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. 2025 Mar 24;16(4):1807–1823. doi: 10.1080/21501203.2025.2478073

Substrate promiscuity catalyzed by an O-glycosyltransferase MrOGT2 from Metarhizium robertsii

Yihan Ma a, Jixia Ren b, Wen-Bing Yin c, Xiaoqing Liu a,✉, Wei Li b,✉
PMCID: PMC12667342  PMID: 41334512

ABSTRACT

Glycosides tremendously extend the wide spectrum of biological activities of flavonoids and phenolics, which are catalysed by the glycosyltransferases (GTs) in diverse plants, bacteria, and fungi. However, the glycosyltransferases identified from fungi are still limited. Herein, one novel O-glycosyltransferase of MrOGT2 from the entomopathogenic fungus Metarhizium robertsii was presented. MrOGT2 exhibited typical substrate promiscuity characteristics towards four uridine diphosphate (UDP) sugar donors and 17 sugar receptors including flavonols, flavanones, flavones, isoflavones, and phenolics five types of compounds. Molecular docking and site-directed mutagenesis revealed the key substrate binding sites in the binding pocket and possible conservative catalytic mechanism of the O-glycosyltransferase MrOGT2. Our research provides an advance in the knowledge of glycosyltransferase in fungi and contributes the application potential for the efficient biocatalyst of O-glycosylation to both agricultural and pharmaceutical industries.

KEYWORDS: Glycosyltransferase, Metarhizium robertsii, flavonoids, promiscuity, molecular docking

GRAPHICAL ABSTRACT

graphic file with name TMYC_A_2478073_UF0001_OC.jpg

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1. Introduction

Flavonoids, a significant class of phenolic natural products, exhibit a broad spectrum of health benefits, such as anti-inflammatory, antitumor, antioxidative, cardioprotective, and neuroprotective effects (Rakha et al. 2022; Teng et al. 2023). Numerous studies have demonstrated that certain flavonoid glycosides exhibit higher activity and better absorption than their corresponding aglycone forms (Veitch and Grayer 2011). Moreover, the bioactivity, bioavailability, and metabolism of flavonoid glycosides are significantly influenced by the type and position of the attached sugar moiety (Veitch and Grayer 2011; Shah et al. 2023). Glycosylation notably improves the aqueous solubility and drug-like properties of molecules (Liang et al. 2015; Yang et al. 2018). Glycosylation, a common modification of small molecules, is catalysed by glycosyltransferases (GTs) to modulate their physicochemical and biological properties (Yang et al. 2018). GTs facilitate the interaction between sugar donors and acceptor molecules (Lairson et al. 2008). In known GT families, a conserved UDP-sugar binding motif is located at the C-terminal domain, utilising the activated uridine diphosphate (UDP) sugars including UDP-glucose, UDP-xylose, UDP-galactose, and UDP-rhamnose as donors (Lairson et al. 2008). Various GTs with distinct catalytic properties have been identified and characterised across diverse plants, bacteria, and fungi.

Filamentous fungi, as the effective microbial biotransformation systems, possess significant potential for glycosylating a range of substrates, including terpenes and flavonoids (Cao et al. 2015; Leonard et al. 2021). Recently, numerous O-glycosyltransferases (OGTs) have been identified as biocatalysts for the O-glycosylated modification of phenol, polyketides, and terpene compounds in fungi. In which, the known OGTs catalysing phenolic compounds covered the MhGT1 from Mucor hiemalis (Feng et al. 2017), UGT58A1 from Rhizopus japonicus (Xie et al. 2017), UGT59A1 from Absidia coerulea (Xie et al. 2017), BbGT86 from Beauveria bassiana (Xie et al. 2018), IfGT (CfGT) from Isaria fumosorosea, CmGT from Cordyceps militaris, and CpGT from Claviceps purpurea (Xie et al. 2019). The OGTs participated in the glycosylation of polyketides included CIOCT from Chalara lognjipes, PcOCT1 and PcOCT2 from Phaeomoniella chlamydospora identified through genome mining (Chen et al. 2024). In addition, the OGTs identified to catalyse the glycosylation of terpenes involved in SdnJ from Sordaria araneosa (Kudo et al. 2016), BuaB from Aspergillus bumettii (Li et al. 2019), AfumC from Aspergillus fumigatus (Ma et al. 2019), EriJ from Hericium erinaceus (Liu et al. 2019), BraB from Annulohypoxylon truncatum (Feng et al. 2020), GFUGT88A1 from Grifola frondose (Liang et al. 2022) and EpiB from Epicoccum nigrum (Fan et al. 2024). Moreover, unique flavonoid biosynthetic gene clusters have been identified in filamentous fungi, which confirmed the ability of fungi to synthesise flavonoids. It suggested that glycosyltransferases for post-modification of flavonoids do exist in fungal resources (Zhang et al. 2022, 2023). Characterizing these fungal OGTs significantly advances our understanding of enzymatic O-glycosylation and indicates that numerous novel functional OGTs remain to be discovered in fungi.

