Embedded Files
Substrate Specificity Determinant of C-Deglycosidase from Human Gut Bacterium Dorea sp. MRG-IFC3
Heji Kim1, Huynh Thi Ngoc Mi1, Joong-Hoon Ahn2, and Jaehong Han1*
1. Metalloenzyme Research Group and Department of Plant Science and Technology, Chung-Ang University, 4726 Seodong-daero, Anseong 17546, Republic of Korea
2. Department of Integrative Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University, Seoul 05029, Republic of Korea
Corresondence to: Jaehong Han, jaehongh@cau.ac.kr
Received: May 19, 2025; Revised: July 14, 2025; Accepted: July 16, 2025; Published: July 21, 2025
NATPRO J. 2025, 2, 9-12
https://doi.org/10.23177/NJ025.501
Copyright © 2025 The Authors, Published by the Asian Society of Natural Products
Abstract
The C-deglycosidase from the human gut bacterium Dorea sp. MRG-IFC3 shows high substrate specificity and only cleaves the C-glycosidic bond of puerarin. To elucidate the origin of the peculiar substrate specificity of the C-deglycosidase from the MRG-IFC3 strain, the genes of the C-deglycosidase d3dgpBC were cloned from the MRG-IFC3 strain, and the amino acid sequence was compared with those from the closely related PUE strain. The primary protein structure comparison revealed that four amino acid differences at the positions of 105 and 118 in D3dgpB and at the positions of 196 and 200 in D3dgpC. Except[JH1] the residue Q118B, all three were found at the surface of the X-ray protein structure of the closely related DgpBC, PDB:7BVR. The residue Q118B was found near the catalytic Mn(II) site at the substrate binding pocket. The modified C-deglycosidase, D3dgpBC with the Q118BP mutation, was prepared to investigate the substrate specificity. The modified enzyme catalyzed the cleavage of the glycosidic C-C bond of various C-glycoside including vitexin, orientin, and puerarin. Thus, Q118B was identified as a key residue preventing flavone C-glycosides from binding to the active site. This work emphasizes the genomics study of gut metabolism through high-throughput sequencing cannot fully reflect the health-promoting effects of the human gut microbiota.
Keywords
gut metabolism, C-deglycosidase, Dorea sp. MRG-IFC3, puerarin, substrate specificity
Dietary natural products benefit human health and are often subjected to gut metabolism [1,2]. In addition to the health-related effects, the contribution of gut microbiota to the metabolism of plant natural products has emerged as a new opportunity for the discovery of novel biochemical conversions [3]. For example, strong phytoestrogenic S-equol is produced from daidzin by a series of reductions and radical dehydroxylation reactions of gut bacteria. In detail, stereospecific reductive dihydroxylation of tetrahydrodaidzein results in the formation of S-equol, and the new radical enzyme reaction was proposed for the conversion [4]. Furthermore, the study on the gut metabolism of puerarin, an isoflavone C-glycoside, has led to the discovery of new C-deglycosidase which cleaves the C-glycosidic bond [5-7] (Figure 1).
Figure 1. Biochemical degradation of puerarin in human gut. Puerarin is subjected to the regiospecific oxidation on the 3”-position by the NAD+-dependent oxidoreductase (D3dpgA), and then the C-glycosidic bond of 3”-oxo-puerarin is cleaved by Mn(II)-dependent C-deglycosidase (D3dgpBC) to form daidzein and hexose enediolone.
To investigate the biochemical C-C bond cleavage reaction, we have isolated a new human gut bacterium Dorea sp. MRG-IFC3 from healthy Korean woman [8]. Interestingly, our MRG-IFC3 strain metabolized only the isoflavone C-glycoside puerarin, but did not convert other C-glycosides, such as vitexin, orientin, and isovitexin [9]. The previously reported other gut bacteria can metabolize various flavone C-glycosides, including the closely related same species of Dorea sp. PUE strain [6]. Through the previous cell-free biotransformation study, it was found that the C-deglycosidase in the MRG-IFC3 strain only reacted with puerarin [10]. Given that a major difference between flavone C-glycoside and isoflavone C-glycoside is the position of the B-ring, we hypothesized that the substrate binding site of the C-deglycosidase of the MRG-IFC3 strain differs from that of the PUE strain.
To test this hypothesis, we cloned the genes, d3dgpBC, responsible for the synthesis of C-deglycosidase and over-expressed the hexahistidine-tagged protein from Escherichia coli BL21. The amino acid sequence of D3dgpBC was deduced from the cloned genes and compared to those of the PUE strain (GenBank number: LC422372.1) (Figure 2). The primary structure of D3dgpB (GenBank number: OR238369.1) was different from the PUE strain at positions of G105B and Q118B. In the case of D3dgpC (GenBank number: OR238370.1), two residues of E196C and K200C were also different from those of DgpC. The corresponding amino acid residues of DgpBC in the PUE strain are E105B, P118B, K196C and E200C, respectively [11].
