Shikimate Pathway

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ABSTRACT
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The shikimate pathway links metabolism of carbohydrates to biosynthesis of aromatic compounds. In a
sequence of seven metabolic steps, phosphoenolpyruvate and erythrose 4-phosphate are converted to
chorismate, the precursor of the aromatic amino acids and many aromatic secondary metabolites. All pathway
intermediates can also be considered branch point compounds that may serve as substrates for other metabolic
pathways. The shikimate pathway is found only in microorganisms and plants, never in animals. All enzymes of
this pathway have been obtained in pure form from prokaryotic and eukaryotic sources and their respective
DNAs have been characterized from several organisms. The cDNAs of higher plants encode proteins with
amino terminal signal sequences for plastid import, suggesting that plastids are the exclusive locale for
chorismate biosynthesis. In microorganisms, the shikimate pathway is regulated by feedback inhibition and by
repression of the first enzyme. In higher plants, no physiological feedback inhibitor has been identified,
suggesting that pathway regulation may occur exclusively at the genetic level. This difference between
microorganisms and plants is reflected in the unusually large variation in the primary structures of the respective
first enzymes. Several of the pathway enzymes occur in isoenzymic forms whose expression varies with
changing environmental conditions and, within the plant, from organ to organ. The penultimate enzyme of the
pathway is the sole target for the herbicide glyphosate. Glyphosate-tolerant transgenic plants are at the core of
novel weed control systems for several crop plants.
INTRODUCTION
In this review the shikimate pathway is defined as the seven metabolic steps beginning with the condensation of
phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P) and ending with the synthesis of chorismate.
This pathway was elucidated by BD Davis and DB Sprinson and their associates about 40 years ago. In the
past decade a monograph (73) and a comprehensive review of the shikimate pathway (16) have appeared.
During the same period, summaries of specific aspects emphasizing the role of the pathway in the biosynthesis
of plant aromatic secondary products were published (84, 85, 168,184). After a few brief historical remarks, we
focus on the research results of the past ten years.
The shikimate pathway, the common route leading to production of the aromatic amino acids phenylalanine,
07 March 2015 Page 1 of 26 ProQuest

tyrosine, and tryptophan, constitutes a part of metabolism that is found only in microorganisms and plants,
never in animals. The pathway is therefore an important target for herbicides (100), antibiotics, and live
vaccines (141). The penultimate step in this pathway is inhibited by N-[phosphonomethyl]glycine, the active
ingredient of the broad-spectrum, nonselective herbicide glyphosate (190). Much effort has been put forward to
understand not only the interaction of this herbicide with its target but also to seek other compounds with similar
inhibitory capabilities. Chemical compounds that interfere with any enzyme activity in this pathway are
considered "safe" for humans when handled in reasonable concentrations. In fact, glyphosate has been tested
successfully in mice as a therapeutic agent against pathogenic protozoans that cause diseases like
toxoplasmosis or malaria (161).
The seven enzymes of the shikimate pathway were originally discovered through studies on bacteria, mainly
Escherichia coli and Salmonella typhimurium. Although the substrates and products of these enzymes, and thus
the intermediates of the pathway, are identical for prokaryotic and eukaryotic organisms, sometimes great
differences are found in the primary structure and properties of the prokaryotic and eukaryotic enzymes
themselves. Also, how aromatic compounds serve as signals in the regulation of the pathway is well understood
for some microorganisms, but is only now beginning to be investigated in higher plants. Therefore, we
considered it useful to outline briefly the properties of bacterial enzymes, particularly when they differ a great
deal from their plant homologues.
Chorismate, the endproduct of the shikimate pathway, is the precursor of the three aromatic amino acids and
several other aromatic compounds of primary metabolism. In addition, the three aromatic amino acids are
precursors to a large variety of plant secondary metabolites (34). Finally, the intermediates of the main trunk of
the shikimate pathway also serve as starting points for biosynthesis of secondary products. Clearly, the
shikimate pathway is of eminent importance to the biosynthesis of many compounds of commercial interest.
THE ENZYMES OF THE MAIN TRUNK
3-Deoxy-D-Arabino-Heptulosonate 7-Phosphate Synthase
The first step of the shikimate pathway is the condensation of PEP and E4P yielding 3-deoxy-D-arabino-
heptulosonate 7-phosphate (DAHP) and inorganic phosphate. The basic structure of DAHP was confirmed by
several different chemical syntheses. Fine structure analysis identified DAHP as 1-carboxy-2deoxy-alpha-D-
glucose-6-phosphate (49).