Metarhizium species are renowned entomopathogenic fungi used in biocontrol, and they have served as hosts for biotransformation processes, including the glycosylation of cucurbitacin E (Martin and Schroder 2000), quercetin (Tronina et al. 2023), and flavokawain B (Chlipala et al. 2024) as well as hydroxylation of 13-ethylgon-4-ene-3,17-dione (Feng et al. 2014) and progesterone (Panek et al. 2023). Kaempferol, a model flavonol compound, contains multiple free hydroxyl groups serving as potential glycosylation sites. During the biotransformation of kaempferol in the fermentation culture of Metarhizium robertsii ARSEF 23, three monomethylglucosides of kaempferol were detected (Xie et al. 2019). However, only the 7-O-β-D-(4-O-methyl)-glucopyranoside and 4’-O-β-D-(4-O-methyl)-glucopyranoside were confirmed to be the products catalysed by the functional module of MrGT1 (glucosyltransferase, MAA_06259)-MrMT1(methyltransferase) (Xie et al. 2019). This suggests that another unidentified glycosyltransferase in M. robertsii may catalyse the glycosylation of the 3-hydroxyl group in kaempferol. The MrGT1-MrMT1 complex from M. robertsii was also confirmed to catalyse the production of monomeric naphtho-γ-pyrone glycosides, known as indigotides (Sun et al. 2022). In bacteria, two glycosyltransferases (BcGT-1 and BcGT-3) from Bacillus cereus were discovered to catalyse the glycosylation of kaempferol at 3-hydroxyl group (Hyung Ko et al. 2006; Ahn et al. 2009). Flavonoid 3-O-glucosyltransferases are probably the most thoroughly studied in plants. There have been at least four 3-O-glucosyltransferase reported to be responsible for glycosylation modification at the same site of kaempferol, including UGT73A16 from Withania somnifera (Singh et al. 2013), UGT78K6 from Clitoria ternatea (Hiromoto et al. 2015), ArGT3 from Arabidopsis thaliana (Pandey et al. 2015) and MaUFGT from Morus alba (Yu et al. 2020). To our knowledge, there have no 3-O-glucosyltransferase for the modification of kaempferol to be found in fungi. In addition, eight isocoumarin O-glycosides, glycosylated at two distinct hydroxyl sites, were derived from the soil fungus Metarhizium anisopliae DTH12-10 (Tian et al. 2016). These findings suggest there are the presence of additional, functionally uncharacterised OGTs in Metarhizium species. In this study, a novel O-glycosyltransferase MrOGT2 in M. robertsii was identified and characterised. MrOGT2 displayed substrate promiscuity, accepting various sugar donors and multiple sugar receptors. The putative catalytic mechanism of this O-glycosyltransferase was also analysed using molecular docking and site-directed mutagenesis. Our research performed one novel O-glycosyltransferase in fungi for the glycosylation of flavonoids.

2. Materials and methods

2.1. Strains and culture conditions

Metarhizium robertsii ARSEF 23 was cultured on Potato Dextrose Agar (PDA, BDTM, USA) at 25 °C for 7 d (Fan et al. 2017). The spores were harvested using 1% Tween 80 in sterile water and then cultured in Potato Dextrose Broth (PDB, BDTM, USA) with shaking at 150 r/min and at 25 °C for 2 d. The hyphae were harvested and stocked at −80 °C for total RNA extraction. Escherichia coli BL21 (DE3) was cultured in Luria-Bertani (LB) medium supplemented with 1% NaCl, 1% tryptone (Oxoid™, UK), and 0.5% yeast extract (Oxoid™, UK) at 37 °C. For plasmid amplification, E. coli BL21 (DE3) was cultivated in LB medium with 100 μg/mL kanamycin at 37 °C with shaking at 200 r/min overnight.

2.2. RNA preparation and cDNA synthesis

Total RNA was isolated from the mycelia of M. robertsii ARSEF 23 using TransZolTM Kit (Transgen Biotech, China) following the manufacturer’s protocol. RNA concentration and quality were determined using a Quawell Q3000 nucleic acid analyser (Quawell, USA). The RNA was reverse transcribed into cDNA using the Fast Quant RT Kit (Tiangen Biotech, China). To remove residual genomic DNA, the cDNA was synthesised using oligo-dT primers with the First Strand cDNA Synthesis Kit (Tsingke Biotech, China), adhering to the manufacturer’s guidelines.

2.3. PCR amplification, molecular cloning, and plasmid construction

The coding region of MrOGT2 was amplified from the cDNA using the primers containing EcoRI and HindIII restriction sites (MrOGT2f_EcoRI and MrOGT2r_HindIII) by PCR (Table S4 and Table S5). PCR was conducted with Q5 high-Fidelity DNA Polymerase (New England Biolabs, USA) according to the manufacturer’s protocol. The PCR products and pET28a vector were double-digested with EcoRI and HindIII, and then the MrOGT2 coding sequence was integrated into pET28a vector. The resulting expression vector pMYH1 was transformed into E. coli BL21 (DE3) competent cells following the manufacturer’s protocol. The plasmid containing the MrOGT2 gene was then replicated and confirmed by restriction digestion analysis and sequencing.

2.4. Protein expression and purification

The overexpression and purification of the His-tagged N-terminal MrOGT2 protein were conducted using a His-tagged MrOGT2 construct. The plasmid was transformed into E. coli BL21 (DE3) component cells for protein expression following the manufacturer’s protocol. Individual bacterial colonies containing either pET28a or pET28a-MrOGT2 were cultured overnight at 37 °C with shaking at 250 r/min in 20 mL LB medium supplemented with 100 μg/mL kanamycin. The culture was then inoculated into 1 L LB medium, and when OD600 reached 0.4–0.6, protein expression was induced with isopropylthio-β-D-galactoside (IPTG) to a final concentration of 0.5 mol/L, and the culture continued at 16 °C for 20 h. The cells were harvested by centrifugation at 4,000 r/min at 4 °C for 20 min, and resuspended in lysis buffer (20 mmol/L NaH2PO4, 500 mmol/L NaCl, 5 mmol/L imidazole, adjust pH to 7.5 adjusted with NaOH). For a 25 mL resuspension, cells were lysed using ultrasonication for 40 min. The supernatant was incubated and then clarified by centrifugation at 10,000 r/min for 40 min. The supernatant was incubated with ProteinlsoTM Ni-NTA Resin (TransGen Biotech, China) at 4 °C for 2 h. The mixture was loaded onto a column pre-equilibrated with lysis buffer. The resin was thoroughly washed with at least 5 column volumes of wash buffer (20 mmol/L NaH2PO4, 500 mmol/L NaCl, 20 mmol/L imidazole, pH 7.5 adjusted with NaOH). The target protein was eluted using the elution buffer containing 250 mmol/L imidazole. Elution fractions containing the target protein were concentrated to 2.5 mL using a 15 mL Ultra-10 KD (Millipore) centrifugal filter, and then exchanged with storage buffer (20 mmol/L NaH2PO4, 100 mmol/L NaCl, pH 7.5 adjusted with NaOH). The purified protein was stored at −80 °C.

