A uridine diphosphate-glycosyltransferase GFUGT88A1 derived from edible mushroom Grifola frondosa extends oligosaccharide chains

Liang, Yingying; Zan, Xinyi; Sun, Lei; Fu, Xin; Cui, Fengjie; Tan, Ming; Shao, Ze-Yu; Sun, Wenjing

doi:10.1016/j.procbio.2021.11.024

Cited by 9 publications

(10 citation statements)

References 48 publications

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“…Generally, the glycosyltransferases belonging to the GT1 family prefer the high DPs of oligosaccharides as the acceptors to the transfer of the glucose moiety from donor UDP-glucose. For example, a recombinant UDP-lycosyltransferase, GFUGT88A1, showed an increased specific activity with the increase of DPs of the oligosaccharide as acceptor Figure A,B presents the substrate preference and the primer requirement of partially purified CMGLS and recombinant CMGLS-CHCD by using UDP-glucose; UDP-glucose and oligosaccharide glucose; LAM2, LAM6, and LAM9 representing DPs of 1, 2, 6, and 9, as the substrate, respectively.…”

Section: Resultsmentioning

confidence: 99%

“…Figure A,B presents the substrate preference and the primer requirement of partially purified CMGLS and recombinant CMGLS-CHCD by using UDP-glucose; UDP-glucose and oligosaccharide glucose; LAM2, LAM6, and LAM9 representing DPs of 1, 2, 6, and 9, as the substrate, respectively. Generally, the glycosyltransferases belonging to the GT1 family prefer the high DPs of oligosaccharides as the acceptors to the transfer of the glucose moiety from donor UDP-glucose . Interestingly, CMGLS has strict substrate specificity to UDP-glucose with the highest specific activity of 87.72 pmol/min/μg compared to other groups using glucose or oligosaccharides with DPs of 2, 6, and 9 as substrates.…”

Section: Resultsmentioning

confidence: 99%

“…The columns were operated at 30 °C and eluted with 57% of acetonitrile isocratic at a flow rate of 1.0 mL/min. The calibration curve of retention time vs DPs of oligosaccharide chains was developed by using LAM2, LAM6, and LAM9 as standards and applied to predict the DPs of synthesized products by CMGLS …”

Section: Methodsmentioning

confidence: 99%

“…The Glycosyltransferase Activity Kit (R&D System, Minneapolis, MN, USA) was used to determine the β-1,3-glucan synthase activity of partially purified CMGLS and recombinant CMGLS-CHCD by measuring the levels of inorganic phosphate released from UDP after the glycosylation reaction with UDP-glucose (Sigma-Aldrich, St. Louis, USA) as substrate. 27 The specific activity of β-1,3-glucan synthase was defined as the amount of 1 pmol of phosphate released per microgram of protein (μg) per unit time (min) (pmol/min/μg).…”

Section: Recombination Of Central Hydrophilic Cytoplasmic Domains Of ...mentioning

confidence: 99%

“…The calibration curve of retention time vs DPs of oligosaccharide chains was developed by using LAM2, LAM6, and LAM9 as standards and applied to predict the DPs of synthesized products by CMGLS. 27 For NMR analysis, the 24 h reaction solution was mixed with 200 μL of dimethyl sulfoxide-d6 (DMSO-d 6 , 99.9% isotropic purity, Sigma-Aldrich) and transferred to an NMR tube. 1 H NMR and 13 C NMR spectra were recorded at the frequency of 400 and 101 MHz on an ARX 600 MHz spectrometer (Bruker, Karlsruhe, Germany) at a temperature of 70 °C, respectively.…”

Section: Recombination Of Central Hydrophilic Cytoplasmic Domains Of ...mentioning

confidence: 99%

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Functional Characterization and Structural Basis of the β-1,3-Glucan Synthase CMGLS from Mushroom Cordyceps militaris

