Planta (1999) 207: 325±334
Review
A unique reaction in a common pathway: mechanism and function of
chorismate synthase in the shikimate pathway
Peter Macheroux, JuÈrg Schmid*, Nikolaus Amrhein, Andreas Schaller
Swiss Federal Institute of Technology, Institute of Plant Sciences, UniversitaÈtstr. 2, CH-8092 ZuÈrich, Switzerland
Received: 22 June 1998 / Accepted: 7 August 1998
Abstract. Chorismate synthase, the seventh enzyme in
the shikimate pathway, catalyzes the transformation of
5-enolpyruvylshikimate 3-phosphate to chorismate
which is the last common precursor in the biosynthesis
of numerous aromatic compounds in bacteria, fungi and
plants. The enzyme has an absolute requirement for
reduced FMN as a cofactor, although the 1,4-anti
elimination of phosphate and the C(6proR)-hydrogen
does not involve a net redox change. The role of the
reduced FMN in catalysis has long been elusive.
However, recent detailed kinetic and bioorganic
approaches have fundamentally advanced our understanding of the mechanism of action, suggesting an
initial electron transfer from tightly bound reduced
¯avin to the substrate, a process which results in CAO
bond cleavage. Studies on chorismate synthases from
bacteria, fungi and plants revealed that in these organisms the reduced FMN cofactor is made available in
di€erent ways to chorismate synthase: chorismate synthases in fungi ± in contrast to those in bacteria and
plants ± carry a second enzymatic activity which enables
them to reduce FMN at the expense of NADPH. Yet, as
shown by the analysis of the corresponding genes, all
chorismate synthases are derived from a common
ancestor. However, several issues revolving around the
origin of reduced FMN, as well as the possible regulation of the enzyme activity by means of the availability
of reduced FMN, remain poorly understood. This
review summarizes recent developments in the biochemical and genetic arena and identi®es future aims in this
®eld.
Key words: Aromatic amino acids ± Chorismate synthase
± Flavin reductase ± Shikimate pathway
*Present address: Novartis Crop Protection, P.O. Box 12257,
Research Triangle Park, NC 27709-2257, USA
Abbreviations: DAHP ˆ 3-deoxy-D-arabino-heptulosonate
7-phosphate; EPSP ˆ 5-enolpyruvylshikimate 3-phosphate
Correspondence to: A. Schaller; FAX: +41 (1) 632 1084;
E-mail: [email protected];
Introduction
The shikimate pathway provides the basic building
blocks for the synthesis of the three aromatic amino
acids as well as an array of other aromatic compounds
required for functions as di€erent as UV protection,
electron transport, signaling, communication, plant
defense and the wound response. The pathway is ®rmly
rooted in primary metabolism and forms a major link
between primary and secondary metabolism in higher
plants. The ®rst seven reactions of the pathway lead
from erythrose 4-phosphate and phosphoenolpyruvate
via shikimate to chorismate and are also referred to as
the main trunk of the shikimate pathway, or the
prechorismate pathway (Bentley 1990).
The shikimate pathway is present only in bacteria,
fungi and plants. The absence of the pathway in all other
genera has rendered the enzymes catalyzing these
reactions potentially useful targets for the development
of new antibiotics and herbicides.
Some of these reactions are quite unique in nature: For
example, 5-enolpyruvylshikimate 3-phosphate synthase
(EPSP-synthase), the sixth enzyme of the prechorismate
pathway, catalyzes the transfer of the intact enolpyruvyl
(carboxyvinyl) moiety from phosphoenolpyruvate to
shikimate 3-phosphate. The only other example of this
type of enol ether transfer reaction is found in the ®rst
committed step in bacterial cell wall biosynthesis, i.e. the
reaction catalyzed by UDP-N-acetylglucosamine
enolpyruvyl transferase (Rogers et al. 1980). These two
enzymes are the targets of the broad-range herbicide
glyphosate (SteinruÈcken and Amrhein 1980) and the
antibiotic fosfomycin (Kahan et al. 1974), respectively.
From a biochemical point of view, chorismate synthase is
even more exceptional than EPSP-synthase: the enzyme
and the reaction it catalyzes are unique and the elusive
requirement for reduced FMN proves to be a challenging
problem. The role of the reduced cofactor in catalysis as
well as its origin and availability in vivo with the
implications of a possible regulatory role were central to
this ®eld of research and will be the focus of this article.
326
Reduced FMN is involved in catalysis of the chorismate
synthase reaction
The chorismate synthase reaction (Fig. 1) comprises an
anti-1,4-elimination of the 3-phosphate group and the
C(6proR)-hydrogen (Hill and Newkome 1969; Onderka
and Floss 1969). As such, this elimination reaction does
not involve an overall change in the redox state.
Therefore, the early observation by Morell et al. (1967)
and Welch et al. (1974) that the enzyme has an absolute
requirement for ¯avin, an essential cofactor typically
encountered in many biological redox reactions, was
surprising. Moreover, it could be shown that the ¯avin
cofactor must be present in its (two-electron) reduced
form (Welch et al. 1974) which, according to these
authors, ``is not consumed stoichiometrically during the
catalytic reaction''. Similarly, the second reaction of the
shikimate pathway catalyzed by 3-dehydroquinate
synthase, i.e. the synthesis of 3-dehydroquinate from
3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP),
does not involve an overall change in redox state either.