2.5. Biochemical characterization of MrOGT2

For characterising the MrOGT2, the glycosylation reaction was carried out in a final volume of 100 μL, containing 50 mmol/L Tris-HCl (pH 7.5), 0.1 mmol/L aglycone, 250 mmol/L UDP-glucose, and 50 μg of the purified enzyme. The reaction mixture was incubated at 30 °C for 3 h and terminated by addition of 100 μL methanol (MeOH). The protein was then pelleted by centrifugation at 13,000 r/min for 10 min. The supernatant was subsequently analysed by High Performance Liquid Chromatography (HPLC) and Liquid Chromatography Mass Spectrometry (LC-MS). The enzymatic products were resolved using a linear gradient of 10%–50% solvent B over 30 min (solvent A = 0.1% formic acid in H2O and solvent B = 100% methanol and 0.1% formic acid) followed by a 15-min wash with 100% methanol. The flow rate was 1 mL/min. The total conversion yield was determined by integrating the peak areas and expressed as a percentage.

2.6. Effects of pH, temperature, and divalent metal ions

To determine the optimal pH for MrOGT2 activity, assays were conducted using buffers with pH values from 3.5 to 5.5 (50 mmol/L citric acid-sodium citrate), 6.0 to 8.0 (50 mmol/L Tris-HCl), and 8.5 to 11.0 (50 mmol/L sodium bicarbonate). To assess the optimal temperature for MrOGT2 activity, temperatures from 5 °C to 60 °C were evaluated. To evaluate the effect of metal ions on MrOGT2 activity, reactions were conducted in the presence of 0.1 mmol/L each of the following salts: BaCl2, CaCl2, MgCl2, NiCl2, CoCl2, FeCl2, CuCl2, and ZnCl2. A reaction containing 0.1 mmol/L EDTA served as a control. All reactions were performed using 0.5 mmol/L UDP-glucose as the donor, 0.1 mmol/L kaempferol as the acceptor, 5 μg of enzyme in 100 μL of buffer. All reactions were terminated by adding 100 μL of MeOH and then centrifuged at 13,000 r/min for 10 min before HPLC and LC-MS analysis. Each value is the average of three replicate measurements.

2.7. Enzymatic kinetics study

To determine the kinetic parameters of MrOGT2 with different substrates, assays were conducted in a final volume of 100 μL containing 50 mmol/L Tris-HCl (pH 7.5), 50 μg of MrOGT2, 1 mmol/L UDP-glucose (saturating concentration), and varying concentrations of kaempferol (0, 20, 40, 60, 80, 100, 200, 400, 500, 1,000 μmol/L). For determining the kinetic parameters of the glycosyl donors, assays were conducted in a final volume of 100 μL, consisting of 50 mmol/L Tris-HCl (pH 7.5), 5 μg MrOGT2, 0.1 mmol/L kaempferol (1), and 0.5 mmol/L each of UDP-xylose, UDP-galactose, and UDP-rhamnose. Additionally, assays were conducted in a final volume of 100 μL containing 50 mmol/L Tris-HCl (pH 7.5), 50 μg MrOGT2, 0.5 mmol/L kaempferol, and varying concentrations of UDP-xylose, UDP-galactose, and UDP-rhamnose (5，10，30，60，100，150，250，500 μmol/L). The reaction mixtures were incubated at 30 °C for 3 h. Finally, the reactions were terminated by adding 100 μL of MeOH. The reaction mixtures were centrifuged at 12,000 r/min for 10 min. The enzymatic products were analysed by HPLC. All experiments were conducted in triplicate.

2.8. Preparative scale reactions

The MrOGT2 enzyme was purified from a 5 L culture of E. coli BL21 (DE3) induced with IPTG and stocked at −80 °C. Scaled-up reactions were conducted in a final volume of 20 mL containing 50 mmol/L Tris-HCl (pH 7.5), 120 mg UDP-glucose, 200 mg of MrOGT2 enzyme, and 28.6 mg kaempferol. The reactions were incubated at 37 °C for 3 h. The products were extracted three times using ethyl acetate. The organic phase was concentrated, dissolved in 10 mL MeOH, and centrifuged at 7,000 r/min for 20 min. The glycosylated products were purified using reverse-phase semi-preparative HPLC and characterised by LC-MS.

2.9. Protein structure prediction and molecular docking

The 3D structure of MrOGT2, utilised for the docking study, was predicted using AlphaFold2 on the AlphaFold DB platform (Jumper et al. 2021; Varadi et al. 2024). The analysis was performed using the “Build Homology Models” module in Discovery Studio 2019 (DS 2019) (Pawar and Rohane 2021), based on the crystal structure of Sterol 3-beta-glucosyltransferase (UGT51) from Saccharomyces cerevisiae (PDB ID: 5GL5). The coordinates of ligand (UDP-galactose) complexed with the protein UGT51 were copied during the homology modelling (Chen et al. 2018). Initially, UDP-glucose, UDP-xylose, UDP-galactose, and UDP-rhamnose were docked into the sugar donor pocket of MrOGT2 using GOLD 5.1, respectively. Then, kaempferol was docked into the acceptor substrate pocket of the complexed structure of MrOGT2-UDP-glucose, MrOGT2-UDP-xylose, MrOGT2-UDP-galactose, and MrOGT2-UDP-rhamnose, respectively. The molecular docking conformations of the ligands were analysed through clustering based on Root Mean Square Deviation (RMSD) values, selecting the poses with the lowest binding energy and appropriate stereochemistry within the primary clusters.

