Cloning and characterization of a glycosyltransferase from Catharanthus roseus for glycosylation of cardiotonic steroids and phenolic compounds

Wen, Chao; Huang, Wei; He, Mei; Deng, Wenli; Yu, Hongfeng

doi:10.1007/s10529-019-02756-5

Cited by 6 publications

(10 citation statements)

References 20 publications

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“…UGT74AN2 was shown with a significant preference for UDP-Glc as a sugar donor with a high conversion rate (>98%) (Figure 1D). Unlike the other characterized plant steroid GTs, 18,19 UGT74AN2 possessed glycosylation catalytic activities using UDP-GlcNAc or UDP-Gal as a sugar donor, although the conversion rates were relatively low (13% for UDP-GlcNAc, 6% for UDP-Gal) (Figures S31 and S32). It suggested that the stereo configuration of the C-2 and C-4 groups on the sugar scaffold might be relevant to the sugar donor preference of UGT74AN2.…”

Section: Resultsmentioning

confidence: 98%

“…Many UDP-glycosyltransferases (UGT) from the GT1 family were found in the biosynthesis pathways of natural products . In the past few years, mining and characterization of plant and microbial UGTs have made great progress. − However, only several steroid 3- O -glycosyltransferases (S3GTs) were identified, including the microbial UGT OleD and YjiC1, which were reported with low catalytic efficiency, narrow spectra of the substrates, and poor regioselectivity toward CTS. , The plant UGTs UGT74AN1 and UGT74AN3 from the UGT74AN subfamily were thought to contribute to the biosynthesis of CTS 3- O -glycosides. , UGT74AN1 is a permissive GT identified from Asclepias curassavica and exhibited robust capabilities of the regiospecific C-3 glycosylation of CTSs. UGT74AN3 from Catharanthus roseus showed catalytic efficiency toward eight structurally different CTSs and phenolic compounds.…”

Section: Introductionmentioning

confidence: 99%

“…16,17 The plant UGTs UGT74AN1 and UGT74AN3 from the UGT74AN subfamily were thought to contribute to the biosynthesis of CTS 3-O-glycosides. 18,19 UGT74AN1 is a permissive GT identified from Asclepias curassavica and exhibited robust capabilities of the regiospecific C-3 glycosylation of CTSs. UGT74AN3 from Catharanthus roseus showed catalytic efficiency toward eight structurally different CTSs and phenolic compounds.…”

Section: Introductionmentioning

confidence: 99%

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Functional and Structural Dissection of a Plant Steroid 3-O-Glycosyltransferase Facilitated the Engineering Enhancement of Sugar Donor Promiscuity

Huang

Jiang

et al. 2022

ACS Catal.

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Cardiotonic steroids (CTSs) are ancient and effective clinical drugs for treating heart failure. Glycosylation of the CTS, especially at the C-3 position, is of great significance in nowadays pharmacological applications because it can lead to increased solubility, reduced toxicity, and expanded bioactivity of the CTS. Plant steroid 3-O-glycosyltransferase (S3GT) is a practical tool to achieve C-3 glycosylation of the CTS. However, a S3GT with both substrate and sugar donor promiscuity remains unidentified. Mechanisms of the plant S3GT for CTS recognition and catalysis are still not clear. In this study, a plant steroid 3-O-glycosyltransferase, UGT74AN2, from the medicinal plant Calotropis gigantea was identified and characterized. UGT74AN2 exhibited substrate regiospecificity and moderate sugar donor promiscuity toward the 3-hydroxyl group of structurally diverse CTSs, as well as N-/S-glycosylation activities. To disclose enzymatic mechanisms of the plant S3GT and guide its engineering, a series of complex structures of UGT74AN2 representing various activity states were solved at atomic resolutions. Key residues were identified for the sugar donor recognition and preference, and a unique V-shaped hydrophobic pocket was revealed, accounting for CTS recognition and regiospecificity. In addition, the conserved residue Gly23 has been proven essential for enzyme catalysis. Based on these findings, an engineered variant UGT74AN2 I284R/W390H/V391G was constructed, which showed enhanced sugar donor promiscuity. The mutant exhibited approximately 1.8-, 7.6-, or 11.4-fold increase in the catalytic activities in comparison with the wild type using UDP-Glc, UDP-GlcNAc, or UDP-Gal as the sugar donor, respectively. In addition, UDP-Rha was accepted as a supplemented sugar donor with a conversion rate of around 55%. Overall, this study revealed the in-depth molecular basis for 3-O-glycosylation of CTS and provided a potent biocatalyst capable of generating diverse CTS 3-O-glycosides for drug discovery.

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Section: Resultsmentioning

confidence: 98%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Functional and Structural Dissection of a Plant Steroid 3-O-Glycosyltransferase Facilitated the Engineering Enhancement of Sugar Donor Promiscuity

Huang

Jiang

et al. 2022

ACS Catal.

