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
  • 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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