2.10. Sequence alignment and phylogenetic analysis

The protein sequences of all related GTs were obtained from the GenBank database in NCBI. Evolutionary analyses of MrOGT2 and other 43 glycosyltransferases from various species were conducted with MEGA 7.0 software. A phylogenetic unrooted tree was then generated with 10,000 bootstrap replicates using the Maximum Likelihood method based on ClustalW multiple alignments. Their GenBank accession numbers and source organisms are listed in Table S1. The conservative analysis of substrate binding sites was performed on the MEME Suite program 5.5.7 (https://meme-suite.org/meme/tools/meme). The default values were used for all analysis parameters.

2.11. General experimental procedures

Compound analysis was performed using an Agilent Technologies 1200 Series HPLC system (Agilent ZORBAX 300SB-C18 Stable Bond Analytical, 4.6 mm × 250 mm, 5 mm) equipped with an ODS column. Water (A) containing 0.1% (v/v) formic acid and MeOH (B) were used as the mobile phases at a flow rate of 1 mL/min. The substances were eluted with a linear gradient from 10% to 50% B over 30 min, followed by a wash with 100% (v/v) solvent B for 15 min and re-equilibrated with 10% (v/v) solvent B for 2 min. UV absorption was monitored at 350 nm. LC-MS analyses were conducted on an Agilent 1200 Accurate-Mass QTOF LC/MS system (Agilent Technologies, Santa Clara, CA, USA) equipped with an Agilent ZORBAX Eclipse column (C18 Plus, 2.1 mm × 4.6 mm, 3 mm) and an ESI source. LC-MS analyses of 1a–1d also were determined using a Waters-Vion-IMS-QTof system with an Electrospray ionisation (ESI) source and a Waters ACQUITY UPLC® BEH column (1.7 μm, C18, 2.1 mm × 100 mm ID). Water (A) containing 0.1% (v/v) formic acid and acetonitrile (B) were used as the mobile phases at a flow rate of 1 mL/min. The substances were eluted using a linear gradient from 5% to 100% solvent B over 30 min, followed by a wash with 100% (v/v) solvent B for 5 min and re-equilibration with 5% (v/v) solvent B for 5 min.

2.12. Isolation and identification of compounds

The glycosylated products were isolated using semi-preparative HPLC with a C18 ODS column (YMC-Pack ODS-A/S-5 mm, 250 mm × 10.0 mm) and a gradient of H2O and MeOH (linear gradient of 10% to 50% MeOH over 30 min at 2 mL/min) to yield compounds. The assignments of the two compounds were determined based on published proton LC-MS data. The corresponding figures are presented individually in the same order as referenced in the manuscript.

Nuclear magnetic resonance (NMR) data were obtained using a 500 MHz or 600 MHz Bruker FTNMR spectrometer with DMSO-d6 as the internal standard. Structural assignments were validated with supplementary data from COSY, HSQC, and HMBC experiments. The chromatographic substrates used included Sephadex LH20 (GE Healthcare Bio-Sciences AB, Sweden) and ODS-A (12 nm, S-50 mm; YMC Co., LTD., Japan). HPLC was conducted using an Alltech 426 pump equipped with an Alltech UVIS-200 detector (210 nm) and a semi-preparative reversed-phase column (YMC-packed, C18, 5 mm, 20 mm × 250 mm). UDP-glucose, UDP-xylose, UDP-galactose, and UDP-rhamnose were purchased by Xi’an QiYue Biology (China). Other chemicals utilised in this study were obtained from Macklin Biochemical Technology (China). Ni-NTA was obtained from TransGen Biotech (China). Masses were analysed in the range of m/z 100 to 1,500. Data were analysed using Compass Data Analysis 4.2 software (Bruker Daltonik, Bremen, Germany).

2.13. Site-directed mutagenesis

The construction of site-directed mutagenesis was performed based on the wild-type pMYH1 as the template to construct plasmids of pMYH1.1 to pMYH1.12, respectively (Table S4 and Table S5). All mutated plasmids were sequenced to ensure that the proper mutations were present. Each mutated plasmid of MrOGT2 was introduced to E. coli BL21 and fermented for 2 L LB medium. The mutated recombinant proteins were expressed and purified following the protocols described above. Enzyme reactions were conducted using wild-type MrOGT2 as positive control. For each reaction, assays were conducted in a final volume of 100 μL containing 50 mmol/L Tris-HCl (pH 7.5), 5 μg MrOGT2 mutant proteins, 0.1 mmol/L kaempferol, and 0.1 mmol/L UDP-glucose and incubated at 30 °C for 3 h. All reactions were quenched using equal volume of MeOH, and analysed using HPLC. All experiments were repeated in triplicate.

2.14. Statistical analysis and mapping

All statistical analyses were performed with software of GraphPad Prism 8. The data were represented as the means ± standard deviation (SD) of three replicates. The differences between the multiple groups were analysed by two-way-ANOVA, and were calculated using parametric unpaired t-test. p < 0.0001 was considered statistically significant. The figures were edited with Adobe Illustrator software.

