Functional characterization of squalene epoxidase and NADPH-cytochrome P450 reductase in Dioscorea zingiberensis

• Published in: Biochemical and biophysical research communications 2019-02, Vol.509 (3), p.822-827
• Main Authors: Song, Wei, Yan, Shan, Li, Yi, Feng, Shan, Zhang, Jia-jiao, Li, Jia-ru
• Format: Article
• Language: English
• Subjects: Dioscorea zingiberensis; Enzyme activity; NADPH-Cytochrome P450 reductase; Squalene epoxidase
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2019.01.010

Dioscorea zingiberensis is a perennial medicinal herb rich in a variety of pharmaceutical steroidal saponins. Squalene epoxidase (SE) is the key enzyme in the biosynthesis pathways of triterpenoids and sterols, and catalyzes the epoxidation of squalene in coordination with NADPH-cytochrome P450 reductase (CPR). In this study, we cloned DzSE and DzCPR gene sequences from D. zingiberensis leaves, encoding proteins with 514 and 692 amino acids, respectively. Recombinant proteins were successfully expressed in vitro, and enzymatic analysis indicated that, when SE and CPR were incubated with the substrates squalene and NADPH, 2,3-oxidosqualene was formed as the product. Subcellular localization revealed that both the DzSE and DzCPR proteins are localized to the endoplasmic reticulum. The changes in transcription of DzSE and DzCPR were similar in several tissues. DzSE expression was enhanced in a time-dependent manner after methyl jasmonate (MeJA) treatments, while DzCPR expression was not inducible. •Optimal conditions for expression of key enzymes in diosgenin synthesis in E. coli.•DzSE and DzCPR cooperate with each other to synthesis 2,3-oxidosqualene in vitro.•Endoplasmic reticulum serves as the active site for squalene oxidation.•Tissue expression showed the synthesis, transport and storage of 2,3-oxidosqualene.