Purification and partial characterisation of geranylgeranyl diphosphate synthase, from Taxus baccata cell cultures. An enzyme that regulates taxane biosynthesis.
Geranylgeranyl diphosphate (GGPP) synthase, an enzyme that regulates taxane biosynthesis, was purified to homogeneity from cell cultures of Taxus baccata. The molecular weight of the native protein was estimated to be 76+/-2 kDa, resulting from the association of two apparently identical subunits having a molecular weight of 38 kDa. Farnesyl diphosphate (K(m) 2.46 µM) in combination with isopentenyl diphosphate (K(m) 1.5 µM) was the most effective substrate. Dimethyl allyl diphosphate was a poorer substrate (K(m) 12.7 µM). Mn(2+) ion at 4 mM in combination with Mg(2+) of 2 mM gave the greatest stimulation of activity. The pI of the enzyme was lower than 4 and the pH optimum was between 6.9 and 7.2. The enzyme activity was found in the 20000xg (centrifugal force) pellet and a non-ionic detergent was used for its extraction. The inclusion of detergent was not necessary during subsequent chromatographic steps.
Laskaris
G
Division of Pharmacognosy, Leiden/Amsterdam Centre for Drug Research, Leiden University, P.O. Box 9502, 2300 RA, Leiden, The Netherlands
van der Heijden R
Verpoorte
R