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 for mation of geranyl diphosphate from dimethylallyl diphosphate and isope ntenyl diphosphate, was isolated from Vitis vinifera L. cv Muscat de F rontignan cell cultures. Purification of the enzyme was achieved succe ssively by ammonium sulfate precipitation and chromatography on DEAE-S ephacel, hydroxylapatite, Mono Q, Phenyl Superose, Superose 12, and pr eparative nondenaturing polyacrylamide gels. The enzyme formed only ge ranyl diphosphate as a product. In all cases, neither neryl diphosphat e, the cis isomer, nor farnesyl diphosphate was detected. The enzyme s howed a native molecular mass of 68 +/- 5 kD as determined by gel perm eation. On sodium dodecyl sulfate polyacrylamide gels, geranyl diphosp hate synthase purified to electrophoretic homogeneity migrated with a molecular mass of 66 +/- 2 kD. Michaelis constants for isopentenyl dip hosphate and dimethylallyl diphosphate were 8.5 and 56.8 mum, respecti vely. The enzyme required Mn2+ and Mg2+ as cofactors and its activity was enhanced by Triton X-100. Inorganic pyrophosphate, aminophenylethy l diphosphate, and geranyl diphosphate had inhibitory effects on the e nzyme.