The enzymatic synthesis of DAHP is catalyzed by DAHP synthase (Figure 1), an enzyme discovered in E. coli
and first purified to electrophoretic homogeneity from microorganisms. The most intensively investigated DAHP
synthase is the enzyme from E. coli (83). Wild-type E. coli produces three feedback inhibitor-- sensitive DAHP
synthase isoenzymes: a Tyr-sensitive, a Phe-sensitive, and a Trp-sensitive enzyme. Their corresponding
structural genes, aroF, aroG, and aroH, are scattered over the E. coli chromosome (18). The genes have been
cloned and the primary structures of the encoded isoenzymes have been obtained through a combination of
protein and DNA sequencing efforts. The E. coli isoenzymes are oligomers with a subunit molecular weight of
about 39,000. The Phe-sensitive isoenzyme has been crystallized as a binary complex with PEP or Phe and as
a ternary complex with additional metal ions (179).

Plant DAHP synthases have been obtained in pure form from carrot (198) and potato (153) as oligomers with
subunit molecular weights of about 54,000. Rabbit antibodies raised against the potato enzyme (152) were used
to screen a cDNA library from potato cells grown in suspension culture (40). The resulting cDNA complements
E. coli mutants devoid of DAHP synthase (215). The first cDNA served as a probe to isolate homologues from
tobacco (212), Arabidopsis (95), tomato (58), and Morinda citrifolia (223). The Arabidopsis cDNA complements
yeast mutants devoid of DAHP synthase (95). cDNA clones encoding a second DAHP synthase isoform have
been reported from Arabidopsis (95), potato (227), and tomato (58). M. citrifolia (223) and Arabidopsis (B Keith,
personal communication) actually contain three DAHP synthase isoenzymes. Although, like E. coli, some plants
have three DAHP synthase-encoding genes, the structures of the encoded enzymes are quite different from the
structures of the bacterial DAHP synthases.
Translation of plant cDNAs yields polypeptides with amino terminal signal sequences that direct plastid import.
These sequences are clipped off during import into the plastid. The precise processing site cannot be
determined by Edman degradation of the mature protein because the amino termini of plant DAHP synthases
are blocked. Mature plant enzymes, purified from potato tuber or carrot roots, have subunits that are about 150
amino acid residues longer than their bacterial homologues. Typically, the comparison of the primary amino acid
sequence of a plant DAHP synthase to a bacterial DAHP synthase shows a surprisingly low 20% pairwise
identity. In fact, PRETTYBOX, a program used to make multiple sequence alignments, identifies only 24
invariant residues for all known DAHP synthases.
DAHP synthase in E. coli is a metallo protein that is inhibited by chelating agents (122). In vitro analyses of pure
enzymes indicate that the metal requirement can be satisfied by several divalent cations (191). The metal
content of the native enzyme may depend on general growth conditions. Under some conditions, DAHP
synthase may be a Cu enzyme (7), but Fe and perhaps Zn are most likely the preferred metals in vivo (191).
The metal plays a catalytic and, possibly, a structural role as well. A Cys residue in a Cys-X-X-His motif of the
protein has been identified as part of a metal binding site (192).
A careful in vitro metal analysis comparable to the study on the Phe-sensitive isoenzyme from E. coli (191) has
not yet been performed on a plant DAHP synthase. However, the activities of all plant DAHP synthases are
enhanced by Mn, and plant DAHP synthases also contain a Cys-X-X-His motif. In bacterial enzymes this motif
is near the amino terminus, but in plant enzymes it is near the middle of the carboxy half of the enzyme. Thus,
this motif is not positionally conserved in sequence alignments between bacterial and plant DAHP synthases,
although it may well be part of a metal binding site for both (84).
Early studies on the plant enzymes suggested the existence of Mn- and Coactivated DAHP synthases. While
both of these enzymes use PEP as one substrate, the Mn-activated enzyme has an absolute specificity for E4P
as the second substrate. The Co-activated enzyme, on the other hand, is less specific, preferring
glycolaldehyde as a second substrate (37). Thus it may be more properly called 4,5-dihydroxy-2-oxovalerate
synthase. A correlation has been reported between this enzyme's activity and the biosynthesis of anthocyanin in
suspension cultured cells of Vitis (201). In contrast to this result, it is the Mn-activated DAHP synthase that is
induced in Petroselinum crispum suspension cultures by light (125) or by fungal elicitation, leading to enhanced
phenylpropanoid biosynthesis; the Co-activated enzyme is not affected (124). A similar expression pattern is
seen upon wounding of potato; the Mn-activated enzyme is wound-inducible, whereas the Co-activated enzyme
is not (136). The Co-activated enzyme has recently been purified from cultured carrot cells (200) but its
contribution to the overall synthesis of DAHP remains questionable because of its seemingly unphysiologically
high K^sub m^ for EAP.