This enzyme utilizes the NAD+-cofactor which is
transiently reduced in the oxidation of a secondary
alcohol group in order to facilitate hydrogen abstraction
at the adjacent carbon (Bender et al. 1989). In the case of
chorismate synthase, two explanations appeared plausible for the function of the reduced ¯avin cofacter: (i) the
¯avin has merely a structural and not a functional role
and (ii) the ¯avin restores the reduced form of an
oxidation-sensitive sulfhydryl group in the enzyme
which is crucial for binding of the substrate or possibly
catalytically important (Hasan and Nester 1978c; Walsh
1979). Circumstantial support for the ®rst possibility
was provided by the presence of a ¯avin in a number of
proteins, such as acetolactate synthase (EC. 4.1.3.18),
oxynitrilase (EC 4.1.2.10), and glyoxylate carboligase
(EC 4.1.1.47), that also catalyze non-redox reactions. It
was shown by Schuman Jorns (1979) that the ¯avin
plays a mainly structural role in the ¯avoprotein
oxynitrilase (also called mandelonitrile lyase or hydroxynitrile lyase), an enzyme involved in cyanogenesis,
which catalyses the reversible condensation of HCN
with aldehydes to form D-a-hydroxynitriles in plants.
However, similar experiments performed with 5-deazaFMN and Escherichia coli chorismate synthase showed
that this ¯avin derivative is inactive as a redox cofactor
(Lauhon and Bartlett 1994; Bornemann et al. 1995b).
More detailed studies revealed that chorismate synthase
activity strongly depends on the chemical nature of the
¯avin derivative (Macheroux et al. 1996a). This evidence
indicates a catalytic role for the ¯avin moiety in
Fig. 1. Reaction catalyzed by chorismate synthase
P. Macheroux et al.: A unique reaction in a common pathway
chorismate synthase, as opposed to its obvious structural role in oxynitrilase. This is also supported by the
fact that not all oxynitrilases contain an FAD cofactor
(Xu et al. 1988; Kuroki and Conn 1989) whereas, on the
other hand, all isolated chorismate synthases from
various sources (bacteria, fungi and plants) have a
requirement for reduced ¯avin.
On the other hand, the second possibility mentioned
above was never conclusively substantiated, and an
alignment of all known chorismate synthase sequences
(a total of 18: 3 from plant, 3 from fungal, and 12 from
bacterial species) revealed that none of the cysteine
residues is conserved in chorismate synthases (Macheroux
et al. 1998). The question of the role of reduced FMN in
the chorismate synthase reaction remained unanswered
for a long time. In part, this was a consequence of the very
limited amounts of protein that were extractable from the
natural sources of this low-abundance enzyme. With the
advent of modern expression technology, however, more
protein material became available and this technical
breakthrough was exploited by Coggins and co-workers
to clone the gene coding for the E. coli enzyme and develop
an overexpression system (White et al. 1988). As a
consequence, most of our current knowledge of the
chorismate synthase mechanism of action was derived
from studies with the E. coli enzyme. As will be discussed
in some detail below, the complementary use of ¯avin
(Macheroux et al. 1996a) and substrate analogs (Bartlett
et al. 1986; Lauhon and Bartlett 1994; Bornemann et al.
1995c; Macheroux et al. 1996b, 1997), and the study of the
rapid reaction kinetics with normal and deuterated
substrates (Balasubramanian et al. 1990, 1995; Bornemann et al. 1995a) laid the foundation for a better
understanding of this puzzling reaction.
Chorismate synthase in microorganisms and plants
Initially, chorismate synthase was described from three
microbial sources: E. coli and Bacillus subtilis, i.e. a
gram-negative and a gram-positive bacterium, as well as
from the fungus Neurospora crassa (Morell et al. 1967;
Gaertner and Cole 1973; Welch et al. 1974; Hasan and
Nester 1978a,b). Surprisingly, these enzymes appeared
to have very di€erent biochemical properties and also
di€erent Mr values of 38 000, 24 000 and 50 000 had
been described for the three enzymes, respectively.
Therefore, it seemed as if nature had created three
di€erent protein catalysts to perform the chemically
demanding transformation of 5-enolpyruvylshikimate
3-phosphate to chorismate. However, a common theme
was apparent from the beginning, namely the requirement of all three enzymes for reduced ¯avin. On the
other hand, there were also striking di€erences with
respect to the ¯avin requirement: the activity of E. coli
chorismate synthase could only be detected under
anaerobic conditions in the presence of either chemically
or enzymatically reduced ¯avin, while the enzymes from
N. crassa and B. subtilis appeared to be associated with a
second enzymatic activity that generated the required
reduced ¯avin at the expense of NADPH. It turned out
P. Macheroux et al.: A unique reaction in a common pathway
327
Table 1. Comparison of chorismate synthases from di€erent organisms
E. coli
B. subtilis
S. aureus
S. cerevisiae
N. crassa
E. gracilis
C. sempervirens
L. esculentum CS1
L. esculentum CS2
Molecular
mass (Da)
Quarternary
structure
Km [EPSP]
Km [FMNH2]
Flavin reductase
activity
Isoelectric
point
pH
optimum
39
39
43
40
46
41
41
41
41
tetramer
heterotrimer
tetramerf
di/tetramer
di/tetramer
oligomer
dimer
di/tetramer
di/tetramer
1.3±2.2 lM
n.d.