Figure 2. Primary protein structure comparison between D3dpgBC from Dorea sp. MRG-IFC3 (up, D3dgpB and D3dgpC) and DgpBC from the strain PUE (down, 7BVR_1 and 7BVR_2). White character in the red box: strict identity, black bold character in yellow: similarity across groups, TT: strict β-turns. It is noteworthy that the Blastp search of D3dgpB resulted in the percent identity of more than 98% with the PUE strain. The other protein sequences from different bacteria were found to have less than 70% of percent identity to D3dgpB. In the case of D3dgpC, it showed more than 99% of sequence identity to the PUE strain, but others showed less than 76% of sequence identity, except one human gut bacterium Ruminococcus sp. AF12-5.
Next, the locations of the different amino acid residues in the tertiary protein structure were inspected to determine whether any of these four residues were in the substrate binding site, by using the available protein X-ray crystallographic structure of DgpBC from the PUE strain (PDB:7BVR) [6]. Three different residues, E105B, K196C, and E200C, of the PUE strain were found on the surface of the protein structure, but the residue P118B corresponding to the Q118B of the MRG-IFC3 was located near the Mn(II) active site (Figure 3). Therefore, the Q118B residue of D3DgpB was suggested to be the key residue preventing flavone C-glycosides from binding to the active site.
Figure 3. The position of different amino acids (labeled with residue) from the X-ray protein crystallographic structure of DgpBC cloned from the strain PUE. The asymmetric unit of DgpBC is produced by Chimera 1.16 software [12]. Pro118B near the substrate binding site, represented as P118β in the Figure to adopt crystallographic symbol, is changed to Gln118B in the D3DgpBC of Dorea sp. MRG-IFC3. The Greek symbols in the Figure were adopted to represent the crystallographic symmetric units. The polypeptide chain of DgpB corresponds to β and DgpC to α. Therefore, Pro118B was represented as P118β in the Figure.
To confirm this, a mutant of D3dgpBC was constructed, of which the Gln118B residue of D3dgpBC was changed to proline. The mutant and wild-type enzymes of D3dgpBC were purified from the E. coli overexpression system using Ni-NTA affinity column chromatography. The reactivity of both C-deglycosidases was tested with vitexin in the presence of D3dgpA which provides the substrate of C-deglycosidase, 3”-oxo-vitexin, in situ [5,13].
From both reactions, D3dgpA produced 3”-oxo-vitexin, observed at the retention time of 12 min. 2”-Oxo-vitexin shown at the retention time of 13 min was also formed from the isomerization of 3”-oxo-vitexin [5]. While the wild-type D3dgpBC was unable to produce apigenin, the Q118BP mutant D3dgpBC was able to metabolize vitexin, as evidenced by the formation of apigenin at the retention time of 17.5 min (Figure 4). The Q118BP D3dgpBC also converted orientin and puerarin to luteolin and daidzein (see the Supporting Information). Therefore, it was evident that the substrate specificity of Dorea sp. MRG-IFC3 in C-deglycosylation is endowed by the Gln118B residue. The results can be further rationalized by the alteration of the β-sheet secondary structure of the substrate-binding region of MRG-IFC3. The mutation of Gln to Pro at the 118B position in the altered D3dgpBC likely forced the loop to move away from the substrate binding site, creating more space for substrate binding at the active site (Figure 5).
Figure 4. HPLC chromatograms (270nm) showing the reaction of vitexin (vix) by Q118βP mutant and wild type D3dgpBC. and The mixture of D3dgpA (11.1 μM) and D3dgpBC (10.95 μM mutant, or 10.81 μM wild type D3dgpBC), methyl β-D-3-oxo-glucopyranoside (1 mM) and vitexin in 50 mM potassium phosphate buffer (pH 5.8) was incubated for 60 min at 37 ºC. The reaction was terminated with 100 µL MeOH, filtered through a 0.2 µL PTFE filter (Advantec, Japan), and analyzed by HPLC.
Figure 5. Proposed structure of the Q118BP mutant D3dgpBC based on the protein structure of DgpBC (PDB:7BVR). The polypeptide chain loop near the substrate binding site of the wild type of D3dgpBC would have been moved away due to the mutation.
Our findings provide a significant insight regarding the substrate specificity of glycosidic C-C bond cleavage to the molecular-level investigation. Additionally, the finding that different metabolites can be produced even from the same species of gut bacteria highlights that the genomics study of gut metabolism through high-throughput sequencing does not fully reflect the health-promoting effects of the gut microbiota. We have previously reported that the same species of gut bacterium exhibited the different biochemical activity to produce different gut metabolites [14]. Therefore, the metabolomic study of natural products focusing on the biochemical pathway of the secondary metabolites should be included.
Declarations
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Competing interests
The authors declare that they have no competing interests.
Funding
This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (RS-2024-00449169).
