Name: Structural Biology and Analytical Chemistry Approaches for Characterizing C-Glycoside Metabolic Enzymes in Human Gut Microbiota (Video) | JoVE Journal
Uploaded: 2025-05-23T12:50:00.000+00:00
Duration: 13 min 35 s
Description: Learn structural biology and analytical chemistry methods to characterize C-glycoside metabolic enzymes in human gut microbiota. This integrated approach reveals enzymatic functions using protein crystallography and LC-MS/MS techniques.

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5 minutes

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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The authors declare no competing interests.

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,<sup class="xref"...

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&#39;&#39;-oxo-puerarin, which then undergoes a &#946;-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.

<table><tbody><tr><td>2,6-dichloroindophenol (DCPIP)</td><td>Solarbio Science and Technology</td><td>D6350</td><td></td></tr><tr><td>2-Mercaptoethanol(&amp;beta;-ME)</td><td>Sigma-Aldrich</td><td>08168</td><td></td></tr><tr><td>&amp;nbsp; 5 &amp;times; Protein Loading Dye</td><td>Sangon Biotech</td><td>C508320</td><td></td></tr><tr><td>Acetic acid</td><td>Shanghai MACKLIN</td><td>A111247</td><td></td></tr><tr><td>Acetonitrile</td><td>Thermo Fisher Scientific&amp;trade;?Invitrogen</td><td>A12147</td><td></td></tr><tr><td>Bruker Avance III HD 700 MHz NMR Spectrometer</td><td>Bruker</td><td>AVANCE III HD 700MHz</td><td></td></tr><tr><td>Bruker Ultrashield 400 Plus 400MHz</td><td>Bruker</td><td>AVANCE III 400 MHz</td><td></td></tr><tr><td>Centrifuge 5424R</td><td>Eppendorf</td><td>022620601</td><td></td></tr><tr><td>Chirascan&amp;trade; plus CD spectrometer</td><td>Applied Photophysics</td><td>Chirascan&amp;trade;&amp;nbsp;</td><td></td></tr><tr><td>Daidzein</td><td>Shanghai Yuanye Bio-Technology</td><td>B20227</td><td></td></tr><tr><td>Daidzin</td><td>Shanghai Yuanye Bio-Technology</td><td>B20226</td><td></td></tr><tr><td>Dithiothreitol(DTT)</td><td>Solarbio Science and Technology</td><td>D8220</td><td></td></tr><tr><td>E. coli BL21 (DE3) competent cells</td><td>Thermo Fisher Scientific&amp;trade;?Invitrogen</td><td>EC0114</td><td></td></tr><tr><td>Genistein</td><td>Shanghai Yuanye Bio-Technology</td><td>S31565</td><td></td></tr><tr><td>Genistein-8-C-glucoside</td><td>Shanghai Yuanye Bio-Technology</td><td>BP2095</td><td></td></tr><tr><td>Genistin</td><td>Shanghai Yuanye Bio-Technology</td><td>S31591</td><td></td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G8270</td><td></td></tr><tr><td>GraphPad Prism 9</td><td>GraphPad Software</td><td>Version?Prism 9</td><td>Calculate the K&lt;sub&gt;m&lt;/sub&gt; and k&lt;sub&gt;cat&lt;/sub&gt; kinetic parameters</td></tr><tr><td>HiLoad Superdex 200 pg preparative SEC columns</td><td>Cytiva</td><td>28989335</td><td></td></tr><tr><td>HisTrap HP His tag protein purification columns</td><td>Cytiva</td><td>17524802</td><td></td></tr><tr><td>HiTrap Q FF anion exchange chromatography column</td><td>Cytiva</td><td>17515601</td><td></td></tr><tr><td>HKL-3000</td><td>HKL research Inc</td><td>&amp;nbsp;version 723</td><td></td></tr><tr><td>HPLC Column TC-C18(2), 170&amp;Aring;, 5 &amp;micro;m, 4.6 x 250 mm</td><td>Agilent Technologies</td><td>518925-902</td><td></td></tr><tr><td>Isopropyl &amp;beta;-D-1-thiogalactopyranoside?IPTG)</td><td>Sigma-Aldrich</td><td>PHG0010</td><td></td></tr><tr><td>Lactose</td><td>Sigma-Aldrich</td><td>17814</td><td></td></tr><tr><td>LC-20AT, high-performance liquid chromatography</td><td>Shimadzu</td><td>LC20AT</td><td></td></tr><tr><td>Maltose</td><td>Sigma-Aldrich</td><td>47288</td><td></td></tr><tr><td>Maltotriose</td><td>Sigma-Aldrich</td><td>443713</td><td></td></tr><tr><td>Manganese chloride</td><td>Shanghai Yuanye Bio-Technology</td><td>S31112</td><td></td></tr><tr><td>Methanol</td><td>Thermo Fisher Scientific&amp;trade;?Invitrogen</td><td>M23142</td><td></td></tr><tr><td>NAD&lt;sup&gt;+&lt;/sup&gt;</td><td>Solarbio Science and Technology</td><td>N8110</td><td></td></tr><tr><td>Olympus SZX12 Stereo Microscope</td><td>OLYMPUS&amp;nbsp;</td><td>SZX7</td><td></td></tr><tr><td>phenylmethylsulfonyl fluoride (PMSF)</td><td>Sigma-Aldrich</td><td>P7626&amp;nbsp;</td><td></td></tr><tr><td>Phenix</td><td>The Phenix development team</td><td>1.21.2</td><td>Phenix is a comprehensive software suite for protein crystal structure determination, incorporating SAD (Single-wavelength Anomalous Dispersion) and MR (Molecular Replacement) methods with Phaser software.