3. Results and discussion

3.1. Identification of glycosyltransferase MrOGT2

The putative glycosyltransferases MrOGT2 (GenBank: MAA_05649) in M. robertsii ARSEF 23 was identified using the GenBank database (Hu et al. 2014). MrOGT2 featured an open reading frame (ORF, 1,383 bp) that encodes 460 amino acids. Both the conserved domains and transmembrane domains of MrOGT2 were predicted using online software tools of NCBI’s Conserved Domains Module Database, Deep TMHMM, and TMHMM 2.0, respectively (Chen et al. 2003). The results showed that MrOGT2 belongs to the GTB-type superfamily as well as YjiC or MGT superfamily without transmembrane helices. Blasting with the known glycosyltransferase in fungi and bacteria, MrOGT2 has less than 40% sequence homology with them (Table S1). These findings indicate that MrOGT2 is a member of the GT-B type glycosyltransferase superfamily and lacks transmembrane domains, classifying it as an intracellular protein (Figure S1). Then the recombinant pET28a plasmid of MrOGT2 was constructed and expressed in E. coli BL21(DE3). The recombinant MrOGT2 protein, with a molecular weight of 50.32 kDa, catalysed the conversion of kaempferol (1) to 1a under reaction conditions at pH 8.0 for 3 h (Figure 1(a,b)). This was confirmed by HPLC-MS analysis, which showed ion peaks for [M+H]+ and [M+Na]+ at m/z 449.1103 and 471.0898, respectively (Figure 1(c)). The product of enzyme-catalysed reaction was identified through comprehensive analysis of the 1H NMR, 13C NMR, and HRESIMS data (Table S2, Figure S20–S23). Product 1a was identified as kaempferol-3-O-glucoside (astragalin) by comparison to a reference standard (Elejalde-Palmett et al. 2019; Xie et al. 2019). This indicates that MrOGT2 catalyzes the kaempferol glycosylation at the 3-hydroxy group, acting as an O-glycosyltransferase. The 3-OH position is superior to 7-OH position for glycosylation to enhance the insecticidal activity of kaempferol (Xie et al. 2019), but the detail mechanism is still unclear. MrGT-MrMT from M. robertsii has no strict limitation of the site of hydroxyl group to synthesise the mixture products of monoglucoside and monomethylglucosides (Feng et al. 2017). Consequently, MrOGT2 represents an effective option for the glycosylation of flavonoids like kaempferol at the 3-OH position.

Figure 1.

Figure 1.

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3-O-glycosylation of Kaempferol (1) catalysed by MrOGT2. (a) SDS-page of the recombinant MrOGT2 fused with His tag. M: protein maker; recombinant MrOGT2 (predicated MW: 50.32 kDa). (b) Formation of kaempferol-3-O-glucose (1a) from Kaempferol (1). (c) HPLC analysis of the reaction mixture at 350 nm and LC/MS analysis of product 1a. The reaction system includes kaempferol (1) and denatured MrOGT2 as a control (ck), the pure compound 1a as another control (ck), and kaempferol (1) with MrOGT2 in the reaction.

3.2. Enzymatic characteristics of MrOGT2

To explore the enzymatic characterisation and dynamic parameters, the recombinant MrOGT2 was purified in a large-scale culture. Firstly, the biochemical characteristics of MrOGT2 were investigated using UDP-glucose as the sugar donor and kaempferol (1) as the sugar acceptor substrate at temperature from 5 °C to 60 °C and at pH from 3.5 to 10 for 3 h, respectively. The MrOGT2 reaction was found to have an optimised temperature at 30 °C, pH of 7–8.5 (Figure 2(a,b)). To determine the other enzymatic properties of MrOGT2, the enzyme reaction systems were incubated in 100 μL of 50 mmol/L Tris-HCl buffer (pH 7.5) at 37 °C for 3 h, respectively. The Km and kcat values of MrOGT2 were 151.2 μmol/L and 17.389 min−1 with UDP-glucose and different concentrations of kaempferol (1), respectively (Figure 2(c)). And the metal ion inhibition analysis showed that catalytic ability of MrOGT2 is not influenced by divalent metal ions such as EDTA, Ba2+, Mg2+, or Ca2+. However, the enzyme activity was significantly inhibited by Fe2+, Cu2+, Co2+, Ni2+, or Zn2+ (Figure 2(d)). These results revealed that MrOGT2 has advantageous thermal stability, pH adaptation range as well as the metal ion compatibility range. In comparison, Km values of apple (Malus domestica) fruits UDP-glucosyltransferase MdUGT75B1 and MdUGT71B1 were 19.8 μmol/L and 38.2 μmol/L for its substrate kaempferol, and kcat values of them were 0.59 S−1 and 0.35 S−1 for substrate kaempferol (Xie et al. 2020). Km values of plant Vaccinium corymbosum UDP-glucosyltransferase VcGT was 36.3 μmol/L for its substrate kaempferol (Kukk 2024).

Figure 2.

Figure 2.

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Enzyme characterisation of MrOGT2. (a) The thermostability of MrOGT2. (b) The pH stability of MrOGT2. (c) The kinetic parameters of MrOGT2. (d) Effect of divalent metal ions on enzyme activity. UDP-glucose and Kaempferol were used for the determination of MrOGT2.

3.3. MrOGT2 exhibited sugar donor promiscuity

To investigate the sugar donor specificity of MrOGT2, assays were conducted with UDP-glucose, UDP-xylose, UDP-galactose, and UDP-rhamnose as sugar donors along with kaempferol (1) as the sugar acceptor (Figure 3, and Figure S2). HPLC and LC/MS analyses indicated that MrOGT2 could utilise not only UDP-glucose, but also UDP-xylose, UDP-galactose and UDP-rhamnose as sugar donors for the O-glycosylation (Figure 3(a)). The LC/MS analysis showed that kaempferol-3-O-Xyl (1b) product had [M+H]+ and [M+Na]+ ion peaks at m/z 419.0987 and 441.0819, respectively. Kaempferol-3-O-Gal (1c) exhibited [M+H]+ and [M+Na]+ ion peaks at m/z 449.1303 and 471.0898, respectively. Kaempferol-3-O-Rha (1d) exhibited [M+H]+ and [M+Na]+ ion peaks at m/z 433.1132 and 455.0955, respectively (Figure 3(a)). Significant differences in the conversion rates were observed among the four substrates. MrOGT2 showed a broad specificity for its sugar donors, and exhibited a preference for the four UDP-sugars in the following order, from highest to lowest: UDP-rhamnose (70.4%), UDP-glucose (68.1%), UDP-xylose (34.6%), and UDP-galactose (23.0%) (Figure 2(b)). The kinetic parameters of MrOGT2 for the four accepted sugar substrates were determined under optimal conditions to evaluate these substrates’ preference with the same sugar acceptor kaempferol. As shown in Figure 3(c), MrOGT2 exhibited the highest Km value and Kcat value for UDP-galactose (316.2 μmol/L and 0.298 S−1), and the Km value for UDP-xylose (110.4 μmol/L) was similar to the Km values for UDP-rhamnose (110.6 μmol/L). The Kcat value for UDP-rhamnose (0.094 S−1) was lower among the four sugar donors. Accordingly, the calculated catalytic efficiency (Kcat/Km) was the lowest for UDP-rhamnose (0.0008 μmol/L−1 S−1) and UDP-galactose (0.0009 μmol/L−1 S−1) (Figure 3(c)). The Km and Kcat values of AgUCGalT1 were 9.21 μmol/L and 6.16 S−1, respectively. By comparison, the cyanidin 3-O-galactosyltransferase AgUCGalT1 from Apium graveolens has been documented to catalyse the glycosylation of various substrates, including cyanidin, peonidin, quercetin, and kaempferol with UDP-galactose (Feng et al. 2018).