12.7 lM
9.7 lM
2.7±7 lM
27 lM
53 lM
11 lM
80 lM
n.d.
12.5 lMb
4.8 lMc
42 nMd
66 nMd
76 nMd
37 nMd
n.d.
n.d.
absent
present
absentf
present
present
present
absent
absent
absent
n.d.a
5.5
n.d.
n.d.
4.9
5.5
5.0
n.d.
n.d.
6.5±8.5
n.d
n.d.
n.d.
7.0±8.5
8.2
8.0
6.0±8.0
6.0±8.0
138
971
026
800
400
700
771e (48 100)
902e (47 722)
605e (46 871)
a
n.d., not determined
Assayed with enzymatically reduced FMN, exploiting the associated ¯avin reductase
c
Assayed with photoreduced FMN
d
Assayed with chemically reduced FMN
e
The molecular masses of the predicted mature chorismate synthases are indicated. The molecular masses of the larger precursor proteins
are given in parentheses
f
The protein was analyzed after overexpression in E. coli. Therefore, the proper conditions may not have been established for formation of
the heterotrimer that was observed in the closely related B. subtilis enzyme
The following references were used to compile these data: E.coli, Morell et al. (1967), White et al. (1988), Charles et al. (1990), Ramjee et al.
(1994); B. subtilis, Hasan and Nester (1978b,c); S. aureus, Horsburgh et al. (1996); S. cerevisiae, Jones et al. (1991), Henstrand et al. (1996);
N. crassa, White et al. (1988), Schaller et al. (1991b), Lauhon and Bartlett (1994), Henstrand et al. (1995a); E. gracilis, Schaller et al.
(1991b); C. sempervirens, Schaller et al. (1990, 1991a), Henstrand et al. (1995b); L. esculentum, GoÈrlach et al. (1993), Braun et al. (1996)
b
that these two activities in the case of the N. crassa
enzyme were located on the same polypeptide chain
(henceforth termed ``bifunctional'' chorismate synthase),
while in the case of B. subtilis chorismate synthase, a
heterotrimeric complex in association with a ¯avin
reductase and 3-dehydroquinate synthase was discovered. Interestingly enough, as noted before, the latter
enzyme shares the requirement for a redox-active
cofactor for an overall redox-neutral reaction with
chorismate synthase.
A chorismate synthase from a higher plant was ®rst
reported in 1986, when Mousdale and Coggins detected
the enzyme's activity in tissue extracts and in chloroplast
preparations of pea (Mousdale and Coggins 1986). A
few years later a plant chorismate synthase was puri®ed
and characterized from a cell-suspension culture of
Corydalis sempervirens (Schaller et al. 1990). Surprisingly, the properties of plant chorismate synthases seemed
to resemble those of the E. coli enzyme rather than those
of the N. crassa enzyme. Like the E. coli chorismate
synthase, they lacked an intrinsic ¯avin reductase
activity, i.e. bifunctionality, and the Mr of 42 000, as
determined by denaturing PAGE, was close to that of
the E. coli enzyme. In the unicellular alga Euglena
gracilis, a further variation of this theme seemed to exist:
chorismate synthase in Euglena, like the plant chorismate synthase, exhibited an Mr of about 42 000, as
found by denaturing gel electrophoresis, but unlike the
plant enzyme, it co-puri®ed with a ¯avin reductase
activity (Schaller et al. 1991b).
The apparent diversity in the molecular organization
of chorismate synthase was at least partially resolved
when, in two studies, a detailed comparison of the
known chorismate synthases was performed (White et al.
1988; Schaller et al. 1991b). It became apparent that
chorismate synthase, when assayed in the presence of
chemically reduced FMN, had very similar characteristics in E. coli, N. crassa, E. gracilis and C. sempervirens
(cf. Table 1). Furthermore, these chorismate synthases
were also found to be related in their primary structure:
sequences of proteolytically generated peptides of
N. crassa chorismate synthase were found in the E. coli
sequence and all four enzymes cross-reacted with
antisera directed against chorismate synthases puri®ed
from E. coli and C. sempervirens. Since the basic
physico-chemical properties of the chorismate synthases
are similar, the main di€erence appears to be the way in
which they recruit the reduced cofactor. The ``monofunctional'' chorismate synthases (as exempli®ed by the
E. coli and plant enzymes) rely on the presence of
reduced FMN in their environment while ``bifunctional''
chorismate synthases possess an intrinsic ¯avin reductase activity which enables them to utilize NADPH for
the reduction of the FMN cofactor (White et al. 1988).
Yet another mechanism is operative in the B. subtilis
enzyme which associates with a separate and apparently
speci®c NADPH:FMN oxidoreductase (Hasan and
Nester 1978c; White et al. 1988). It was originally
proposed that the di€erence in molecular weight
between the bifunctional N. crassa enzyme (50 000)
and the monofunctional enzymes (39 000±42 000) is
accounted for by an additional domain carrying the
¯avin reductase activity (White et al. 1988). This
concept, however, was not borne out by recent studies
with the small (Mr 40 800) yet bifunctional Saccharomyces cerevisiae enzyme (Henstrand et al. 1995a, 1996),
and hence it is not feasible to predict mono/bi-functionality based on the size of the protein.
