, and analytical techniques, including liquid chromatography-tandem mass spectrometry (LC-MS/MS) and nuclear magnetic resonance (NMR), to systematically characterize these enzymatic activities. This comprehensive approach enables precise characterization of C-glycoside metabolism at molecular, enzymatic, and metabolic levels.

Introduction

Recently, the human gut microbiota has gained considerable attention for its crucial role in supporting host health1,2. These microbial communities metabolize host-derived compounds, producing biologically active metabolites1,2,3. For instance, flavonoids, among the most abundant natural compounds in our daily diet, primarily exist as O- or C-glycosides, which themselves are poorly absorbed in the human gut4. However, gut microbiota can transform these glycosides into more bioactive derivatives, such as secondary glycosides or aglycones, reducing their solubility and enhancing their absorption, thereby improving their bioactivities compared to their native forms5,6.

Historically, the hydrolysis mechanisms of O-glycosides have been extensively studied7,8. The C-glycosides, characterized by their C-C glycosidic bonds, exhibit greater chemical stability and resistance to cleavage9,10. Despite this, certain gut bacteria have evolved the ability to deglycosylate C-glycosides11,12. In 1988, Hattori et al. first demonstrated that some gut bacteria could remove glycosyl groups from C-glycosides13. Subsequent studies isolated the first bacterial strain capable of this activity, enabling further exploration of C-glycoside cleaving mechanisms14. To date, however, only a limited number of intestinal bacterial strains and C-glycoside-metabolizing enzymes have been identified and characterized14,15,16,17,18,19,20,21,22,23,24,25,26. Recent studies have shown that the deglycosylation puerarin (daidzein-8-C-glucoside) enzymes (DgpA/B/C) in the PUE strain are able to cleave puerarin into daidzein and glucose through a two-step processes: DgpA, a Gfo/Idh/MocA family oxidoreductase, oxidizes puerarin to 3''-oxo-puerarin, which then undergoes a β-elimination reaction mediated by the DgpB/C complex to yield the aglycone and glucose10,26. Nevertheless, the structural and functional characteristics of C-glycoside-metabolizing enzymes, particularly the DgpA/B/C system, remain incompletely understood, and there is a need for more advanced methodologies, particularly the integration of biological and chemical strategies, to advance research in this field24.

To address this gap, we employ integrated multidisciplinary approaches to investigate the human gut microbiota DgpA/B/C-catalyzed C-glycoside metabolic reaction. In this reaction, DgpA catalyzes the glycosyl oxidation of C-glycosides, subsequently, DgpB and DgpC form a DgpB/C complex that specifically cleaves the oxidized C-glycoside products10,26,27. We first performed recombinant expression and purification of DgpA and DgpB/C separately. The purified DgpA and DgpB/C proteins were then crystallized, and their three-dimensional structures were determined by X-ray crystallography. Finally, we reconstituted the C-glycoside cleavage function using the complete DgpA/B/C enzyme system, with comprehensive metabolic product analysis conducted via coupled LC-MS/MS and NMR spectroscopy27. Based on these approaches, we have studied C-glycoside metabolism catalyzed by the DgpA/B/C enzyme system.

