Purification and cDNA Cloning of Isochorismate Synthase from
Elicited Cell Cultures of Catharanthus roseus |
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Authors: | Léon JP van Tegelen Paolo RH Moreno Anton F Croes Robert Verpoorte and George J Wullems |
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Institution: | Léon J.P. van Tegelen, Paolo R.H. Moreno, Anton
F. Croes, Robert Verpoorte, and George J. Wullems |
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Abstract: | Isochorismate is an important
metabolite formed at the end of the shikimate pathway, which is
involved in the synthesis of both primary and secondary metabolites. It
is synthesized from chorismate in a reaction catalyzed by the enzyme
isochorismate synthase (ICS; EC 5.4.99.6). We have purified ICS to
homogeneity from elicited Catharanthus roseus cell
cultures. Two isoforms with an apparent molecular mass of 64 kD were
purified and characterized. The Km values
for chorismate were 558 and 319 μm for isoforms I and II,
respectively. The isoforms were not inhibited by aromatic amino acids
and required Mg2+ for enzyme activity. Polymerase chain
reaction on a cDNA library from elicited C. roseus cells
with a degenerated primer based on the sequence of an internal peptide
from isoform II resulted in an amplification product that was used to
screen the cDNA library. This led to the first isolation, to our
knowledge, of a plant ICS cDNA. The cDNA encodes a protein of 64 kD
with an N-terminal chloroplast-targeting signal. The deduced amino acid
sequence shares homology with bacterial ICS and also with anthranilate
synthases from plants. Southern analysis indicates the existence of
only one ICS gene in C. roseus.The shikimate pathway is a major pathway in primary and secondary
plant metabolism (Herrmann, 1995). It provides chorismate for the
synthesis of the aromatic amino acids Phe, Tyr, and Trp, which are used
in protein biosynthesis, but also serves as a precursor for a wide
variety of aromatic substances (Herrmann, 1995; Weaver and Hermann,
1997; Fig. a). Chorismate is also the starting point of a biosynthetic
pathway leading to phylloquinones (vitamin K1)
and anthraquinones (Poulsen and Verpoorte, 1991). The first committed
step in this pathway is the conversion of chorismate into
isochorismate, which is catalyzed by ICS (Poulsen and Verpoorte, 1991;
Fig. b). Its substrate, chorismate, plays a pivotal role in the
synthesis of shikimate-pathway-derived compounds, and its distribution
over the various pathways is expected to be tightly regulated. Elicited
cell cultures of Catharanthus roseus provide an example of
the partitioning of chorismate. Concurrently, these cultures produce
both Trp-derived indole alkaloids and DHBA (Moreno et al., 1994). In
bacteria DHBA is synthesized from isochorismate (Young et al.,
1969). Elicitation of C. roseus cell cultures with a fungal
extract induces not only several enzymes of the indole alkaloid
biosynthetic pathway (Pasquali et al., 1992) but also ICS
(Moreno et al., 1994). Information concerning the expression and
biochemical characteristics of the enzymes that compete for available
chorismate (ICS, CM, and AS) may help us to understand the regulation
of the distribution of this precursor over the various pathways. Such
information is already available for CM (Eberhard et al., 1996) and AS
(Poulsen et al., 1993; Bohlmann et al., 1995) but not for ICS.
a, Position of ICS in the plant metabolism. SA,
Salicylic acid, OSB, o-succinylbenzoic acid. b, Reaction
catalyzed by ICS.Isochorismate plays an important role in bacterial and plant metabolism
as a precursor of o-succinylbenzoic acid, an intermediate in
the biosynthesis of menaquinones (vitamin K2)
(Weische and Leistner, 1985) and phylloquinones (vitamin
K1; Poulsen and Verpoorte, 1991). In bacteria
isochorismate is also a precursor of siderophores such as
DHBA (Young et al., 1969), enterobactin (Walsh et
al., 1990), amonabactin (Barghouthi et al., 1991), and salicylic acid
(Serino et al., 1995). Although evidence from tobacco would indicate
that salicylic acid in plants is derived from Phe via benzoic acid
(Yalpani et al., 1993; Lee et al., 1995; Coquoz et al., 1998), it
cannot be excluded that it is also synthesized from isochorismate. In
the secondary metabolism of higher plants, isochorismate is a precursor
for the biosynthesis of anthraquinones (Inoue et al., 1984; Sieweke and
Leistner, 1992), naphthoquinones (Müller and Leistner, 1978),
catalpalactone (Inouye et al., 1975), and certain alkaloids in orchids
(Leete and Bodem, 1976).ICS was first extracted and partially purified from crude extracts of
Aerobacter aerogenes (Young and Gibson, 1969). Later, ICS
activity was detected in protein extracts of cell cultures from plants
of the Rubiaceae, Celastraceae, and Apocynaceae families (Ledüc
et al., 1991; Poulsen et al., 1991; Poulsen and Verpoorte, 1992). Genes
encoding ICS have been cloned from bacteria such as Escherichia
coli (Ozenberger et al., 1989), Pseudomonas aeruginosa
(Serino et al., 1995), Aeromonas hydrophila (Barghouthi et
al., 1991), Flavobacterium K3–15
(Schaaf et al., 1993), Hemophilus influenzae
(Fleischmann et al., 1995), and Bacillus subtilis
(Rowland and Taber, 1996). Both E. coli and B.
subtilis have two distinct ICS genes; one is involved in
siderophore biosynthesis and the other is involved in menaquinone
production (Daruwala et al., 1996, 1997; Müller et al., 1996;
Rowland and Taber, 1996). The biochemical properties of the two ICS
enzymes from E. coli are different (Daruwala et al., 1997;
Liu et al., 1990). Sequence analysis has revealed that the bacterial
ICS enzymes share homology with the chorismate-utilizing
enzymes AS and p-aminobenzoate synthase, suggesting that
they share a common evolutionary origin (Ozenberger et al.,
1989).Much biochemical and molecular data concerning the shikimate pathway in
plants have accumulated in recent years (Schmid and Amrhein, 1995;
Weaver and Hermann, 1997), but relatively little work has been done on
ICS from higher plants. The enzyme has been partially purified from
Galium mollugo cell cultures (Ledüc et al., 1991,
1997), but purification of the ICS protein to homogeneity has remained
elusive, probably because of instability of the enzyme.Our interests focus on the role of ICS in the regulation of chorismate
partitioning over the various pathways. Furthermore, we studied ICS in
C. roseus to gain insight into the biosynthesis of DHBA in
higher plants (Moreno et al., 1994). In this paper we report the first
purification, to our knowledge, of ICS to homogeneity from a plant
source and the cloning of the corresponding cDNA. |
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