The eventual similarity between chorismate synthases
from di€erent sources was con®rmed when the ®rst
genomic and cDNA sequences of chorismate synthases
became available (a phylogenetic tree comparing all
328
chorismate synthase sequences known to date is shown
in Fig. 2). The chorismate synthase amino acid sequences of the two closely related gram-negative bacteria
E. coli and Salmonella typhimurium are 95% identical
(Charles et al. 1990). The ®rst eukaryotic sequence to be
identi®ed was that of a C. sempervirens chorismate
synthase cDNA, which was isolated by screening of an
expression library with a monospeci®c antibody (Schaller et al. 1991a). The amino acid sequence deduced from
the open reading frame in this cDNA was 48% identical
to the bacterial sequences. An even higher degree of
sequence identity of 53% was observed with Saccharomyces cerevisiae (Jones et al. 1991). The size of yeast
chorismate synthase (Mr 40 800), as well as a similarity
to the E. coli sequence that extends over the entire open
reading frame, led the authors to suggest that S. cerevisiae chorismate synthase is also monofunctional. The
NADPH-dependent chorismate synthase activity detectable in crude extracts from yeast could very well be
explained by the presence of a general ¯avin reductase in
the extract. However, it was later shown that this ¯avin
reductase activity co-puri®es with chorismate synthase
activity and, furthermore, that it is an intrinsic component of the protein (Henstrand et al. 1996). Therefore,
S. cerevisiae chorismate synthase, despite its smaller size,
is bifunctional as is the N. crassa enzyme and possibly all
other fungal enzymes.
P. Macheroux et al.: A unique reaction in a common pathway
In an e€ort to identify the hypothetical ¯avin
reductase domain in N. crassa chorismate synthase, a
cDNA of this enzyme was cloned by complementation
of a chorismate-synthase-de®cient E. coli strain (Henstrand et al. 1995a). This cDNA encodes a 46.4-kDa
protein which, when expressed in E. coli, possesses both
a chorismate synthase as well as a ¯avin reductase
activity. Based on sequence comparisons with monofunctional chorismate synthases, two regions of 29
C-terminal and 18 internal amino acid residues were
identi®ed that account for the larger size of the N. crassa
enzyme as compared to the monofunctional chorismate
synthases in plants and bacteria. Deletion of these
regions ± either separately or in combination ± did not
result in loss of chorismate synthase activity. The
polypeptide lacking the C-terminal 29 amino acids was
demonstrated to carry both enzymatic activities
(Henstrand et al. 1995a). A sequence comparison
between mono- and bifunctional chorismate synthases
did not allow the identi®cation of a domain responsible
for bifunctionality. Apparently, the residues that
catalyze NADPH-dependent FMN reduction are
embedded within the primary structures of chorismate
synthases (Henstrand et al. 1996). In the future, it will be
interesting to de®ne exactly which residues these are and
how they relate to the active site catalyzing the chorismate synthase reaction.
Fig. 2. Unrooted phylogenetic tree of chorismate synthases. The tree was constructed using the computational biochemistry research group
(CBRG) server at the Swiss Federal Institute of Technology/ZuÈrich (http://cbrg.inf.ethz.ch). The distance between protein sequences is expressed
in PAM ( ˆ accepted point mutations per 100 aligned positions) units. The sequences used are from the following species (accession numbers in
parentheses): Archaeoglobus fulgidus (O29587), Bacillus subtilis (P31104), Corydalis sempervirens (precursor, X60544), Escherichia coli (M27714),
Haemophilus in¯uenzae (P43875), Helicobacter pylori (P56122), Lycopersicon esculentum (precursor 1, Z21796), Lycopersicon esculentum
(precursor 2, Z21791), Methanococcus jannaschii (Bult et al. 1996; Q58575), Mycobacterium tuberculosis (P95013), Neurospora crassa (U25818),
Plasmodium falciparum (O15864), Saccharomyces cerevisiae (X60190), Salmonella typhimurium (M27715), Staphylococcus aureus (U31979),
Synechocystis sp. (P23353), Toxoplasma gondii (U93689), Vibrio anguillarum (P39198)
P. Macheroux et al.: A unique reaction in a common pathway
The analysis of the phylogenetic relationship among
chorismate synthases reveals that all chorismate
synthases ± mono- as well as bifunctional enzymes ±
are derived from a common ancestor. Thus, plant and
fungal chorismate synthases emerged from a common
ancestral protein after diverging from the monofunctional bacterial proteins. It is dicult to imagine how,
within the given framework of a monofunctional
chorismate synthase, bifunctionality could have arisen.
Therefore, it appears more likely that the common
ancestor of today's chorismate synthases had an intrinsic
¯avin reductase activity. Apparently, this activity was
only maintained under selective pressure, i.e. in organisms where the availability of reduced ¯avin is limiting
for growth. In yeast, this seems to be the case, which
explains why the complementation of a chorismatesynthase-de®cient yeast strain with a monofunctional
plant chorismate synthase did not restore wild-type
growth (Henstrand et al. 1996). In other organisms with
sucient unspeci®c ¯avin reductase activity ± as has
been described for E. coli (Morell et al. 1967) ±
bifunctionality may have been lost due to the lack of
selective pressure.
On the other hand, evolutionary selection of monofunctionality can also be envisioned. The loss of bifunctionality possibly results in a novel mechanism of
regulating chorismate synthase activity and the ¯ux
through the shikimate pathway by controlling the
availability of the reduced cofactor. The regulation of
higher-plant chorismate synthases by this and other
mechanisms will be dicussed below.
