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

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Abstract

Objectives

To characterize a glycosyltransferase (UGT74AN3) from Catharanthus roseus and investigate its specificity toward cardiotonic steroids and phenolic compounds.

Results

UGT74AN3, a novel permissive GT from C. roseus, displayed average high conversion rate (> 90%) toward eight structurally different cardiotonic steroids. Among them, resibufogenin, digitoxigenin, and uzarigenin gave 100% yield. Based on LC–MS, 1H-NMR and 13C-NMR analysis, structure elucidation of eight glycosides was consistent with 3-O-β-d-glucosides. We further confirmed UGT74AN3 was permissive enough to glycosylate curcumin, resveratrol, and phloretin. The cDNA sequence of UGT74AN3 contained an ORF of 1,425 nucleotides encoding 474 amino acids. UGT74AN3 performed the maximum catalytic activity at 40 °C, pH 8.0, and was divalent cation-independent. Km values of UGT74AN3 toward resibufogenin, digitoxigenin, and uzarigenin were 7.0 µM, 12.3 µM, and 17.4 µM, respectively.

Conclusions

UGT74AN3, a glycosyltransferase from a noncardenolide-producing plant, displayed catalytic efficiency toward cardiotonic steroids and phenolic compounds, which would make it feasible for glycosylation of bioactive molecules.

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Acknowledgements

The study was financially supported by the Doctor Fundation of Jinggangshan University, China (No. JZB1821).

Supporting information

Section 1—MS, 1H NMR, 13C NMR data of compound 1a–8a.

Supplementary Table 1—The Genebank accession numbers and the primers of CrGT1-5.

Supplementary Table 2—Genebank accession numbers of the GTs used for phylogenetic analysis.

Supplementary Fig. 1—Alignment of five glycosyltransferases from Catharanthus roseus with UGT74AN1.

Supplementary Fig. 2—HPLC chromatogram of UGT74AN3 enzymatic products of bufadienolide aglycons.

Supplementary Fig. 3—HPLC chromatogram of UGT74AN3 enzymatic products of cardenolides aglycons.

Supplementary Fig. 4—HPLC chromatogram of UGT74AN3 enzymatic products of curcumin (9), resveratrol (10), and phloretin (11).

Supplementary Fig. 5—Effects of temperature (A), pH (B), divalent metal ions (C) and reaction time (D) on the activity of UGT74AN3.

Supplementary Fig. 6—Determination of kinetic parameters for UGT74AN3.

Author information

Authors and Affiliations

  1. Department of Medicine, Jinggangshan University, Ji’an, 343009, People’s Republic of China

    Chao Wen, Miao-Miao He, Wen-Li Deng & Hai-Hong Yu

  2. Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, Guangzhou, 510632, People’s Republic of China

    Wei Huang

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  1. Chao Wen
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  2. Wei Huang
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  3. Miao-Miao He
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  4. Wen-Li Deng
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Correspondence to Chao Wen.

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Wen, C., Huang, W., He, MM. et al. Cloning and characterization of a glycosyltransferase from Catharanthus roseus for glycosylation of cardiotonic steroids and phenolic compounds. Biotechnol Lett 42, 135–142 (2020). https://doi.org/10.1007/s10529-019-02756-5

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