Citation: Jia-Jing Zhou, Zi-Long Wang, Meng Zhang, Yang-Oujie Bao, Guo-Wei Chang, Yun-Gang Tian, Min Ye. Characterization of two highly specific O-rhamnosyltransferases involved in the biosynthesis of steroidal saponins from Paris polyphylla var. yunnanensis[J]. Chinese Chemical Letters, ;2025, 36(11): 110805. doi: 10.1016/j.cclet.2024.110805 shu

Characterization of two highly specific O-rhamnosyltransferases involved in the biosynthesis of steroidal saponins from Paris polyphylla var. yunnanensis

Jia-Jing Zhou ^a ,
Zi-Long Wang ^a ,
Meng Zhang ^a ,
Yang-Oujie Bao ^a ,
Guo-Wei Chang ^d ,
Yun-Gang Tian ^a ,
Min Ye ^{a, b, c, *,}

a.
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China
b.
Peking University-Yunnan Baiyao International Medical Research Center, Beijing 100191, China
c.
Southwest United Graduate School, Kunming 650092, China
d.
Key Laboratory of Seed Innovation, Institute of Genetics and Developmental Biology, The Innovative Academy of Seed Design, Chinese Academy of Sciences, Beijing 100101, China

yemin@bjmu.edu.cn

Received Date: 16 August 2024
Revised Date: 23 December 2024
Accepted Date: 26 December 2024
Available Online: 26 December 2024

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Steroidal saponins are major bioactive compounds of the medicinal plant Paris polyphylla var. yunnanensis. In this work, two O-rhamnosyltransferases PpRhaGT1 and PpRhaGT2 with strict substrate specificity were characterized from this plant. These enzymes could catalyze the synthesis of paris saponins Ⅱ and Ⅶ, and realized semi-biosynthesis of a series of paris steroidal saponins in tobacco leaves. Molecular dynamics simulation revealed the substrate specificity of PpRhaGT1 was due to interactions between the 2′-O-rhamnosyl group and surrounding amino acids particularly S382 and E383.
- Paris polyphylla var. yunnanensis,
- Glycosyltransferase,
- Paris saponin Ⅱ,
- Paris saponin Ⅶ,
- Biosynthesis
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Figure 1. Bioinformatic analyses of candidate rhamnosyltransferase genes from Paris polyphylla var. yunnanensis. (A) Putative biosynthetic pathway of paris saponin Ⅱ (9) and paris saponin Ⅶ (10). (B) Expression levels of candidate genes in the transcriptome of different parts of the plant. (C) Cluster analysis of five candidate genes with complete reading frames.

Figure 2. Functional characterization of PpRhaGT1. (A) Glycosylation of paris saponin Ⅴ (5) and paris saponin Ⅵ (6) catalyzed by recombinant PpRhaGT1 using UDP-Rha as sugar donor. (B) Total ion chromatograms (TICs) of the enzymatic reaction mixture and the reference standards. (C) LC/MS analysis of the glycosylated products 7 and 8.

Figure 3. Functional characterization of PpRhaGT2. (A) Glycosylation of dioscin (7) and pennogenin 3-O-chacotrioside (8) catalyzed by recombinant PpRhaGT2 using UDP-Rha as sugar donor. (B) TICs of the enzymatic reaction mixture and the reference standards. (C) LC/MS analysis of the glycosylated products 9 and 10.

Figure 4. Substrate selectivity of PpRhaGT1. (A) Two putative biosynthetic pathways of dioscin (7) and pennogenin 3-O-chacotrioside (8). (B) TICs of the enzymatic reaction mixture and the reference standards. (C) Distance between the oxygen atom and anomeric carbon in 50 ns molecular dynamics simulation. (D) Structural analysis of PS Ⅴ (5) with surrounding amino acids. (E) The frequency of hydrogen bonds formed by 2′-O-rhamnosyl group and surrounding amino acids in MD.

Figure 5. Semi-biosynthesis of dioscin (7), pennogenin 3-O-chacotrioside (8), paris saponin Ⅱ (9) and paris saponin Ⅶ (10) in tobacco. (A) The experimental procedure. (B) The engineered biosynthetic pathway of 7, 8, 9 and 10 in Nicotiana benthamiana. (C) Semi-biosynthesis of 7, 8, 9 and 10. (D) LC/MS analysis of the glycosylated products. The structures of 7, 8, 9 and 10 were identified by comparing with reference standards.