With more and more data becoming available,
chorismate synthases from E. coli, N. crassa, Euglena
gracilis and C. sempervirens, which earlier appeared to
be very distinct enzymes, are now known to be in fact
very similar to each other, as well as to the more recently
characterized enzymes from S. cerevisiae (Johnston et al.
1994; Henstrand et al. 1996), Lycopersicon esculentum
(Braun et al. 1996) and Staphylococcus aureus (Horsburgh et al. 1996), in their enzymatic and molecular
properties (cf. Table 1). Where there are still signi®cant
di€erences, as for example in the case of the apparent
Km for the ¯avin cofactor, these are most likely due to
the experimental methods employed. A Km for FMN in
the micromolar range was observed for the bifunctional
N. crassa chorismate synthase when assayed under
aerobic conditions exploiting the intrinsic NADPH:
FMN oxidoreductase activity (Hasan and Nester
1978b). A Km derived from an aerobic measurement
with a bifunctional enzyme is more likely to re¯ect the
anity for FMN to the NADPH:FMN oxidoreductase
activity rather than the chorismate synthase activity.
Whenever the Km was determined under anaerobic
conditions in the presence of chemically reduced FMN
it was found to be in the nanomolar range (Schaller et al.
1991b; Henstrand et al. 1996). This is in good agreement
with the dissociation constant for reduced FMN recently
determined for the E. coli chorismate synthase (Kd ˆ 18
nM; Macheroux et al. 1996b).
The similarities within the chorismate synthase family
are re¯ected by the close phylogenetic relationship
329
(Fig. 2) as well as by the fact that two exclusive
®ngerprint sequences can be de®ned which allow all
deposited chorismate synthases to be retrieved from the
Swissprot and Trembl data bases. These ``signatures''
are characterized by (i) R-P-[GS]-H-[AG]-D-x(5)-K
and (ii) R-x-S-[AG]-R-[EV]-[ST]-x(3)-V-x(2)-G-x(6)-L,
where x denotes any amino acid, and the amino acids
shown in brackets depict the option at a given position.
Owing to the excellent availability of the E. coli
chorismate synthase, most of the e€orts to elucidate the
reaction mechanism have concentrated on this enzyme.
However, since the enzymes from plants and E. coli seem
closely related (phylogeny, monofunctionality) we are
con®dent that most of the kinetic and mechanistic
®ndings, discussed brie¯y in the next section, can be
readily extrapolated to chorismate synthases from
plants.
The chemical mechanism of the chorismate synthase
reaction
In addition to explaining the role of reduced FMN, a
mechanism for the chorismate synthase reaction must
also provide a rationale for the stereochemical course
and for the cleavage of the stable C(6proR)-hydrogen
bond. Since molecular orbital calculations and model
studies with cyclohexene systems have shown that
concerted 1,4-elimination reactions preferably proceed
with syn-stereochemistry (Fukui 1965; Anh 1968; Hill
and Bock 1978), a non-concerted, stepwise reaction
mechanism for the chorismate-synthase-catalyzed reaction appears most attractive. On the other hand, the
C(6)-hydrogen bond is not activated, i.e. this CAH bond
has a very high pKa value (probably around 30), and
hence such a non-concerted mechanism has to ensure
that this CAH bond becomes substantially weaker. The
most probable mechanistic possibilities have recently
been reviewed (Bornemann et al. 1996a). The small
kinetic isotope e€ect of 1.13 using (6R)-[6-2H]-EPSP
(Bornemann et al. 1995a) indicates that this step is not
rate-limiting, a ®nding which was unexpected considering the non-activated nature of this carbon-hydrogen
bond. However, as was discussed by these authors, the
small isotope e€ect could also be a secondary isotope
e€ect, in other words, the deuteriation at C-6 may a€ect
bond breakage of the CAO bond. In order to investigate
this intriguing alternative explanation of their data,
measurements using [4-2H]-EPSP have been carried out
recently (S. Bornemann and R.N.F. Thorneley, personal
communication), con®rming this interpretation. This
implies that the ®rst bond to be broken in the substrate
molecule is not the CAH bond but the CAO bond, i.e.
phosphate cleavage occurs prior to CAH bond cleavage.
The observation of a secondary tritium-isotope e€ect
with the enzyme from N. crassa is also in line with such a
non-concerted, stepwise bond cleavage (Balasubramanian et al. 1995).
How is this phosphate cleavage achieved by chorismate synthase? Although one could postulate a loss of
phosphate to generate a transient carbocation which is
330
then deprotonated by an active-site base (the proton is
much more acidic, i.e. activated, and deprotonation is
facilitated in the cationic species), such a mechanism
does not provide a driving force for CAO bond cleavage
(Fig. 3, route A). Also, in this mechanism, there is no
role for the reduced ¯avin cofactor. Reaction route B
(Fig. 3) reconciles the need for a driving force to bring
about CAO bond breakage and the requirement for
reduced ¯avin (Bartlett et al. 1989; Bornemann et al.