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Protocol

1. Protein production and purification

  1. Construction of expression vector
    NOTE: In this study, the DgpA, DgpB, and DgpC genes from the human gut microbiota, PUE strain, serve as a model system. The first step involves using expression plasmids to produce the corresponding recombinant proteins.
    1. Obtain the DgpA/B/C gene cluster sequence (GenBank: LC422372.1) from NCBI. Then, commercially synthesize the DgpA/B/C DNA and individually clone DgpA, DgpB, and DgpC genes into a modified pET-28a vector, which will produce the target protein with the N-terminal SUMO and 6×His tag (His-SUMO tag)28.
    2. Transform each of the DgpA, DgpB, or DgpC plasmids into E. coli BL21 (DE3) competent cells. Subsequently, culture bacteria in LB medium with 50 µg/mL kanamycin at 37 °C.
    3. Add 0.2 mM IPTG to the medium when the bacterial density (OD600) reaches 0.6. Culture the bacteria at 16 °C for 16 h.
    4. Centrifuge each of the DgpA, DgpB, and DgpC expressing bacterial culture at 4,000 x g for 20 min at 4 °C. Resuspend the cell pellet in 20 mL of Buffer A (Table 1) for every 1 L of bacterial culture.
  2. Purification of protein
    NOTE: High-purity, sufficient quantities of recombinant enzymes are essential for functional characterization and structural biology studies. DgpB and DgpC form a stable protein complex (spontaneously form DgpB/C complex during the lysis procedure) for functionality10,26. The purification of DgpA protein and DgpB/C complex is then performed.
    1. Add 1 mM PMSF to the bacterial resuspension. Mix the DgpB and DgpC bacterial suspension from step 1.1.4 together and then lyse the DgpA bacterial suspension or the DgpB and DgpC mixture (to form functional DgpB/C complex) using an ultrasonic cell disruptor.
    2. Centrifuge the bacterial lysate at 48,000 x g for 40 min at 4 °C. Retain the supernatant containing the protein and discard the bacterial pellet.
    3. Purify DgpA or DgpB/C complex from the supernatant using a Nickel affinity chromatography (Ni-NTA) column. Elute the bound proteins by applying a 0 - 50% imidazole concentration gradient with Buffer A and Buffer B (Table 1) in the ÄKTA purifier.
    4. Take 10 µL of eluted samples and mix them with 2 µL of 5 × protein dye, then perform SDS-PAGE electrophoresis at 220 V for 45 min using a vertical electrophoresis system, followed by Coomassie Blue staining to visualize protein bands. Collect fractions containing DgpA or the DgpB/C complex based on SDS-PAGE analysis for future use.
      NOTE: The DgpA (44 kDa) with a His-SUMO tag (17 kDa) band migrates at around 61 kDa (Figure 2D), while DgpC (33 kDa) with a His-SUMO tag (17 kDa) migrates around 50 kDa, and DgpB (15 kDa) with a His-SUMO tag (17 kDa) migrates around 33 kDa (Figure 2J)..
    5. Dialyze the DgpA or DgpB/C complex samples from step 1.2.3 using 10 kDa molecular weight cutoff dialysis membrane against Buffer C to remove imidazole, and with Ubiquitin-like protease 1 (ULP1; Table of Materials) enzyme to remove the His-SUMO tag29.
    6. Load the dialyzed protein sample in Buffer C (Table 1) through the ion-exchange chromatography column (Q column). Then, elute the bound protein from the column using a sodium chloride concentration gradient ranging from 100 mM to 600 mM, prepared with Buffer C and Buffer D (Table 1) in the ÄKTA purifier.
    7. Perform the SDS-PAGE analysis of DgpA and DgpB/C complex eluted from the Q column as the step 1.2.3, noting the molecular weights: His-SUMO tag (17 kDa), DgpA (44 kDa), oligomeric bands of DgpA (above 95 kDa) (Figure 2E), DgpB (15 kDa), DgpC (33 kDa), oligomeric bands of DgpB and DgpC (between 53 kDa and 95 kDa) (Figure 2K).
    8. Purify the protein using a size-exclusion column (SEC) with Buffer E (Table 1). Perform SDS-PAGE analysis using proteins from the elution fraction, followed by Coomassie Blue staining to visualize protein bands. Collect the fractions containing the target protein DgpA or DgpB/C complex after analysis by SDS-PAGE.
      NOTE: DgpA band is seen at around 44 kDa with oligomerized DgpA band above 95 kDa (Figure 2F), while DgpC is at around 33 kDa, and DgpB is at around 15 kDa with oligomerized DgpB and DgpC (between 53 kDa and 95 kDa) (Figure 2L).
    9. Measure the protein concentration of DgpA or DgpB/C complex using a nanodrop at 280 nm. Then, concentrate the DgpA or DgpB/C complex to about 20 mg/mL with ultrafiltration tubes, aliquot them, and flash freeze them with liquid nitrogen. Store DgpA or DgpB/C complex at -80 °C for future use.
  3. Circular dichroism (CD) spectrum data collection
    NOTE: Since the glycosyl oxidation catalyzed by DgpA serves as the crucial initiation step in the metabolic pathway, CD spectroscopy was specifically employed to verify both the proper folding state and secondary structural integrity of DgpA. These structural parameters are critically important for both enzymatic activity and successful protein crystallization.
    1. Dilute DgpA protein to 0.2 mg/mL with 1 x PBS buffer (pH 7.4) (Table 1).
    2. Load 1x PBS buffer (pH 7.4) into a quartz cuvette and record the baseline CD spectrum over 200 - 260 nm using a CD spectrometer to sensitively detect secondary structure and folding state differences27.
    3. Replace the buffer cuvette with protein samples from 1.3.1, record the CD spectrum over 200 - 260 nm, and subtract the buffer baseline to get the CD spectrum for the protein sample.
    4. Collect three scans for accuracy and analyze the data for secondary structure features and protein folding state (Figure 3G, 3H).