Figure 3.

Figure 3.

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The promiscuity of sugar donors of MrOGT2. (a) HPLC analysis at 350 nm and LC/MS analysis of products 1b, 1c, and 1d. (b) Conversion rates of kaempferol (1) to its glycosides with different sugar donors. (c) The kinetic parameters of MrOGT2 with kaempferol and four sugar donors.

3.4. MrOGT2 showed sugar acceptor promiscuity

To assess the sugar acceptor promiscuity of MrOGT2, along with its synthetic biological ability in vitro, we evaluated 44 representative compounds, including 30 flavonoids (1–15, S1–S15), 10 phenolic compounds (16–17, S16–S23) and four additional compounds (S24–S27) using UDP-glucose as sugar donor (Figure 4(a), Figure S3, and Table S3). Among them, only compounds 1–17 were demonstrated with O-glycosylation activity by MrOGT2. And these glycosylated products were evaluated by LC/MS analysis (Figure S4–S19). These compounds include 8 flavonols (1–8), 3 flavanones (9–11), 2 flavones (12–13), 2 isoflavones (14–15), 2 phenols (16–17). In which, the flavonols (2–3, 5), flavanone (9), and flavones (12–13) have been glycosylated with over 80% conversion rates, and flavanones (10–11), isoflavones (14–15) and phenols (16–17) showed lower conversion rates of less than 20% (Figure 4(b)). Moreover, other 27 compounds including the favonols (S1–S3), flavanones (S4–S8), flavones (S9–S12), isoflavones (S13–S15), phenols (S16–S23), and the four additional compounds (S24–S27) were not accepted by MrOGT2 (Figure S5, with no LC/MS data shown). It was determined that MrOGT2 exhibits a wider range of specificity for its sugar acceptors with hydroxyl group at the different sites. Besides catalysing the glycosylation of 3-hydroxyl position in kaempferol (1), MrOGT2 could also at least glycosylate both the 3’-hydroxyl position in 4’-hydroxyflavanone (9) and the 5’-hydroxyl position in 2’-hydroxyflavanone (11). In comparison to the fungal glycosyltransferases of MhGT1 from M. hiemalis and UGT58A1 from A. coerulea, which accepted flavonoids, coumarins, and phenolic compounds, respectively (Feng et al. 2017; Xie et al. 2017), MrOGT2’s acceptors were assessed. And the results revealed that there is the significant difference of acceptors between MrOGT2 and UGT58A1.

Figure 4.

Figure 4.

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Substrate promiscuity of MrOGT2 with the various sugar receptors. (a) Structures of the compounds to be glycosylated as sugar receptors (1–17). (b) Conversion rates using UDP-glucose with the 17 compounds as the sugar receptors. The reactions were incubated at 30 °C for 3 h.

3.5. Analysis of the proposed substrate binding mechanism of MrOGT2

The AlphaFold2-generated model has been considered as the sufficient tactics to explore the catalytic mechanism of enzymes (Varadi et al. 2024). To further investigate the proposed mechanism of substrate specificity of MrOGT2, molecular docking study was performed. Structure of MrOGT2 was predicted as E9F050 using AlphaFold2 on AlphaFold Protein Structure Database (AlphaFold DB, https://alphafold.ebi.ac.uk) (Figure S7) (Varadi et al. 2024). Glycosyltransferase UGT51 from S. cerevisiae served as a template due to the observed sequence similarity of 36.4% between MrOGT2 and Uridine diphosphate-glycosyltransferase (UDPGT) domain of UGT51 (Chen et al. 2018). So, the three dimensions (3D) structure of MrOGT2 was reliably constructed using the homology modelling approach. Firstly, kaempferol (1) was simultaneously docked into the postulated acceptor substrate binding pocket of the complex structure. Subsequently, UDP-glucose, UDP-xylose, UDP-galactose, and UDP-rhamnose were respectively positioned within the sugar donor binding pocket, which was highly conserved with reported OGT structures from fungi (Chen et al. 2018; Liang et al. 2022). The sugar donors binding pocket is formed by a serial of amino acid residues in the N-terminal domain of MrOGT2, including TYR345, VAL346, ALA347, TYR348, ASP349, ALA350, ALA362, TYR364, ALA366 and PHE367 (Figure 5(a)). HIS-ASP or ASN-ASP catalytic dyad has been considered as the critical catalytic dyad during glycosylation of GTs from plant (Bao et al. 2022). In the conserved narrow binding pocket of MrOGT2, HIS20-ASP125 dyad near the active centre was revealed to play a crucial role in deprotonation of substrate (Figure 5(a) and Figure S25) (Breton et al. 2012). The four amino acid residues, including HIS20, ASP125, GLU384, and GLU385, constituted the binding pocket between the sugar donors and sugar acceptor kaempferol. In addition, the analysis of spatial arrangements for one oxygen atom linking the UDP sugar and different sugar donors revealed that UDP-rhamnose exhibits the optimal pose to facilitate glycosyl group transfer to sugar receptor kaempferol, whereas the pose of UDP-galactose is least conducive to glycogen transfer (Figure 5(b)). These characteristics were consistent with the conversion rate of these different sugar donors catalysed by MrOGT2.

