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

摘要

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.