2. Protein crystallography and structure determination

NOTE: After confirming that DgpA maintained proper folding and structural integrity, structural biology approaches were employed to determine the three-dimensional structures of both DgpA. Given that the catalytic competence of the DgpB/C complex had been biochemically confirmed through previous functional assays10,26,27, CD spectral verification step was omitted.

  1. Protein crystal growth and optimization
    1. Mix 0.5 µL of purified DgpA (20 mg/mL) or DgpB/C complex (20 mg/mL) with an equal volume (0.5 µL) of reservoir solution from crystallization kits on 48-well crystal plates in a sitting-drop method30. Transfer the crystal plates to 16 °C for crystal growth.
      NOTE: Observe these sitting drops with a microscope to identify the conditions (including protein concentration and precipitants, such as PEG 3350, magnesium formate, etc.) for DgpA or DgpB/C complex crystal growth.
    2. Perform crystallization optimization for both DgpA and the DgpB/C complex using the hanging-drop vapor diffusion method30.
    3. For DgpA, begin with the condition identified at step 2.1.1 (20 mM magnesium formate, 20% PEG3350) and apply an orthogonal optimization approach by testing six PEG3350 concentration gradients (17%, 18%, 19%, 20%, 21%, and 22%) against four magnesium formate concentration gradients (16 mM, 18 mM, 20 mM, and 22 mM)30.
    4. Prepare crystallization drops by mixing 0.8 µL of DgpA protein solution (10 or 20 mg/mL) with 0.8 µL of reservoir solution on silanized glass coverslips. Then invert the coverslips over 24-well crystallization plates and incubate at 16 °C for crystal growth.
    5. For the DgpB/C complex optimization, follow the same approach: mix 0.8 µL of protein complex (10 or 20 mg/mL) with 0.8 µL of reservoir solution containing magnesium acetate (16, 18, 20, or 22 mM) and PEG 8000 (8%, 9%, 10%, 11%, 12%, or 13%). Similarly, set up hanging drops on silanized coverslips over 24-well plates and incubate at 16°C.
    6. Let the protein crystals of the DgpA and DgpB/C complex grow to full size until they cease to grow, and then transfer the crystals to a soaking solution containing reservoir buffer and 5 mM of the substrate compounds (glucose and puerarin) of the DgpA and DgpB/C complex at 16 °C for 30 min.
    7. Transfer the crystals, including DgpA (with glucose or substrate-free) and DgpB/C complex (substrate-free), to a cryoprotectant solution containing the soaking buffer condition supplemented with 25% glycerol and immediately store them in liquid nitrogen for data collection.
      NOTE: Although both DgpA and the DgpB/C complex crystals were soaked with substrates (glucose and puerarin), only glucose was successfully incorporated into the catalytic center of DgpA. Consequently, three distinct crystal forms were obtained for structural analysis: DgpA with glucose (Figure 3E); substrate-free DgpA (Figure 3B-C); and substrate-free DgpB/C complex (Figure 3D). The cryoprotectant solution safeguards protein crystals by preventing ice formation during flash-cooling in liquid nitrogen, thereby preserving their structural integrity and minimizing radiation-induced damage. Typically formulated using the mother liquor as a base, the solution is supplemented with optimized concentrations of cryoprotectants (e.g., glycerol or ethylene glycol).
  2. Data collection and structure determination
    1. Collect the crystal's X-ray diffraction data for DgpA with glucose or in substrate-free forms, and DgpB/C complex in substrate-free form at the Shanghai Synchrotron Radiation Facility (SSRF) of the Shanghai National Protein Science Facility (NFPS) with an incident wavelength of 0.978 Å.
    2. Process the three sets of X-ray diffraction data, including DgpA and DgpB/C complex, using a crystallographic data processing software31. First, load the raw data, then sequentially execute the index, integrate, and scale functions by clicking the corresponding buttons in the software. Generate a processed output file (output.sca) suitable for subsequent structure determination.
    3. Determine the structures of DgpA with glucose or in substrate-free forms (Figure 3B-C, 3E), and DgpB/C complex in substrate-free (Figure 3D) using the molecular replacement method (Table of Materials). And then refine the structures and electron maps by incorporating relevant ligands (such as glucose, Mn2+, H2O, etc.)31.

