Plant Science 51 2000 97 – 105
Purification and partial characterisation of geranylgeranyl diphosphate synthase, from Taxus baccata cell cultures
An enzyme that regulates taxane biosynthesis
Gregory Laskaris , Robert van der Heijden, Rob Verpoorte
Di6ision of Pharmacognosy, LeidenAmsterdam Centre for Drug Research, Leiden Uni6ersity, P.O. Box
9502
,
2300
RA Leiden, The Netherlands Received 8 September 1999; received in revised form 2 November 1999; accepted 6 November 1999
Abstract
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 9 2 kDa, resulting from the
association of two apparently identical subunits having a molecular weight of 38 kDa. Farnesyl diphosphate K
m
2.46 mM in combination with isopentenyl diphosphate K
m
1.5 mM was the most effective substrate. Dimethyl allyl diphosphate was a poorer substrate K
m
12.7 mM. 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 20 000 × g
centrifugal force pellet and a non-ionic detergent was used for its extraction. The inclusion of detergent was not necessary during subsequent chromatographic steps. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords
:
Taxus baccata; GGPP synthase; Purification www.elsevier.comlocateplantsci
1. Introduction
Geranylgeranyl diphosphate GGPP synthase E.C. 2.5.1.29 provides the C-20 building block
for various classes of compounds, i.e. diterpenes, carotenoids and chlorophylls in plants.
Paclitaxel is a diterpene originating from vari- ous Taxus species and is one of the most impor-
tant anticancer agents. The efficacy of paclitaxel has urged scientists to gain insight in the pathway
flux towards taxane formation in order to use this knowledge to increase taxane production by
means of metabolic engineering. The three first steps of paclitaxel biosynthesis are known: cyclisa-
tion of geranylgeranyl diphosphate to taxa- 45,1112-diene
[1], hydroxylation
to taxa-
420,1112-dien-5a-ol [2] and the conversion of this alcohol to the corresponding acetate ester [3]
Fig. 1.
Taxadiene synthase,
the enzyme
catalysing the cyclisation of GGPP, has been purified [4] and the corresponding cDNA cloned
[5]. Although the low level of taxadiene synthase activity in Taxus bre6ifolia stem disks suggests a
crucial role for the regulation of the pathway, this could not be confirmed by using T. canadensis cell
cultures [6]. An insight into the molecular mecha- nisms governing taxane biosynthesis is important
in order to ensure an increased production of paclitaxel and to achieve biotransformations of
various other taxoids i.e. taxines to pharmaceuti- cally important taxanes. Thus far, GGPP synthase
is the sole enzyme having shown to control the rate of taxane skeleton formation [7,8] and conse-
quently further characterisation seemed necessary. In this paper the purification and some properties
Abbre6iations
:
NAA, naphthalene acetic acid; IPP, isopentenyl n pOp isopentenyl diphosphate; FPP, farnesyl n pOp farnesyl diphos-
phate; GGPP, geranylgeranyl n pOp geranylgeranyl diphosphate. Corresponding author. Present address: Laboratory of Pharma-
cognosy, Department of Pharmacy, Aristotle University of Thessa- loniki, 540 06 Thessaloniki, Greece. Tel.: + 30-31-997617, 997619;
fax: + 30-31-997645. E-mail address
:
laskarispharm.auth.gr G. Laskaris 0168-945200 - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 1 6 8 - 9 4 5 2 9 9 0 0 2 6 3 - 0
of this key enzyme from Taxus baccata cell cul- tures are presented.
2. Methods