Authors' contributions
All authors contributed to the preparation of manuscript by providing study conception, performing the experiments, collecting and analyzing data, preparing figures and tables, and writing drafts of manuscripts. Project directing and the first draft of the manuscript was written by JH, material preparation, data collection and analysis were performed by HK, HM, and JA. All authors read and approved of the final manuscript.
Supporting Information
The Supporting Information is available free of charge on the Journal Website. Figures for the structure of C-deglycosidase showing the substrate accessing channel and substrate binding at the active site. HPLC analysis of puerarin and orientin conversion by the Q118BP mutant C-deglycosidase.
References
1. Fan Y.; Pedersen O. Gut microbiota in human metabolic health and disease. Nat. Rev. Microbiol. 2021, 19, 55-71. DOI: 10.1038/s41579-020-0433-9.
2. Rowland I.; Gibson G.; Heinken A.; Scott K.; Swann J.; Thiele I.; Tuohy K. Gut microbiota functions: metabolism of nutrients and other food components. Eur. J. Nutr. 2018, 57, 1-24. DOI: 10.1007/s00394-017-1445-8.
3. Han J. Chemical aspects of gut metabolism of flavonoids. Metabolites, 2019, 9, 136. DOI: 10.3390/metabo9070136.
4. Kim M.; Marsh E. N.; Kim S. U.; Han J. Conversion of (3S,4R)-tetrahydrodaidzein to (3S)-equol by THD reductase: proposed mechanism involving a radical intermediate. Biochemistry, 2010, 49, 5582-5587. DOI: 10.1021/bi100465y.
5. Nakamura K.; Zhu S.; Komatsu K.;, Hattori M.; Iwashima M. Deglycosylation of the isoflavone C-glucoside puerarin by a combination of two recombinant bacterial enzymes and 3-oxo-glucose. Appl. Environ. Microbiol. 2020, 86, e00607-20. DOI: 10.1128/AEM.00607-20.
6. Mori T.; Kumano T.; He H.; Watanabe S.; Senda M.; Moriya T.; Adachi N.; Hori S.; Terashita Y.; Kawasaki M.; Hashimoto Y.; Awakawa T.; Senda T.; Abe I.; Kobayashi M. C-Glycoside metabolism in the gut and in nature: Identification, characterization, structural analyses and distribution of C-C bond-cleaving enzymes. Nat. Commun. 2021, 12, 6294. DOI: 10.1038/s41467-021-26585-1.
7. He P.; Wang S.; Li S.; Liu S.; Zhou S.; Wang J.; Tao J.; Wang D.; Wang R.; Ma W. Structural mechanism of a dual-functional enzyme DgpA/B/C as both a C-glycoside cleaving enzyme and an O- to C-glycoside isomerase. Acta Pharm. Sin. B. 2023, 13, 246-255. DOI: 10.1016/j.apsb.2022.05.022.
8. Kim M.; Lee J.; Han J. Deglycosylation of isoflavone C-glycosides by newly isolated human intestinal bacteria. J. Sci. Food Agric. 2015, 95, 1925-1931. DOI: 10.1002/jsfa.6900.
9. Mi H. T. N.; Chaiyasarn S.; Kim H.; Han J. C-Glycoside-metabolizing human gut bacterium, Dorea sp. MRG-IFC. J. Microbiol. Biotechnol. 2023, 33, 1606-1614. DOI: 10.4014/jmb.2308.08021.
10. Mi H. T. N.; Kim H.; Lee J. S.; Eser B. E.; Han J. Flavonoids biotransformation by human gut bacterium Dorea sp. MRG-IFC3 cell-free extract. J. Microbiol. Biotechnol. 2024, 34, 1270-1275. DOI: 10.1016/j.jbiosc.2015.05.008.
11. Nakamura K.; Zhu S.; Komatsu K.; Hattori M.; Iwashima M. Expression and characterization of the human intestinal bacterial enzyme which cleaves the C-glycosidic bond in 3″-oxo-puerarin. Biol. Pharm. Bull. 2019, 42, 417-423. DOI: 10.1248/bpb.b18-00729.
12. Pettersen E. F.; Goddard T. D.; Huang C. C.; Couch G. S.; Greenblatt D. M.; Meng E. C.; Ferrin T. E. UCSF Chimera--a visualization system for exploratory research and analysis. J. Comput. Chem. 2004, 25, 1605-1612. DOI: 10.1002/jcc.20084.
13. Kim H.; Mi H. T. N.; Ahn J. H.; Lee J. S.; Eser B. E.; Choi J.; Han J. Glycoside-metabolizing oxidoreductase D3dgpA from human gut bacterium. Front. Bioeng. Biotechnol. 2024, 12, 1413854. DOI: 10.3389/fbioe.2024.1413854.
14. Kim M.; Kim N.; Han J. Deglycosylation of flavonoid O-glucosides by human intestinal bacteria Enterococcus sp. MRG-2 and Lactococcus sp. MRG-IF-4. Appl. Biol. Chem. 2016, 59, 443-449. DOI: 10.1007/s13765-016-0183-6.
Page updated
Google Sites
Report abuse