3. Reaction activity monitoring and determination of kinetic parameters

NOTE: The DgpA/B/C system mediates C-glycoside transformations through a defined two-step cascade: DgpA initiates catalysis by oxidizing the sugar moiety, generating an intermediate that the DgpB/C complex subsequently cleaves or isomerizes10,26,27. In this study, DgpA/B/C refers to the enzymes responsible for this reaction cascade, while it should be noted that no direct interaction occurs between DgpA and the DgpB/C complex. This coupled activity can be rigorously quantified using three complementary approaches: HPLC product analysis, DCPIP-coupled spectrophotometric assays monitoring electron transfer, or detailed kinetic characterization of the reaction parameters27,32. Specifically, the C-glycoside cleavage products (daidzein) generated by DgpA/B/C catalysis were analyzed by HPLC (UV detection at 265 nm). Product formation was quantified using a standard calibration curve (The method for establishing the standard curve is described in detail in the note under Step 3.3.2.), and kinetic parameters were determined by fitting the product formation rates to the Michaelis-Menten model through nonlinear regression analysis27,32. The inhibition assay measuring glucose's effect on puerarin C-glycosidic bond cleavage provided corroborating evidence that saccharides can serve as substrates for DgpA. Subsequently, the DCPIP assay monitors sugar oxidation reactions by measuring the reduction of blue-colored 2,6-dichlorophenolindophenol (DCPIP) to colorless leuco-DCPIP as it accepts electrons from oxidized sugars33, thereby reflecting the catalytic activity of DgpA.