1995c). Here, reduced ¯avin serves as a reductant for the
substrate molecule which upon one-electron reduction
splits o€ phosphate, probably in a synchronous fashion,
to yield an intermediate allylic radical. This intermediate
then decays to the product. This latter process could
either proceed via one-electron back-donation to the
¯avin semiquinone to yield a cationic substrate-derived
intermediate and the anionic reduced ¯avin as in route
B, upper path (Fig. 3). The C(6)AH bond is now very
labile and deprotonation, perhaps facilitated by an
active-site base, will occur rapidly. Alternatively, deprotonation could occur prior to the back transfer of an
electron to the ¯avin as shown in route B, lower path
(Fig. 3). The mechanism shown in route B is supported
by kinetic data as well as by the properties of the
enzyme-bound reduced ¯avin. One of the key observations in the investigation of the chorismate synthase
reaction was the discovery of a transient ¯avin-derived
intermediate (Ramjee et al. 1991). Detailed kinetic
characterisation of this transient species showed that
its formation precedes the chemical steps, i.e. CAO and
CAH bond cleavage (Bornemann et al. 1996b). Therefore, this species can not be a reaction intermediate
between reduced ¯avin and the substrate. However,
formation of this ¯avin species is associated with binding
of the substrate, i.e. formation of a ternary complex
between enzyme, reduced ¯avin and EPSP. Accompa-
P. Macheroux et al.: A unique reaction in a common pathway
nying studies concerning the binding of the ¯avin
cofactor in its di€erent redox states showed that the
reduced ¯avin is bound in its deprotonated (monoanionic) form to the enzyme in the absence of substrate
(Macheroux et al. 1996b). Using chemically modi®ed
¯avin derivatives it could be shown that chorismate
synthase preferably binds the ¯avin in a neutral form
(Macheroux et al. 1996a) when the substrate or a
substrate analog is bound. Hence the occurrence of the
¯avin intermediate re¯ects the protonation of the deprotonated reduced ¯avin upon binding of the substrate.
This protonation of the reduced ¯avin indicates that the
¯avin experiences a di€erent polarity in the active site
when substrate binds to the enzyme. In such an
environment the redox potential of reduced ¯avin is
thought to be more negative, i.e. the reduced ¯avin
becomes a better reductant as required for the mechanism presented above (Fig. 3). Compelling additional
evidence was obtained recently from the reaction with
the EPSP analog (6R)-6-¯uoro-EPSP (Macheroux et al.
1997). When this analog is reacted with chorismate
synthase, cleavage of phosphate and the formation of
the neutral ¯avin semiquinone is observed. According to
this result, it appears that the initial reaction proposed
above in route B (Fig. 3) still occurs with the analog.
However, due to the replacement of the (6R)-hydrogen
by ¯uorine, the reaction cannot proceed as with EPSP
(unlike hydrogen, ¯uorine is unlikely to be split o€ as a
radical or cation) resulting in the generation of the ¯avin
semiquinone and a substrate-derived radical (see Fig. 3).
The (6R)-6-¯uoro-EPSP therefore elicits the same reaction as the natural substrate; however, this reaction leads
to a stable enzyme-bound (inactive) ¯avin semiquinone.
Finally, it is interesting to note the mechanistic
similarity between 3-dehydroquinate synthase and
chorismate synthase: both enzymes utilize the redox
Fig. 3. Possible reaction mechanisms assuming CAO bond cleavage as the initial step in the chorismate synthase catalyzed reaction. For details
see text
P. Macheroux et al.: A unique reaction in a common pathway
properties of a tightly bound cofacter (NAD+ and
reduced ¯avin, respectively) to activate a CAH bond in
an overall redox-neutral chemical reaction.
Regulation of chorismate synthase activity in higher plants
Feedback inhibition of the prechorismate pathway in
bacteria and plants. In bacteria, almost all of the
aromatic amino acids produced via the shikimate
pathway are consumed in protein biosynthesis. This
situation is re¯ected by the fact that aromatic biosynthesis in bacteria is controlled by the three aromatic
amino acids at the transcriptional level by repression
mediated by the tyrosine- and tryptophan-repressors,
and at the metabolic level by feedback inhibition of
individual DAHP-synthase isozymes, which catalyze the
®rst reaction in the shikimate pathway (for a review, see
Herrmann 1995). The situation is di€erent in higher
plants where the shikimate pathway not only provides
the building blocks for protein synthesis but also for
numerous secondary metabolites of various functions
(reviewed in Bentley 1990; Schmid and Amrhein 1995).
In woody plants, up to 30% of the photosynthetically
®xed carbon can be incorporated into lignin via
phenylalanine-derived hydroxycinnamyl alcohols (Higuchi 1985). As a consequence, the ¯ux into the pathway is
not regulated by the levels of aromatic amino acids.
While DAHP-synthase was found to be activated by
tryptophan in carrot cells (Suzich et al. 1985) and to be
inhibited by L-arogenate in several plant species (Rubin
and Jensen 1985; Doong et al. 1993), feedback inhibition
by aromatic amino acids could not be shown conclusively in plants for either DAHP-synthase, or for any
other enzyme of the prechorismate pathway. Likewise,
chorismate synthase is insensitive to feedback inhibition
by the three aromatic amino acids or anthranilate
(Schaller et al. 1991b). It seems as if the demand of
aromatic amino acids for protein biosynthesis is met by
a constitutive activity of shikimate pathway enzymes.
However, the relative ¯uxes into the pathways leading to
tryptophan on the one side and to phenylalanine and
tyrosine on the other side are controlled by feedback
regulation of the anthranilate synthase and chorismate
mutase activities, respectively. For example, chorismate
mutase (plastidic isoform), the committed enzyme of the
branch leading to phenylalanine and tyrosine, is activated by tryptophan and inhibited by phenylalanine and
tyrosine.
