Purification and Characterization of Geranyl Diphosphate Synthase from Vitis vinifera L. cv Muscat de Frontignan Cell Cultures.

A geranyl diphosphate synthase (EC 2.5.1.1), which catalyzes the formation of geranyl diphosphate from dimethylallyl diphosphate and isopentenyl diphosphate, was isolated from Vitis vinifera L. cv Muscat de Frontignan cell cultures. Purification of the enzyme was achieved successively by ammonium sulfate precipitation and chromatography on DEAE-Sephacel, hydroxylapatite, Mono Q, Phenyl Superose, Superose 12, and preparative nondenaturing polyacrylamide gels. The enzyme formed only geranyl diphosphate as a product. In all cases, neither neryl diphosphate, the cis isomer, nor farnesyl diphosphate was detected. The enzyme showed a native molecular mass of 68 [plus or minus] 5 kD as determined by gel permeation. On sodium dodecyl sulfate polyacrylamide gels, geranyl diphosphate synthase purified to electrophoretic homogeneity migrated with a molecular mass of 66 [plus or minus] 2 kD. Michaelis constants for isopentenyl diphosphate and dimethylallyl diphosphate were 8.5 and 56.8 [mu]M, respectively. The enzyme required Mn2+ and Mg2+ as cofactors and its activity was enhanced by Triton X-100. Inorganic pyrophosphate, aminophenylethyl diphosphate, and geranyl diphosphate had inhibitory effects on the enzyme.

Evidence for a geranyl-diphosphate synthase located within the plastids of Vitis vinifera L. cultivated in vitro.

Intact plastids from cell suspensions of Vitis vinifera L. cv. Muscat de Frontignan, free of detectable contamination by other particles as judged by the distribution of organelle-specific marker enzymes and by electron microscopy, exhibit geranyl-diphosphate synthase activity (EC 2.5.1.1). This synthase activity remains stable after tryptic digestion of unlysed organelles and is enhanced by plastid disruption. We conclude that the enzyme is located within the organelle. The possibility of an isopentenyl diphosphate/dimethylallyl diphosphate translocating system which would play a major role in the regulation of monoterpene metabolism is discussed.

Prenyltransferase compartmentation in cells of Vitis vinifera cultivated in vitro.

Two prenyltransferases were located in cell cultures of Vitis vinifera. A geranyl pyrophosphate synthase (EC 2.5.1.1) was associated with plastid-like membranes whereas a farnesyl pyrophosphate synthase (EC 2.5.1.10) was found to be soluble.

Extractive bioconversion of geraniol by a Vitis vinifera cell suspension employing a two-phase system.

Among the problems associated with the bioconversion of monoterpenes by plant cell suspensions are the toxicity of some substrates and/or products at low concentrations, the transient state of nascent products and the length of time required to obtain the cell suspension. We investigated the extractive bioconversion of geraniol by a Vitis vinifera c.v. Muscat de Frontignan cell suspension in a two-phase system consisting of an aqueous nutrient phase surmounted by a lipid (Miglyol 812) phase. This system proved to be advantageous as it allowed a five-fold increase in the substrate load without causing any detrimental effect on the cell suspension, it improved the persistence of nascent products and it permitted the recycling or re-use of the cell suspension in a monoterpene bioconversion process.

Biogenesis of monoterpenes : Bioconversion of citral by a cell suspension culture of muscat grapes.

Cell suspension cultures of "Muscat de Frontignan" grapes Vitis vinifera L. are able to convert citral (a mixture of neral and geranial) into the corresponding monoterpenic alcohols, nerol and geraniol. The geraniol formed is esterified into geranyl acetate. Bioconversion of nerol or geraniol added alone to the cell suspension was also studied. Interconversions between these different monoterpenic compounds are described and discussed.