  1. Monitoring of DgpA/B/C activity by HPLC
    1. Conduct the DgpA/B/C-catalyzed C-glycoside cleavage or isomerization reaction in 500 µL of Buffer F (Table 1) containing 20 µM DgpA and DgpB/C complex (from Step 1.2.7), and 0.1 mM of either: puerarin or genistein-8-C-glucoside for cleavage reactions, or daidzin or genistin for isomerization reactions27. Carry out the reactions at 37 °C for 8 h and terminate them by adding 1.5 mL of methanol to the reaction mixture.
    2. Centrifuge the reaction solution at 16,000 × g for 15 min to remove the protein precipitate formed due to methanol-induced enzyme denaturation. Filter the supernatant using a 0.22 µm filter membrane.
    3. Perform HPLC analysis with gradient elution, at a flow rate of 1 mL/min with an injection volume of 10 µL and detection wavelength of UV 265 nm.
      NOTE: The mobile phases were acetonitrile (Mobile Phase A) and 0.1% aqueous acetic acid (Mobile Phase B), with gradient elution. The gradient of the mobile phase elution is as follows: 5%-20% A at 0-5 min, 20%-45% A at 5-15 min, 45%-52% A at 15-18 min, 52%-55% A at 18-19 min, 55%-5% A at 19-21 min, and 5% A at 21-23 min. The DgpA/B/C enzyme system exhibits markedly different catalytic efficiencies toward substrates such as daidzin and genistin, resulting in substantial variations in their respective product formation rates. To quantitatively assess the substrate conversion kinetics and product accumulation dynamics, a comprehensive time-course analysis was performed using HPLC at 0, 2, 4, 6, 12, 16, 20, 24, 36, and 48 h intervals.
  2. Monitoring of DgpA activity with 2,6 -dichlorophenol indophenol (DCPIP) assay
    NOTE: The DCPIP assay monitors sugar oxidation by measuring the reduction of blue DCPIP to colorless leuco-DCPIP as it accepts electrons from oxidized sugars33. Here, DCIPIP reporter assay was utilized to record the DgpA oxidation activity toward different glycosylated compounds, monosaccharides, disaccharides, or polysaccharides. Wildtype DgpA and its sugar binding defective mutants including D182A, Δ(308-316) and Δ(311-316) were used27.
    1. Incubate 0.8 mM DCPIP, 50 µM protein including wildtype DgpA and its three sugar-binding defective mutants (DgpA D182A, DgpA Δ(308-316), or DgpA Δ(311-316)), with 10 mM substrates including puerarin, vitexin, genistin, soluble starch, maltotriose, lactose, sucrose, glucose, or maltose in 1x PBS buffer (pH 7.4).
    2. After 20 h of reaction, record the absorbance change of DCPIP at 600 nm using a microplate reader33.
  3. Determination of the inhibition rate of glucose on the C- glycosidic bond cleavage reaction of puerarin
    NOTE: DgpA belongs to the sugar oxidoreductase enzyme family, capable of oxidizing the sugar moiety of C-glycosides. Figure 3E demonstrates that glucose is bound to the DgpA catalytic center, and the protocol to determine the DgpA structure is detailed in step 2.2.3. To examine the competitive binding of DgpA between glucose and the C-glycoside puerarin, the following experiments were conducted.
    1. Carry out the reaction in 1 × PBS buffer (pH 7.4). Add different concentrations of glucose (1 mM, 5 mM, 10 mM, 20 mM, 50 mM, 80 mM, and 100 mM) to the C-glycoside (puerarin) cleavage reaction system as described in step 3.1. Perform the reaction at 37 °C for 12 h.
    2. Perform quantitative analysis of the product (daidzein) using the HPLC calibration curve for the reference compound (standard daidzein). Calculate the inhibition rate by first determining the difference in product yield between reactions containing varying glucose concentrations and uninhibited C-glycoside cleavage reactions (without glucose), and then dividing this difference by the product yield from the uninhibited reaction.
      NOTE: To generate a calibration curve for quantitative HPLC analysis, prepare a serial dilution of a reference standard (e.g., puerarin or daidzein) across a concentration range (e.g., 0.1-1 mM, in 0.1 mM increments). Analyze each concentration by HPLC, record the peak areas, and plot them against the corresponding standard concentrations. Perform linear regression analysis (y = mx + b, where y = peak area, x = concentration) to establish the standard curve. The regression equation can then be used to interpolate unknown sample concentrations from their measured peak areas.
  4. Determination of kinetic parameters
    NOTE: The kinetic parameters are widely recognized as critical gold standards for quantitatively evaluating enzymatic catalytic efficiency. To quantitatively assess DgpA/B/C-catalyzed reactions, the kinetic parameters for the C-glycoside puerarin cleavage reactions catalyzed by DgpA/B/C were determined.
    1. Prepare substrate C-glycoside (puerarin) concentration gradients (ranging from 0.01 to 4 mM in 2-fold increments) for the DgpA/B/C-catalyzed C-glycoside cleavage reaction.
    2. Carry out the reaction at 37 °C for 8 h according to the method described in step 3.1. Perform quantitative analysis of the product (daidzein) using the HPLC calibration curve of standard daidzein. Then calculate the reaction rate (product formation rate) by dividing the quantified amount by the reaction time.
    3. Determine the Km and kcat kinetic parameters by fitting the calculated reaction rate and substrate concentration to the Michaelis-Menten model using statistical software (Table of Materials).

