Geranylhydroquinone 3′′-hydroxylase, a cytochrome P-450 monooxygenase from Lithospermum erythrorhizon cell suspension cultures

Abstract

Geranylhydroquinone 3"-hydroxylase, which is likely to be involved in shikonin and dihydroechinofuran biosynthesis, was identified in cell suspension cultures of Lithospermum erythrorhizon Sieb. et Zucc. (Boraginaceae). The enzyme hydroxylates the isoprenoid side chain of geranylhydroquinone (GHQ), a known precursor of shikonin. Proton/proton correlation spectroscopic and proton/proton long-range correlation spectroscopic studies confirmed that hydroxylation takes place specifically at position 3", i.e. at the methyl group involved in the cyclization reaction. The enzyme is membrane-bound and was found in the microsomal fraction. It requires NADPH and molecular oxygen as cofactors, and is inhibited by cytochrome P-450 inhibitors such as cytochrome c and CO. The inhibitory effect of CO is reversed by illumination. These data suggest that the enzyme is a cytochrome P-450-dependent monooxygenase. The optimum pH of GHQ 3"-hydroxylase is 7.4, and the apparent K(m) value for GHQ is 1.5 microM. The reaction velocity obtained with 3-geranyl-4-hydroxybenzoic acid was more than 100 times lower than that obtained with geranylhydroquinone.

<i>Lithospermum erythrorhizon</i> cell cultures: Present and future aspects

“…Two key precursors of shikonin, geranyl diphosphate (GPP) derived via the mevalonate pathway and PHB derived via the shikimate pathway (Inouye et al 1979), are coupled by a geranyltransferase to yield the intermediate, m-geranyl-p-hydroxybenzolic acid (GBA) (Heide and Tabata 1987b). GBA is subsequently converted to geranylhydroquinone, followed by the hydroxylation of the geranyl chain (Yamamoto et al 2000a). The formation of a naphthalene ring by this intermediate yields shikonin, whereas cyclicization of the geranyl moiety to form a furan ring yields dihydroechinofuran (Yazaki et al 1986a).…”

“…Geranylhydroquinone 3″-hydroxylase, which provides a common intermediate for shikonin and dihydroechinofuran from geranylhydroquinone (GHQ), was identified in suspension cultures of non-pigmented cells (Yamamoto et al 2000a). This enzyme, which introduces a hydroxyl residue to the isoprenoid side chain of GHQ at position 3″, localizes to the microsomal fraction.…”

“…Proteins involved in this pathway include GPP synthase (Sommer et al 1995), PHB O-glucosyl transferase (Bechthold et al 1991;Li et al 1997; and geranylhydroquinone 3″-hydroxylase (Yamamoto et al 2000a). Molecular biological approaches are required to isolate these genes, including their regulation by light in cultured L. erythrorhizon cells.…”

Cytochromes P450 in phenolic metabolism

“…Shikonin is a red naphthazarin pigment derived from the roots of Lithospermum erythorhizon. Investigations of the metabolism of geranylhydroquinone in cell-free extracts from L. erythrorhizon cells, in order to identify the reactions preceding the final cyclization step in shikonin biosynthesis, led Yamamoto et al (2000) to the discovery of geranylhydroquinone 3¢-hydroxylase (GHQ3¢H), a cytochrome P450 monooxygenase catalyzing the conversion of GHQ to its hydroxylated derivative 3¢-hydroxy-geranylhydroquinone, which is an intermediate in the biosynthesis of shikonin (Fig. 7).…”

High Level Expression of Chorismate Pyruvate-Lyase (UbiC) and HMG-CoA Reductase in Hairy Root Cultures of Lithospermum erythrorhizon

“…GBA undergoes decarboxylation and oxidation to geranyl hydroquinone, which is subsequently hydroxylated (Yamamoto et al 2000) prior to cyclization to the naphthalene ring system. Further oxidation reactions lead to shikonin.…”