Protopanaxadiol 6‐hydroxylase and its role in regulating the ginsenoside heterogeneity in Panax notoginseng cells

Abstract

Various structure-similar plant secondary metabolites like ginseng saponins (ginsenosides) possess different or even totally opposite biological activities. Intentional manipulation of the ginsenoside heterogeneity in cellular biosynthesis is of great interest and significance [Zhong and Yue (2005); Adv Biochem Eng Biotechnol 100:53-88]. In this work, CO-binding spectra of microsomes prepared from the suspended cells of Panax notoginseng showed increases in absorption at 450 nm compared with the control without CO sparging, and protopanaxadiol 6-hydroxylase (P6H), a new enzyme catalyzing the conversion of ginsenoside aglycone protopanaxadiol into protopanaxatriol, was found. P6H was dependent on NADPH and molecular oxygen. The enzymatic reaction was inhibited by carbon monoxide and partially reversible upon illumination with blue light, and sensitive to cytochrome P450 inhibitors. The results supported the contention that P6H was a cytochrome P450-dependent hydroxylase, whose catalytic product was confirmed to be protopanaxatriol by HPLC-MS. Induction of P6H activity by phenobarbital, a cytochrome P450 inducer, was observed. A maximal activity of P6H was obtained with addition of 0.5 mM phenobarbital on day 4 of shake-flask cultivation. The maximum content of protopanaxatriol-type ginsenosides (Rg(1) and Re, Rg group) and the maximum ratio of the content of protopanaxatriol: protopanaxadiol reached 6.88 +/- 0.21 mg g(-1) dry weight and 7.0, respectively, which was about 1.4 and 2.0-fold that of respective controls (without addition of phenobarbital). Oxidative burst was also observed in the cell cultures with addition of phenobarbital. P6H was concluded as a key enzyme in regulating Rg-group ginsenoside biosynthesis in P. notoginseng cells.

Upregulation of ginsenoside and gene expression related to triterpene biosynthesis in ginseng hairy root cultures elicited by methyl jasmonate

“…Two triterpene aglycons, protopanaxadiol and protopanaxatriol, were synthesized by the consecutive reaction of dammarenediol 12-hydroxylase (D12H) and protopanaxadiol 6-hydroxylase (P6H), respectively. These enzymes responsible for hydroxylation were putatively Recently, P6H enzyme, which catalyzes the biosynthesis of protopanaxatriol from protopanaxadiol, has been identified using microsomal proteins of P. notoginseng cell suspension cultures (Yue et al 2008). Hu and Zhong (2007) have revealed that the P6H activity was increased with MJ exposure time in cell suspension cultures.…”

“…However, there is currently no genetic information available regarding the next step in ginseng, possibly hydroxylation or glycosidation. Recently, protopanaxatriol has been successfully biotransformed from protopanaxadiol using microsomal proteins evidencing protopanaxadiol 6-hydroxylase (P6H) activity (Yue et al 2008). Choi et al (2005) have reported that the EST (expressed sequence tag) data of this plant and the candidate genes encoding for cytochrome P450-dependent hydroxylase and glucosytransferase have been selected.…”

Key Enzymes of Triterpenoid Saponin Biosynthesis and the Induction of Their Activities and Gene Expressions in Plants

“…PDMOs mediate the oxidation of inert methylene and methyl groups of triterpene skeletons [60,61]. They include several concrete enzymes, for example, protopanaxadiol hydroxylase (P6H) which catalyzes the biosynthesis of protopanaxatriol from protopanaxadiol [62], and cytochrome P 450 -dependent hydroxylase [63] NADPH and O 2 , and that the enzymatic reaction was inhibited by carbon monoxide, was partially reversible upon illumination with blue light, and sensitive to cytochrome P 450 inhibitors [62].…”

Advances in ginsenoside biosynthesis and metabolic regulation

“…In 2008, protopanaxadiol 6‐hydroxylase (P6H), a new enzyme catalyzing the conversion of ginsenoside aglycone protopanaxadiol into protopanaxatriol was found by Yue et al. . In 2012, Han et al.…”

“…At present, more than 40 genes encoding ginsenoside biosynthesis related enzymes have been cloned and identified from Panax genus, and the basic biological elements have been provided for the production of ginsenosides by biosynthesis, which laid a good foundation for the study. In 2008, protopanaxadiol 6-hydroxylase (P6H), a new enzyme catalyzing the conversion of ginsenoside aglycone protopanaxadiol into protopanaxatriol was found by Yue et al [43]. In 2012, Han et al [2] identified CYP716A47 from ginseng to catalyze the synthesis of protopanaxadiol (PPD) at the C-12 hydroxylation of dammarane; CYP716A53v2 catalyzes the further synthesis of protopanaxadiol (PPT) through C-6 hydroxylation of PPD.…”