4. Preparation and detection of reaction intermediate product

NOTE: Beyond C-glycoside cleavage activity, DgpA/B/C also catalyzes the isomerization of O-glycosides to C-glycosides. Therefore, when O-glycosides are used as substrates for DgpA/B/C, the intermediate products will be produced as the isomerized C-glycosides. To characterize the intermediate product (puerarin), the intermediate compound was purified and subsequently analyzed using analytical chemistry tools, including LC-MS/MS and NMR spectroscopy.

  1. Preparation of reaction intermediate product in DgpA/B/C-catalyzed C -glycoside isomerization reaction 27
    1. Prepare the DgpA/B/C-catalyzed O- to C-glycoside isomerization reaction using 0.2 mM O-glycoside (daidzin) as substrates and 20 µM DgpA and DgpB/C complex in a 90 mL reaction volume, following the protocol described in the above step 3.1.
    2. Purify the intermediate product C-glycoside (puerarin) using a preparative HPLC at the gradient elution strategy (NOTE).
      NOTE: The mobile phases were acetonitrile (Mobile Phase A) and 0.1% aqueous acetic acid (Mobile Phase B), with gradient elution. The gradient of the mobile phase elution is as follows: 5%-20% A at 0-5 min, 20%-45% A at 5-15 min, 45%-52% A at 15-18 min, 52%-55% A at 18-19 min, 55%-5% A at 19-21 min, and 5% A at 21-23 min.
    3. Calculate the concentration of the purified intermediate product using the standard calibration HPLC curve in step 3.3.2, get about 6 mg puerarin the 90 mL reaction system at step 4.1.1, and then dry the purified intermediate products with a vacuum centrifugal concentrator for future use.
  2. Characterization of reaction intermediate product with LC-MS/MS
    1. Collect the dried intermediate products from step 4.1.3. Weigh 100 µg of the daidzin-derived intermediates, then dissolve it in 2 mL of methanol to achieve a final concentration of 50 µg/mL. Centrifuge at 13,500 x g for 10 min and collect the supernatant for LC-MS/MS analysis.
    2. Use the same HPLC gradient elution program at step 4.1.2 for LC-MS/MS analysis with an injection volume of 5 µL.
  3. Characterization of reaction intermediate product with NMR
    1. Dissolve 4 mg of the purified intermediate products from the daidzin reaction in 340 µL of dimethyl sulfoxide-d6 for 1H and 13C NMR spectroscopy. Set instrument parameters as follows: temperature at 298 K, shim 3 times, pulse width 18 µs, relaxation delay 1.5 s, 48 scans, with other parameters at default settings27.
    2. Collect the NMR 1H and 13C spectra using a 600 MHz NMR instrument.

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Results

Following the procedures outlined in Figure 1, we first expressed the DgpA, DgpB, and DgpC genes in E. coli BL21(DE3) cells. Since DgpB and DgpC form a stable enzyme complex (DgpB/C complex)10,26,27,34, cells expressing these two proteins were combined for co-purification. After cell lysis, the supernatant was collected by ...

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Discussion

Currently, the structural and functional characteristics of C-glycoside metabolism enzymes in the field remain unclear, and appropriate research methods are lacking10,24,26. By leveraging the significant advantages of combined chemistry and biotechnology40,41,42,43,

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Disclosures

The authors declare no competing interests.