Figure 5.

Figure 5.

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Proposed substrate binding mechanism of MrOGT2 revealed by molecular docking. (a) Details of the binding sites of MrOGT2 with four sugar donors and kaempferol (1) as sugar acceptor. (b) Distance analysis of oxygen atom in the UDP-sugar donors relative to the UDP-sugar receptor of kaempferol (1). The colours of the oxygen atoms were pink, light blue, green, and purple in the structures of UDP-rhamnose, UDP-glucose, UDP-galactose, and UDP-xylose, respectively. (c) The key sites analysis in the binding pocket of MrOGT2 with UDP-glucose and flavonol and flavanone sugar acceptors. Sugar donors and sugar acceptors were docked into MrOGT2 using discovery studio software.

Based on the 3D structure of MrOGT2-UDP-glucose complex, the seven flavonoid compounds and one phenol were docked into the substrate binding pocket, respectively. Structural analysis revealed that a conserved narrow binding pocket formed by amino acid residues, including PHE15, PRO69, and GLU384 along with HIS20 and ASP125 for sugar acceptors recognition. The spatial positions of the glycosylated hydroxyl groups of different glycosylated receptors in the complexes, as depicted in Figure 5(c), exhibited a significant correlation with the catalytic efficiency of multiple substrates (Figure 4(a)). The B-ring of both flavonols 3-hydroxyflavone (4) and isorhamnetin (5) and flavones 4’-hydroxyflavanone (9) and 2’-hydroxyflavone (11) exhibited similar binding conformations, respectively (Figure 5(c)).

By comparing the stereospecific conformation of 3-OH in flavones, we found that it is opposing orientation and far distance from the active sites of MrOGT2. It may be due to the position of methoxymethyl and 4’-OH at the B ring between flavonol substrates 3-hydroxyflavone (4) and isorhamnetin (5). This led to a prominent difference in the reaction efficiency between them. A similar phenomenon has also occurred in the flavones 4’-hydroxyflavanone (9) and 2’-hydroxyflavone (11). The difference of hydroxyl position in B-ring in them leads to the difference in their stereospecific conformation, and then the catalytic efficiency of 4’-hydroxyflavanone was 14.3 times than the catalytic efficiency of 2’-hydroxyflavone (Figure 5(c)). Comparing the other type sugar acceptor substrates, including flavonol kaempferol (1), flavone baicalein (12), isoflavone genistin (15), and phenol isoliquiritigen (17), the difference in binding conformation of hydroxyl group impeded the catalytic reactions for sugar transfer to these glycosylated hydroxyl groups (Figure S26). These results probably implied the significant mechanism for the regio-specificity of MrOGT2 to catalyse the sugar acceptors with different sites of hydroxyl groups and other modifying group.

3.6. Conservatism of the active sites in O-glycosyltransferase

To our knowledge, 27 O-glycosyltransferase and C-glycosyltransferase have been identified for the glycosylation of natural products in fungi. There were only 19% to 45% sequence identity between MrOGT2 and the other fungal representative glycosyltransferases (Table S1). Phylogenetic analysis was performed to explore the conservative property of these known fungal glycosyltransferases with O-glycosyltransferases in bacteria. There were six distinct clades in the phylogenetic tree (Figure 6(a)). MrOGT2 is the most closely related to the OGTs in clade IV involved in the O-glycosylation of some flavonoids and phenols in fungi, which suggests similar substrate specificity mechanisms among them. The other OGTs in the same clade were identified from the Hypocreales fungi with a wide range of substrates including different classes of phenolic natural products (Xie et al. 2019). Orthologous enzymes in this clade typically exhibited similar structures and functions in closely related species. The predicted structure of MrOGT2 closely matched those of six OGTs in its clade, including CmGT (AF DB: G3JUG1), CpGT (AF DB: M1W861), PcOGT2 (AF DB: A0A0G2EC47), BbGT86 (AF DB: J4VV61), CfGT (AF DB: A0A162IGC1), and MrGT (AF DB: E9F261). Remarkably, 3D structures of these OGTs in complex with UDP-glucose and kaempferol were highly conserved, displaying the characteristic GT-B fold (Figure 6(b)). In addition to conserved amino acid residues in the sugar donor binding pocket, MrOGT2’s active site features an HIS-ASP dyad (HIS20 and ASP125). The HIS20 residue was highly conserved, whereas the ASP125 residue was unique to MrOGT2 and PcOGT2, with glutamic acid (GLU) replacing aspartic acid (ASP) in the other five OGTs (Figure 6(c)). However, PcOGT2 from P. chlamydospora catalyses the O-galactosylation of the polyketide phaeomoniecin D (Chen et al. 2024). The amino acid residues in the sugar donor binding pocket belong to UGT conserved motifs, including the nucleotide base binding motif, PPi binding motif, and D/E-Q motif near the C terminal domain (Chen et al. 2018; Liang et al. 2022). This feature is also present in MrOGT2, indicating the high degree of conservation within this GT-B family.

Figure 6.

Figure 6.