Zan

Sun

et al. 2022

J. Agric. Food Chem.

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β-1,3-Glucan synthases play key roles in glucan synthesis, cell wall assembly, and growth of fungi. However, their multi-transmembrane domains (over 14 TMHs) and large molecular masses (over 100 kDa) significantly hamper understanding of their catalytic characteristics and mechanisms. In the present study, the 5841-bp gene CMGLS encoding the 221.7 kDa membranebound β-1,3-glucan synthase CMGLS in Cordyceps militaris was cloned, identified, and structurally analyzed. CMGLS was partially purified with a specific activity of 87.72 pmol/min/μg, a purification fold of 121, and a yield of 10.16% using a product-entrapment purification method. CMGLS showed a strict specificity to UDP-glucose with a K m value of 84.28 μM at pH 7.0 and synthesized β-1,3-glucan with a maximum degree of polymerization (DP) of 70. With the assistance of AlphaFold and molecular docking, the 3D structure of CMGLS and its binding features with substrate UDP-glucose were proposed for the first time to our knowledge. UDPglucose potentially bound to at least 11 residues via hydrogen bonds, π-stacking ,and salt bridges, and Arg 1436 was predicted as a key residue directly interacting with the moieties of glucose, phosphate, and the ribose ring on UDP-glucose. These findings would open an avenue to recognize and understand the glucan synthesis process and catalytic mechanism of β-1,3-glucan synthases in mushrooms.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Recombination Of Central Hydrophilic Cytoplasmic Domains Of ...mentioning

confidence: 99%

Section: Recombination Of Central Hydrophilic Cytoplasmic Domains Of ...mentioning

confidence: 99%

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Functional Characterization and Structural Basis of the β-1,3-Glucan Synthase CMGLS from Mushroom Cordyceps militaris

Zan

Sun

et al. 2022

J. Agric. Food Chem.

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A newly discovered glycosyltransferase gene UGT88A1 affects growth and polysaccharide synthesis of Grifola frondosa

Li,

Wang,

Zhang

et al. 2024

Appl Microbiol Biotechnol

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Grifolafrodosa polysaccharides, especially β-D-glucans, possess significant anti-tumor, antioxidant and immunostimulatory activities. However, the synthesis mechanism remains to be elucidated. A newly discovered glycosyltransferase UGT88A1 was found to extend glucan chains in vitro. However, the role of UGT88A1 in the growth and polysaccharide synthesis of G. frondosa in vivo remains unclear. In this study, the overexpression of UGT88A1 improved mycelial growth, increased polysaccharide production, and decreased cell wall pressure sensitivity. Biomass and polysaccharide production decreased in the silenced strain, and the pressure sensitivity of the cell wall increased. Overexpression and silencing of UGT88A1 both affected the monosaccharide composition and surface morphology of G. frondosa polysaccharides and influenced the antioxidant activity of polysaccharides from different strains. The messenger RNA expression of glucan synthase (GLS), UTP-glucose-1-phosphate uridylyltransferase (UGP), and UDP-xylose-4-epimerase (UXE) related to polysaccharide synthesis, and genes related to cell wall integrity increased in the overexpression strain. Overall, our study indicates that UGT88A1 plays an important role in the growth, stress, and polysaccharide synthesis of G. frondosa, providing a reference for exploring the pathway of polysaccharide synthesis and metabolic regulation. Key points •UGT88A1 plays an important role in the growth, stress response, and polysaccharide synthesis in G. frondosa. •UGT88A1 affected the monosaccharide composition, surface morphology and antioxidant activity of G. frondosa polysaccharides. •UGT88A1 regulated the mRNA expression of genes related to polysaccharide synthesis and cell wall integrity.

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Recent advances in Grifola frondosa polysaccharides: production, properties, and bioactivities

Ren

Shixiang²,

He³

2023

Current Opinion in Food Science

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A uridine diphosphate-glycosyltransferase GFUGT88A1 derived from edible mushroom Grifola frondosa extends oligosaccharide chains

Cited by 9 publications

References 48 publications

Functional Characterization and Structural Basis of the β-1,3-Glucan Synthase CMGLS from Mushroom Cordyceps militaris

Functional Characterization and Structural Basis of the β-1,3-Glucan Synthase CMGLS from Mushroom Cordyceps militaris

A newly discovered glycosyltransferase gene UGT88A1 affects growth and polysaccharide synthesis of Grifola frondosa

Recent advances in Grifola frondosa polysaccharides: production, properties, and bioactivities

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