Functional Expression of Geraniol 10-Hydroxylase Reveals Its Dual Function in the Biosynthesis of Terpenoid and Phenylpropanoid

Abstract

Geraniol 10-hydroxylase (G10H), a cytochrome P450 monooxygenase, has been reported to be involved in the biosynthesis of terpenoid indole alkaloids. The gene for Catharanthus roseus G10H (CrG10H) was cloned and heterologously expressed in baculovirus-infected insect cells. A number of substrates were subjected to assay the enzyme activity of CrG10H. As reported in a previous study, CrG10H hydroxylated the monoterpenoid geraniol at the C-10 position to generate 10-hydroxygeraniol. Interestingly, CrG10H also catalyzed 3'-hydroxylation of naringenin to produce eriodictyol. Coexpression of an Arabidopsis NADPH P450 reductase substantially increased the ability of CrG10H to hydroxylate naringenin. The catalytic activity of CrG10H was approximately 10 times more efficient with geraniol than with naringenin, judged by the k(cat)/K(m) values. Thus, G10H also plays an important role in the biosynthetic pathway of flavonoids, in addition to its previously described role in the metabolism of terpenoids.

Functional expression of a putative geraniol 8-hydroxylase by reconstitution of bacterially expressed plant CYP76F45 and NADPH-cytochrome P450 reductase CPR I from Croton stellatopilosus Ohba

“…Its peak eluted at the retention time of 15.2 min appeared to have a pattern of mass fragmentation (m/z 67, 81, 109, 121 and 152, Fig. 6B) that was very similar to the reported mass fragmentation of 8-OH geraniol (2) (Collu et al, 2001;Sung et al, 2011). However, attempts to purify both CYP76F45 and CsCPR I from the 20,000 g microsomal fractions for kinetic studies were not successful due to the loss of enzyme activity in the process.…”

“…G8H has been suggested to be a potential site for regulation of the production of secologanin, which is involved in the biosynthesis of monoterpenoid indole alkaloids and iridoid monoterpenoids (Hofer et al, 2013). The enzyme from Catharanthus roseus has also been shown to catalyze the 3 0 hydroxylation of naringenin to produce eriodictyol in the flavonoid biosynthetic pathway (Sung et al, 2011).…”

“…Due to the difficulties usually encountered in fractioning individual proteins from endogenous CYP pools, characterization of CYPs has primarily depended on gene cloning and heterologous expression in eukaryotic systems, such as Pichia pastoris yeast (Wang et al, 2010), insect cells (Sung et al, 2011), and Saccharomyces cerevisiae (Tamaki et al, 2005). The use of eukaryotic systems allows CYP enzyme activities to be defined in cell lysates or purified microsomes with endogenous or co-expressed CPRs (Sung et al, 2011). For prokaryotic systems, however, there have been fewer cases where Escherichia coli was successfully used to express functional CYPs and CPRs to characterize their activities (for review, (Duan and Schuler, 2006)).…”

A Review on Pharmacological and Analytical Aspects of Naringenin

“…From the experimental study, it was found that the catalytic activity of CrG10H was approximately 10 times more effi cient with geraniol than with naringenin. (64) In another study, the activity of key enzymes of ammonium assimilation in cell-free extracts of soybean rhizobia characterized with different effectiveness in symbiosis was investigated. The nonspecific flavonoid naringenin has not stimulated glutamine synthetase (GS)-activity of the highly effi cient rhizobia, but has stimulated the activity of glutamate dehydrogenase.…”

A Pair of Tabersonine 16-Hydroxylases Initiates the Synthesis of Vindoline in an Organ-Dependent Manner inCatharanthus roseus

“…To analyze the substrate specificity of CYP71D351 and CYP71D12, microsomes from yeast strains expressing each enzyme were assayed against several different compounds, including MIAs, MIA precursors, and naringenin, which is a hydroxylatable flavonoid with aromatic ring, accumulated in leaf epidermis and substrate of geraniol 10-hydroxylase (Sung et al, 2011). LC-MS analysis of reaction mixtures revealed that, among all the tested compounds, only tabersonine (Aspidospermatype MIA) and to a lesser extent the two closely related compounds, 2,3-dihydro-3-hydroxytabersonine and 2,3-dihydrotabersonine, were metabolized by the two enzymes (Table I).…”