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“…The recombinant plasmid of pET28a-UGT74AN3 was constructed previously for the expression of the full-length protein. 36 For crystallization purposes, the construct of pET28a-UGT74AN3 11−474 was obtained for the expression of a truncated protein with the N-terminal first ten amino acids removed. Expression and purification of UGT74AN3 and UGT74AN3 11−474 followed the methods reported in our previous work.…”

Section: Methodsmentioning

confidence: 99%

“…Expression and purification of UGT74AN3 and UGT74AN3 11−474 followed the methods reported in our previous work. 36 All proteins were concentrated to 5 mg/mL using Amicon Ultra-30k (Millipore, USA), flash-frozen in liquid nitrogen, and kept at −80 °C for future use. The sitedirected mutagenesis of UGT74AN3 was performed by the polymerase chain reaction (PCR) amplification with designed primers (Table S3) using the wild-type pET28a-UGT74AN3 plasmid as the template, followed by the DpnI digestion of the template DNA.…”

Section: Methodsmentioning

confidence: 99%

Substrate Promiscuity, Crystal Structure, and Application of a Plant UDP-Glycosyltransferase UGT74AN3

Huang,

Zhang,

et al. 2023

ACS Catal.

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Glycosyltransferases are effective enzymes for glycosylating natural products (NPs), and some of them have the unusual property of being exceedingly promiscuous catalytically toward a range of substrates. UGT74AN3 is a plant glycosyltransferase identified from Catharanthus roseus in our previous work. In this study, we found that UGT74AN3 exhibits high substrate promiscuity toward 78 acceptors and 6 sugar donors and also exhibits N-/S-glycosylation activity toward simple aromatic compounds. The crystal structures of UGT74AN3 in the complex with various NPs were solved. Sugar donor recognition of UGT74AN3 was altered by structure-based mutagenesis, and the T145V mutant shifted its sugar donor preference from UDP-Glc to UDP-Xyl. Structural analysis reveals that a spacious U-shaped hydrophobic binding pocket accounts for the high substrate promiscuity of UGT74AN3. The residues E85 and F193 might serve as gatekeepers of UGT74AN3 to control substrate binding. In addition, a rare substrate binding mode was discovered in the structure of UGT74AN3, and the process of substrate flipping in the pocket was charted by molecular dynamics simulations. Moreover, a cost-effective one-pot system by coupling UGT74AN3 with AtSuSy, a sucrose synthase, was established for in situ generating and recycling UDP-Glc from sucrose and UDP to glycosylate NPs. Our study reveals the structural basis underlying the substrate promiscuity of UGT74AN3 and provides an efficient and economical enzymatic synthesis strategy for producing valuable glycosides for drug discovery.

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Enzymatic Synthesis of Phloretin α‐Glucosides Using a Sucrose Phosphorylase Mutant and its Effect on Solubility, Antioxidant Properties and Skin Absorption

et al. 2021

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Glycosylation of polyphenols may increase their aqueous solubility, stability, bioavailability and pharmacological activity. Herein, we used a mutant of sucrose phosphorylase from Thermoanaerobacterium thermosaccharolyticum engineered to accept large polyphenols (variant TtSPP_R134A) to produce phloretin glucosides. The reaction was performed using 10% (v/v) acetone as cosolvent. The selective formation of a monoglucoside or a diglucoside (53% and 73% maximum conversion percentage, respectively) can be kinetically controlled. MS and 2D‐NMR determined that the monoglucoside was phloretin 4’‐O‐α‐D‐glucopyranoside and the diglucoside phloretin‐4’‐O‐[α‐D‐glucopyranosyl‐(1→3)‐O‐α‐D‐glucopyranoside], a novel compound. The molecular features that determine the specificity of this enzyme for 4’‐OH phenolic group were analysed by induced‐fit docking analysis of each putative derivative, using the crystal structure of TtSPP and changing the mutated residue. The mono‐ and diglucoside were, respectively, 71‐ and 1200‐fold more soluble in water than phloretin at room temperature. The α‐glucosylation decreased the antioxidant capacity of phloretin, measured by DPPH and ABTS assays; however, this loss was moderate and the activity could be recovered upon deglycosylation in vivo. Since phloretin attracts a great interest in dermocosmetic applications, we analyzed the percutaneous absorption of glucosides and the aglycon employing a pig skin model. Although the three compounds were detected in all skin layers (except the fluid receptor), the diglucoside was present mainly on superficial layers.

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Cloning and characterization of a glycosyltransferase from Catharanthus roseus for glycosylation of cardiotonic steroids and phenolic compounds

Cited by 6 publications

References 20 publications

Functional and Structural Dissection of a Plant Steroid 3-O-Glycosyltransferase Facilitated the Engineering Enhancement of Sugar Donor Promiscuity

Functional and Structural Dissection of a Plant Steroid 3-O-Glycosyltransferase Facilitated the Engineering Enhancement of Sugar Donor Promiscuity

Substrate Promiscuity, Crystal Structure, and Application of a Plant UDP-Glycosyltransferase UGT74AN3

Enzymatic Synthesis of Phloretin α‐Glucosides Using a Sucrose Phosphorylase Mutant and its Effect on Solubility, Antioxidant Properties and Skin Absorption

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