Transcriptional regulation of chorismate synthase. There
are two chorismate synthase genes in tomato, designated
LeCS1, and LeCS2 (GoÈrlach et al. 1993). Yet, three
transcripts are derived from these two genes due to
di€erential splicing of the primary LeCS2 transcript
(Braun et al. 1996). The relative abundance of LeCS1
and LeCS2 transcripts in various tomato tissues was
very similar for the two chorismate synthase genes, with
the LeCS1 transcript being by far more prevalent than
those of LeCS2. Expression levels were highest in
¯owers and roots, followed by stems, leaves and
331
cotyledons. A survey of the literature shows that the
organ-speci®c expression of chorismate synthase genes is
comparable to that of genes encoding other enzymes of
the prechorismate pathway as well as phenylalanine
ammonia-lyase (PAL), chalcone synthase, chalcone
isomerase and 4-coumaryl:CoA ligase, suggesting that
chorismate is mainly used for the synthesis of ¯avonoid
compounds in ¯owers and roots and of lignin in vascular
tissues (Schmid and Amrhein 1995; Weaver and Herrmann 1997). Also after pathogen infection, there appears
to be an increased demand for aromatic precursors of
suberin and lignin as well as of salicylic acid and
phytoalexins involved in defense responses (Weaver and
Herrmann 1997). Transcripts were induced for LeCS1
but not for LeCS2 by a fungal elicitor in cultured tomato
cells and in intact tomato plants after infection with
Phytophthora infestans (GoÈrlach et al. 1995). A similar
induction of transcripts was observed for other enzymes
of the shikimate pathway as well as for PAL (GoÈrlach et
al. 1995). It appears that the increased demand for
aromatic precursors needed for the synthesis of secondary metabolites, as opposed to the requirement for
protein biosynthesis alone, is provided for by a higher
expression level of the biosynthetic enzymes involved. It
has to be emphasized, however, that only steady-state
transcript levels have been determined. These do not
necessarily re¯ect the abundance of the corresponding
enzymes or the levels of enzymatic activity. These will
have to be determined in the future.
The factors involved in transcriptional regulation
have not yet been identi®ed. The levels of soluble
aromatic amino acids within the cell, however, do not
seem to be involved, since increased concentrations of
phenylalanine resulting from in-vivo inhibition of PAL
activity, had no e€ect on the transcript levels of
shikimate-pathway enzymes and did not a€ect the
induction of the corresponding genes after elicitor
treatment (GoÈrlach et al. 1995). Also, glyphosate treatment of tomato cells, resulting in the inhibition of
aromatic amino acid biosynthesis at the site of EPSP
synthase (see above), did not alter the transcript levels of
any of the prechorismate-pathway enzymes, either in the
absence or presence of an elicitor, in a tomato cell
culture (own unpublished results). In contrast, glyphosate was found to induce DAHP-synthase several-fold in
cultured potato cells (Pinto et al. 1988). The identi®cation of the factors that bring about the coordinate
induction of shikimate pathway genes after elicitation or
after pathogen infection will be an interesting problem
of future work.
Metabolic regulation of chorismate synthase activity. For
the discussion of a possible metabolic regulation of plant
chorismate synthase, its subcellular localization has to
be considered. The compartmentalization of the shikimate pathway has long been a matter of debate. While it
was shown many years ago that isolated chloroplasts are
able to synthesize the aromatic amino acids from labeled
CO2 (Bickel et al. 1978; Bagge et al. 1986), it remained
unclear whether or not there exists a full or partial set of
cytosolic shikimate-pathway enzymes as well. Some of
332
the enzymatic activities have been detected in the
cytosolic fraction in cell fractionation studies, but none
of the enzymes has been puri®ed to homogeneity. The
molecular data available today lend no support to the
existence of a complete cytosolic pathway: all cDNAs
for prechorismate-pathway enzymes so far analyzed
encode proteins with an N-terminal, presumably plastidspeci®c transit peptide, and targeting to the chloroplast
has been shown for a number of shikimate-pathway
enzymes in in-vitro import studies (for a detailed
discussion of the subject, see Schmid and Amrhein
1995).
Also for the in-vitro-translated Corydalis sempervirens chorismate synthase, incorporation into isolated
chloroplasts has been demonstrated (own unpublished
results). Do these data preclude enzymatic activity in the
cytosol? It has been suggested that the cytoplasmically
synthesized precursors of shikimate-pathway enzymes
may account for cytoplasmic activity of the corresponding enzymes. For EPSP-synthase (Della-Cioppa et al.
1986) and shikimate kinase (Schmid et al. 1992), the
precursor proteins have been shown to possess catalytic
activity. Furthermore, there is precedent for a single
gene product to be targeted to two di€erent subcellular
locations (Danpure 1995). However, neither one of these
two observations can explain chorismate synthase
activity in the cytosol, since in C. sempervirens (Henstrand et al. 1995b) as well as in tomato (Braun et al.
1996) the precursor of chorismate synthase has been
shown to lack enzymatic activity.
The two tomato chorismate synthase isozymes LeCS1
and LeCS2 were individually expressed in E. coli, both in
their mature forms and with N-terminal transit peptides.
Only the mature forms were found to be active.
