2. Academic Validation
  3. Characterization and protein engineering of a novel UDP-glycosyltransferase involved in pseudoginsenoside Rt5 biosynthesis from Panax japonicus

Characterization and protein engineering of a novel UDP-glycosyltransferase involved in pseudoginsenoside Rt5 biosynthesis from Panax japonicus

  • Int J Biol Macromol. 2024 Oct;277(Pt 4):134537. doi: 10.1016/j.ijbiomac.2024.134537.
Pengfei Li 1,  Chaokang Huang 1,  Tengfei Niu 1,  Xiaolin Yang 1,  Huida Guan 1,  Lili Ding 1,  Li Yang 2,  Zhengtao Wang 2,  Zhongji Pu 3,  Rufeng Wang 4
Affiliations

Affiliations

  • 1 Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China; The MOE Key Laboratory for Standardization of Chinese Medicines, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China.
  • 2 Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China; The MOE Key Laboratory for Standardization of Chinese Medicines, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China; The SATCM Key Laboratory for New Resources and Quality Evaluation of Chinese Medicines, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China.
  • 3 Xianghu laboratory, Hangzhou 311231, China.
  • 4 Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China; The MOE Key Laboratory for Standardization of Chinese Medicines, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China; The SATCM Key Laboratory for New Resources and Quality Evaluation of Chinese Medicines, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China. Electronic address: [email protected].
PMID: 39111463 DOI: 10.1016/j.ijbiomac.2024.134537
Abstract

As one of rare high-value ocotillol (OCT)-type ginsenosides, pseudoginsenoside Rt5 has been identified with significant pharmacological activities. UDP-glycosyltransferases (UGTs) play pivotal roles in catalyzing the transfer of a glycosyl moiety from a donor to an acceptor. In this study, the novel UGT, PjUGT10, was screened from the transcriptome database of Panax japonicus and identified with the enzymatic activity of transferring a glucosyl group on OCT to produce Rt5. The catalytic efficiency of PjUGT10 was further enhanced by employing site-directed mutation. Notably, the variant M7 exhibited a remarkable 6.16 × 103-fold increase in kcat/Km towards 20S,24R-ocotillol and a significant 2.02 × 103-fold increase to UDP-glucose, respectively. Moreover, molecular dynamics simulations illustrated a reduced distance between 20S,24R-ocotillol and the catalytic residue His15 or UDP-glucose, favoring conformation interactions between the enzyme and substrates. Subsequently, Rt5 was synthesized in an engineered Escherichia coli strain M7 coupled with a UDP-glucose synthetic system. This study not only shed light on the protein engineering that can enhance the catalytic activity of PjUGT10, but also established a whole-cell approach for the production of Rt5.

Keywords

OCT-type ginsenosides; Pseudoginsenoside Rt5; UDP-glycosyltransferase.

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