Acknowledgements

This study is funded with Startup fund program at Beijing University of Chinese Medicine (BUCM) (90011451310011) to W.M., the emergency fund against COVID-19 program at BUCM (1000061223476) to W.M., the innovation team and talents cultivation program of national administration of traditional Chinese medicine (ZYYCXTD-C-202006) to W.M. and the independent research project for graduate students at BUCM(ZJKT2023012) to H.P. We thank all of the lab members in the Wenfu Ma lab for helpful discussion.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
2,6-dichloroindophenol (DCPIP)Solarbio Science and TechnologyD6350
2-Mercaptoethanol(β-ME)Sigma-Aldrich08168
  5 × Protein Loading DyeSangon BiotechC508320
Acetic acidShanghai MACKLINA111247
AcetonitrileThermo Fisher Scientific™?InvitrogenA12147
Bruker Avance III HD 700 MHz NMR SpectrometerBrukerAVANCE III HD 700MHz
Bruker Ultrashield 400 Plus 400MHzBrukerAVANCE III 400 MHz
Centrifuge 5424REppendorf022620601
Chirascan™ plus CD spectrometerApplied PhotophysicsChirascan™ 
DaidzeinShanghai Yuanye Bio-TechnologyB20227
DaidzinShanghai Yuanye Bio-TechnologyB20226
Dithiothreitol(DTT)Solarbio Science and TechnologyD8220
E. coli BL21 (DE3) competent cellsThermo Fisher Scientific™?InvitrogenEC0114
GenisteinShanghai Yuanye Bio-TechnologyS31565
Genistein-8-C-glucosideShanghai Yuanye Bio-TechnologyBP2095
GenistinShanghai Yuanye Bio-TechnologyS31591
GlucoseSigma-AldrichG8270
GraphPad Prism 9GraphPad SoftwareVersion?Prism 9Calculate the Km and kcat kinetic parameters
HiLoad Superdex 200 pg preparative SEC columnsCytiva28989335
HisTrap HP His tag protein purification columnsCytiva17524802
HiTrap Q FF anion exchange chromatography columnCytiva17515601
HKL-3000HKL research Inc version 723
HPLC Column TC-C18(2), 170Å, 5 µm, 4.6 x 250 mmAgilent Technologies518925-902
Isopropyl β-D-1-thiogalactopyranoside?IPTG)Sigma-AldrichPHG0010
LactoseSigma-Aldrich17814
LC-20AT, high-performance liquid chromatographyShimadzuLC20AT
MaltoseSigma-Aldrich47288
MaltotrioseSigma-Aldrich443713
Manganese chlorideShanghai Yuanye Bio-TechnologyS31112
MethanolThermo Fisher Scientific™?InvitrogenM23142
NAD+Solarbio Science and TechnologyN8110
Olympus SZX12 Stereo MicroscopeOLYMPUS SZX7
phenylmethylsulfonyl fluoride (PMSF)Sigma-AldrichP7626 
PhenixThe Phenix development team1.21.2Phenix is a comprehensive software suite for protein crystal structure determination, incorporating SAD (Single-wavelength Anomalous Dispersion) and MR (Molecular Replacement) methods with Phaser software.
Preparative high-performance liquid chromatographWaters2545/2998
Protein Crystal Screening KitHampton ResearchHR2-110/HR2-112/HR2-144/HR2-126/HR2-098
PuerarinShanghai Yuanye Bio-TechnologyB20446
Shaking incubatorShanghai Zhichu Instrument Co.,Ltd ZQZY-AF8
SnakeSkin™ Dialysis Membrane, 10 kDa MWCO, 35 mmThermo Fisher Scientific™?Invitrogen88245
Soluble starchSigma-AldrichS9765
Sorvall LYNX 6000 Superspeed CentrifugeThermo Fisher Scientific™75006590
SpectraMax i3x Multi-Mode Microplate ReaderMOLECULAR DEVICESSpectraMax i3x 
SpectraMax QuickDrop Micro-Volume Spectrophotometer SpecificationsMOLECULAR DEVICESQuickDrop
SucroseSigma-AldrichS5016
SunFire C18 OBD Prep ColumnWaters186002741
Thermo Fisher Scientific Q EXACTIVEThermo Fisher Scientific™?InvitrogenIQLAAEGAAPFALGMBDK
Ubiquitin-like protease 1Thermo Fisher Scientific™?Invitrogen12588018
Ultra Low Temperature FreezerThermo Fisher Scientific905GP-ULTS
Vacuum centrifugal concentratorBeijing JM TechnologyCV600
VitexinSigma-Aldrich49513

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C Glycoside MetabolismGut Microbiota EnzymesProtein CrystallographyLC MS AnalysisNMR SpectroscopyEnzyme KineticsChromatography PurificationFlavonoid Metabolism

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