Induction and Characterization of a Cytochrome P-450-Dependent Camphor Hydroxylase in Tissue Cultures of Common Sage (Salvia officinalis)

Abstract

(+)-Camphor, a major monoterpene of the essential oil of common sage (Salvia officinalis), is catabolized in senescent tissue, and the pathway for the breakdown of this bicyclic ketone has been previously elucidated in sage cell-suspension cultures. In the initial step of catabolism, camphor is oxidized to 6-exo-hydroxycamphor, and the corresponding NADPH-and O,-dependent hydroxylase activity was demonstrated in microsomal preparations of sage cells.Severa1 well-established inhibitors of cytochrome P-450-dependent reactions, including cytochrome c, clotrimazole, and C O , inhibited the hydroxylation of camphor, and CO-dependent inhibition was partially reversed by blue light. Upon treatment of sage suspension cultures with 30 mM MnCI2, camphor-6-hydroxylase activity was induced up to 7-fold. A polypeptide with estimated molecular mass of 58 kD from sage microsomal membranes exhibited antigenic cross-reactivity in western blot experiments with two heterologous polyclonal antibodies raised against cytochrome P-450 camphor-5-exo-hydroxylase from Pseudomonas putida and cytochrome P-450 limonene-6s-hydroxylase from spearmint (Mentha spicata). Dot blotting indicated that the concentration of this polypeptide increased with camphor hydroxylase activity in microsomes of Mn'+-induced sage cells. These results suggest that camphor-6-exo-hydroxylase from sage is a microsomal cytochrome P-450 monooxygenase that may share common properties and epitopes with bacterial and other plant monoterpene hydroxylases.The biosynthetic capacity of plant cell cultures to produce various monoterpenoid, sesquiterpenoid, and diterpenoid substances has been demonstrated; however, the accumulation of monoterpenes in cultured cell systems is only rarely observed (Banthorpe et al., 1986; Charlwood et al., 1989). Studies with undifferentiated suspension cultures of common sage (Salvia officinalis) showed that the enzymes directly responsible for the conversion of the ubiquitous isoprenoid precursor, geranyl pyrophosphate (Fig. 1, structure l)', to (+)-camphor (Z), a major monoterpene of the intact plant, were readily detected during the late logarithmic phase of growth. Yet, no significant accumulation of camphor (C0.3 ng/g fresh

A Cytochrome P-450 Monooxygenase Catalyzes the First Step in the Conversion of Tabersonine to Vindoline in Catharanthus roseus

“…In addition, CO inhibition of Cyt P-450-catalyzed reactions is photoreversible (Estabrook et al, 1963). CO inhibited 16-OH (Table I) in the range observed for other Cyt P-450-dependent reactions in plants (Karp et al, 1990;Funk and Croteau, 1993;Gerardy and Zenk, 1993;Rueffer and Zenk, 1994). However, the photoreversibility of this inhibition could not be tested by exposure of the reaction mixture to white light or to light filtered through a 10% CuSO, solution, which transmits light around 450 nm.…”

The Sauromatum guttatum appendix as an osmophore: excretory pathways, composition of volatiles and attractiveness to insects

“…For example, n-hexanal is formed from linoleic acid (Galliard et al, 1977). The ketones, alcohols, and the phenolic derivatives might be oxidative products of cytochrome P450-linked enzymes that have been implicated in different biosynthetic pathways (Donaldson & Luster, 1991;West CA, 1980):terpene hydroxylation (Funk & Croteau, 1993;Funk et al, 1994), fatty acid hydroxylation (Zimmerlin et al, 1992), and phenolic hydroxylation (Benveniste, Salaun & Durst, 1977). Some of the alcohols and ketones might be isoprenoid in orgin, e.g.…”

Cloning of Wound-Induced Cytochrome P450 Monooxygenases Expressed in Pea

“…Those in the core and branched phenylpropanoid pathways are induced by exposure to manganese, funga1 elicitors, phenobarbital, wounding, and some herbicides (Reichhart et al, 1980;Bolwell and Dixon, 1986;Stewart and Schuler, 1989;Frear et al, 1991;Mougin et al, 1991;Fahrendorf and Dixon, 1993;Funk and Croteau, 1993;Orr et al, 1993;Pendharkar and Nair, 1993;Toguri et al, 1993b;Werck-Reichhart et al, 1993;. The magnitude and time course for induction of these enzymatic activities vary greatly depending on the inducer used, the tissue examined, and, in seedlings, which contain a large number of cell types, the developmental state of the cells.…”