LeCS2D, which was derived from the di€erentially
spliced LeCS2 transcript, was found to be unstable in
E. coli and the physiological relevance of this protein is
not clear (Braun et al. 1996). LeCS1 and LeCS2 were
characterized after expression in E. coli and were found
to have very similar enzymatic properties, with the
notable exception of the apparent Km value for EPSP
which was much higher for LeCS2 (80 lM) than for
LeCS1 (11 lM). Like other chorismate synthases, the
tomato isozymes formed oligomers of two to four
subunits (Braun et al. 1996). Assuming that heterooligomers can be formed between LeCS1 and LeCS2, several
di€erent chorismate synthase isozymes are conceivable
with potentially di€erent catalytic properties.
The LeCS1 and LeCS2 isozymes both lack ¯avin
reductase activity and need to recruit either a ¯avin
reductase, or the reduced FMN from their plastidic
environment. If one assumes that the common ancestor
of all chorismate synthases was bifunctional, as pointed
out above, then the question arises why plant chorismate
synthases should have lost bifunctionality. Especially in
the oxygen-rich environment of the chloroplast, either
bifunctionality, or the close association with a ¯avin
reductase as observed in Euglena gracilis (Schaller et al.
1991b), would be expected to eciently ensure the
availability of the reduced cofactor necessary for enzymatic activity. Nevertheless, all higher-plant chorismate
P. Macheroux et al.: A unique reaction in a common pathway
synthases analyzed so far are clearly monofunctional.
An intriguing possibility is that there was selection
pressure toward the loss of ¯avin reductase activity as
higher-plant chorismate synthases evolved, in order to
render the enzyme more susceptible to the availability of
reduced FMN, or in other words to redox regulation.
The question of how the reduced ¯avin is generated in
the plastid is still unresolved. Although photoreduction
of FMN is a convenient method to provide the reduced
cofactor for chorismate synthase in vitro (Schaller et al.
1991b; Ramjee et al. 1994), it seems rather unlikely that
this mode of reduction is feasible in vivo, since photoprotective pigments are abundant in the chloroplasts.
The source of redox equivalents for the reduction of
FMN has not yet been determined, but NADPH as well
as the photosynthetic electron transport chain are
conceivable electron donors. In all three cases, the
availability of reduced FMN and consequently the
activity of chorismate synthase would ultimately depend
on light. The light dependence of the shikimate pathway
has been demonstrated in isolated chloroplasts (Homeyer and Schultz 1988). This phenomenon can possibly
be explained by a regulation of chorismate synthase
activity in planta via light-dependent generation of
reduced FMN. However, leucoplasts isolated from roots
have also been shown to be capable of synthesizing the
three aromatic amino acids (Leuschner and Schultz
1991), and the transcripts of shikimate-pathway enzymes
have been shown to be highly abundant in non-photosynthetic tissues (GoÈrlach et al. 1994; Bischo€ et al.
1996). In these tissues, the reducing power cannot be
directly derived from photosynthetic electron transport.
Hence, it is conceivable that di€erent reducing agents
are employed in photosynthetic and non-photosynthetic
tissues. Likewise, the immediate electron donor for
nitrite reductase within the chloroplast is reduced
ferredoxin. In root plastids, however, there seems to
exist a ferredoxin-like protein which obtains reducing
power from NADPH generated in the oxidative pentose
phosphate pathway (Wray 1993, and references therein).
Many enzymes in the chloroplast are known to be
regulated in their activity via redox-sensitive thiols.
Preliminary results from our laboratory suggest that
chorismate synthase activity is stimulated by reduced
glutathione and other thiol reagents such as dithiothreitol. Within the three plant chorismate synthase
sequences, there are a pair of conserved cysteines (Cys78/
Cys83) and two single cysteines only present in plant
sequences (Cys118 and Cys295; numbers refer to the
LeCS1 sequence), all of which are possible targets for
redox regulation via reduced thiol reagents in vivo. This
might constitute an additional mechanism ± like the
availability of reduced FMN ± to regulate chorismate
synthase activity by linking it to the overall redox status
within the plastid.
Outlook
A prime target for future research e€orts will be the
elucidation of the three-dimensional structure of choris-
P. Macheroux et al.: A unique reaction in a common pathway
mate synthase. It can be expected that a high-resolution
structure will shed further light on the mechanism of
action and the role of reduced ¯avin. Furthermore, a
comparative structural study of mono- and bifunctional
chorismate synthases may help to pinpoint the structural
determinants involved in reduction of the ¯avin cofactor. For the structural biologist, it will be interesting to
see whether an enzyme with such an unusual catalytic
activity has an already-known tertiary structure or
whether this feature is matched by an unprecedented
structural topology. From a physiological point of view,
the possible regulation of chorismate synthase by the
redox potential of the cell (or cell compartment) is an
area for further research. Such a regulation can occur at
two levels: the availability of the required reduced
cofactor and/or by means of a disul®de interchange as
commonly found among plastidic enzymes. In short, this
unusual enzyme presents challenges in areas ranging
from biophysical enzymology to structural biology and
plant physiology.
We would like to express our deep appreciation to Prof. R.N.F.
Thorneley and Dr. S. Bornemann at the Nitrogen Fixation
Laboratory, Norwich, U.K. for their critical advice throughout
the preparation of this manuscript. Support of our work by the
Swiss National Science Foundation is greatly appreciated.
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