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Phylogenetic analysis of MrOGT2 and the conservative property of active sites in MrOGT2. (a) Phylogenetic tree analysis of MrOGT2 and its homologues among known GTs in fungi and bacteria. (b) Structural superposition comparison of the predicted MrOGT2 (green) with CmGT (light blue), CpGT (light purple), PcOGT2 (orange), BbGT86 (light brown), CfGT (light red), and MrGT (light atrovirens) within the clade IV. (c) MEME analysis of the amino acids at the HIS-ASP dyad, UGT conserved motif, and DE-Q motif sites in the OGTs of the clade IV. (d) Conversion rate of substrate kaempferol (1) with proteins of wild-type MrOGT2 (WT) and its mutants. The reactions were incubated at 30 °C for 3 h. Data are presented as mean ± standard deviation, n = 3 in each group. ****p < 0.0001 compared with the control group.

3.7. Verification of site-directed mutagenesis of active sites

To identify the function of aforementioned key sites in binding pocket of MrOGT2 enzyme catalytic activity, we mutated the 12 amino acid residues to hydrophobic alanine by site-directed mutagenesis, respectively. Kaempferol (1) and UDP-glucose were used as substrates for the activity evaluation. The results revealed that all of the catalytic activities of mutants are significantly decreased, especially the TYR348 and ASP349 in the nucleotide base binding motif, GLY363 and TYR364 in PPi binding motif, HIS20 and ASP125 in the conserved dyad, and two binding sites PHE15 and PRO69 for sugar acceptor (Figure 6(d)). These results indicated the importance of both hydrophobicity at sites of PHE15, PRO69, and GLY363 and hydrophily at sites of HIS20, ASP125, TYR364, and TYR348. These differences changed the shape and size of the acceptor binding pocket, which is suitable for their sugar donors and acceptor substrates (Chen et al. 2018). Detailed analysis of the sugar donor and acceptor binding pocket in MrOGT2 mutants, we found that the pocket of MrOGT2 mutants is more compact than the wild-type MrOGT2. There were more hydrophobic interactions between substrates and the mutant than between substrates and the wild-type MrOGT2 (Chen et al. 2018). This could be mainly the important reason to drastically limit the entry efficiency of the substrates of MrOGT2 mutants.

In conclusion, flavonoids are an important class of bioactive compounds, and glycosides tremendously extend the wide spectrum of biological activities of flavonoids, which are catalysed by the glycosyltransferase from plants, bacteria or fungi for both O-glycoside and C-glycoside (Yang et al. 2018). In recent years, an increasing number of glycosyltransferases have been reported in plants and bacteria, but the glycosyltransferases are still relatively limited in fungi. The known O-glycosyltransferases from fungi have unique catalytic activity. MhGT1 from M. hiemalis exhibited capabilities for the regio- and stereospecific O-glycosylation of structures with UDP-glucose as sugar donor, in which the 7-OH group in flavonoids is glycosylated (Feng et al. 2017). The O-glycosyltransferases from serial entomopathogenic fungi glycosylate the hydroxyl groups at different sites in flavonoids, including BbMT86 from B. bassiana for 3-OH and 7-OH groups, MrGT from M. robertsii for 4’-OH and 7-OH groups, IFGT from I. fumosorosea and CpGT from C. purpurea for 3-OH and 4’-OH, CpGT from C. militaris for the 3-OH, 7-OH and 4’-OH groups, respectively (Xie et al. 2018). In our study, MrGT2 typically catalysed the glycosylation of 3-OH of flavonol kaempferol (Figure 1), and it also glycosylated the 4’-OH and 2’-OH groups in flavanone 4’-hydroxylavanone and 2’-hydroxylavanone, respectively (Figure 4). Moreover, hydroxyl or hydroxymethyl modification at 2’ site of flavonols can significantly improve the catalytic efficiency of MrGT2. These discrepancies in regiospecificity were assumed to be related to the divergent binding poses of acceptor flavonoid substrates inside the catalytic chamber of GTs (Figure 5) (Xie et al. 2019). Mutagenesis of key active sites in binding pocket confirmed that the changes of hydrophily and hydrophobicity significantly affected the catalytic efficiency (Figure 6). Nonetheless, crystallographic study still is imperative to shed light on the underlying mechanisms of regiospecific glycosylation of the fungal OGTs (Xie et al. 2019; Liang et al. 2022).

Taken together, we have identified and characterised one novel O-glycosyltransferase of MrOGT2 from M. robertsii. MrOGT2 is a representative O-glycosyltransferase in fungi, capable of recognising a variety of flavonoid receptors and glycosyl donors. Substrate specificity investigations showed that MrOGT2 exhibits sugar donor promiscuity and specificity for the utilisation of four sugar donors in the form of UDP. Moreover, it demonstrated the promiscuity of sugar acceptors, encompassing flavonol, flavone, isoflavone, flavanone, and phenols. We proposed a catalytic mechanism for MrOGT2, informed by its predicted structure, molecular docking, and mutagenesis studies, highlighting the critical roles of key residues in the UGT-defining motif and the HIS-ASP dyad at the OGT active site. This suggests conservation of glycosylation mechanisms among GTs from plants and bacteria. In addition, the O-glycosylation of kaempferol increased observably the antitumor activity of kaempferol. Our work introduces a novel and potent biocatalyst for the O-glycosylation of flavonoids and polyphenols, enriches the understanding of the diversity of OGTs in fungi, and offers potential application in the pharmaceutical and agricultural industries.

Supplementary Material

accept-0303-TMYC-2024-0241_SI__revision3.docx
TMYC_A_2478073_SM4538.docx (4.8MB, docx)

Acknowledgments

We thank Dr. Aili Fan from State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University for LC/MS data collection.

Funding Statement

This work was supported by Key Research Program of Frontier Sciences, Chinese Academy of Sciences [ZDBS-LY-SM016], Biological Resources Program, Chinese Academy of Sciences [KFJ-BRP-009-005], and Research Start-up Fund for High-level Talents and Doctors of the Department of Science and Technology, Liaocheng University [318052344].

Disclosure statement

No potential conflict of interest was reported by the author(s).

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/21501203.2025.2478073

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