</td></tr><tr><td>Preparative high-performance liquid chromatograph</td><td>Waters</td><td>2545/2998</td><td></td></tr><tr><td>Protein Crystal Screening Kit</td><td>Hampton Research</td><td>HR2-110/HR2-112/HR2-144/HR2-126/HR2-098</td><td></td></tr><tr><td>Puerarin</td><td>Shanghai Yuanye Bio-Technology</td><td>B20446</td><td></td></tr><tr><td>Shaking incubator</td><td>Shanghai Zhichu Instrument Co.,Ltd</td><td>&amp;nbsp;ZQZY-AF8</td><td></td></tr><tr><td>SnakeSkin&amp;trade; Dialysis Membrane, 10 kDa MWCO, 35 mm</td><td>Thermo Fisher Scientific&amp;trade;?Invitrogen</td><td>88245</td><td></td></tr><tr><td>Soluble starch</td><td>Sigma-Aldrich</td><td>S9765</td><td></td></tr><tr><td>Sorvall LYNX 6000 Superspeed Centrifuge</td><td>Thermo Fisher Scientific&amp;trade;</td><td>75006590</td><td></td></tr><tr><td>SpectraMax i3x Multi-Mode Microplate Reader</td><td>MOLECULAR DEVICES</td><td>SpectraMax i3x&amp;nbsp;</td><td></td></tr><tr><td>SpectraMax QuickDrop Micro-Volume Spectrophotometer Specifications</td><td>MOLECULAR DEVICES</td><td>QuickDrop</td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>S5016</td><td></td></tr><tr><td>SunFire C18 OBD Prep Column</td><td>Waters</td><td>186002741</td><td></td></tr><tr><td>Thermo Fisher Scientific Q EXACTIVE</td><td>Thermo Fisher Scientific&amp;trade;?Invitrogen</td><td>IQLAAEGAAPFALGMBDK</td><td></td></tr><tr><td>Ubiquitin-like protease 1</td><td>Thermo Fisher Scientific&amp;trade;?Invitrogen</td><td>12588018</td><td></td></tr><tr><td>Ultra Low Temperature Freezer</td><td>Thermo Fisher Scientific</td><td>905GP-ULTS</td><td></td></tr><tr><td>Vacuum centrifugal concentrator</td><td>Beijing JM Technology</td><td>CV600</td><td></td></tr><tr><td>Vitexin</td><td>Sigma-Aldrich</td><td>49513</td><td></td></tr></tbody></table>

structural biology and analytical chemistry approaches for characterizing c-glycoside metabolic enzymes in human gut microbiota

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

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.

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 ...

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.

The gut microbiota has emerged as a key modulator of host health, particularly because of its capacity to metabolize dietary glycosides. While ...

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Biochemistry

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Analysis of Interactions between Endobiotics and Human Gut Microbiota Using In Vitro Bath Fermentation Systems

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Open searching enables the identification of glycopeptides decorated with previously unknown glycan compositions. Within this article, a streamlined approach for undertaking open searching and subsequent glycan-focused glycopeptide searches are presented for bacterial samples using Acinetobacter baumannii as a model.

The Application of Open Searching-based Approaches for the Identification of Acinetobacter baumannii O-linked Glycopeptides

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Learn structural biology and analytical chemistry methods to characterize C-glycoside metabolic enzymes in human gut microbiota. This integrated approach reveals enzymatic functions using protein crystallography and LC-MS/MS techniques.

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

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