Structurally distinct true DAHP synthases with potentially different functions exist in the form of Mn-activated
isoenzymes. These Mn-activated DAHP synthases, the Co-activated 4,5-dihydroxy-2-oxovalerate synthase, and
the metal ion independent 3-deoxy-D-manno-octulosonate 8-phosphate synthase (224) all share PEP as one
substrate and an aldehyde as the other. They catalyze similar condensation reactions that proceed with a C-O

rather than a P-O bond cleavage on PEP (79) and appear to be evolutionarily related (196).
Because of the large differences in the primary structure, Walker and coworkers made a distinction between a
small bacterial type I DAHP synthase of 39 kD and a large plant type II enzyme of 54 kD (210). However, a type
II DAHP synthase was purified to homogeneity from the bacterium Streptomyces rimosus (193) and partial
sequence data from this protein show substantial similarity to plant DAHP synthases (210). Also, both yeast
DAHP synthases (108,146,148) are type I enzymes. Thus, the "plant type II" DAHP synthase is also found in
prokaryotes and the "bacterial type I" in eukaryotic microorganisms.
DAHP synthase in E. coli is regulated at the transcriptional level by repression and at the protein level by
feedback-inhibition. Both the Phe- and the Tyr-sensitive isoenzymes can be completely inhibited by about 0.1
mM of the corresponding amino acid. In contrast, the Trp-sensitive isoenzyme is only partially inhibited by Trp
(1,158). The inability of Trp to totally inhibit DAHP synthase apparently ensures a sufficient supply of chorismate
for the biosynthesis of other aromatic compounds when Tyr, Phe, and Trp are present in excess in the growth
medium. Amino acid residues within the enzymes' allosteric sites have been identified through structural
analysis of feedback-insensitive mutant enzymes (54, 96,159, 213,214). It appears that these sites partially
overlap the enzymes' active sites, in good agreement with kinetic data (1,123,173). Recently, an overlap of
active and allosteric sites has also been shown for the Phe-- and the Tyr-sensitive DAHP synthases from
Saccharomyces cerevisiae (172).
Like their bacterial counterparts, plant DAHP synthases are oligomers and subject to metabolic regulation.
Surprisingly, plant DAHP synthases are not feedback inhibited by any aromatic amino acid. In fact, tryptophan is
a hysteretic activator of the enzymes (198). Arogenate, a post-chorismate intermediate of phenylalanine and
tyrosine biosynthesis (93), inhibits the enzyme; 155 (mu)M arogenate causes 50% inhibition of the bean DAHP
synthase (163). However, since arogenate does not accumulate in plants to detectable levels, it seems unlikely
that it can play a role as a physiological regulator (160). No classical allosteric inhibitor of a plant DAHP
synthase has yet been identified.
The expression of the three E. coli genes encoding DAHP synthases is subject to repression by the tyr- and trp-
repressors complexed to the aromatic amino acids (67, 228). Cis-acting regulatory mutants with lesions in the
repressor target sites were identified in the regulatory regions of aroH ( 104, 228) and aroF (50). Furthermore,
the tyr- and the trp-regulons are functionally connected by aroH, because the expression of this gene is
controlled by both the trp- and the tvr-repressors (137). Transcriptional control for the expression of the three
genes encoding DAHP synthases is complex; however, feedback inhibition of the three isoenzymes is
quantitatively the major regulatory mechanism in vivo, as demonstrated by noninvasive nuclear magnetic
resonance spectroscopy on whole living E. coli cells (142).
In plants, metabolic regulation of DAHP synthase appears to occur preferentially at the genetic level. Sublethal
doses of glyphosate cause an increase in DAHP synthase activity in vivo (152). The target of the herbicide is
the penultimate enzyme in the pathway. In vitro, the herbicide has no effect on the DAHP synthase enzyme
activity. However, by inhibiting the penultimate enzyme, the herbicide reduces chorismate production in vivo,
thereby initiating a signal that leads to increased carbon flow into the shikimate pathway through elevation of
DAHP synthase activity. The nature of the signal is not known.
This elevation of the enzyme activity is due to an increase in the amount of the polypeptide chain. Modulation of
the overall DAHP synthase activity is the result of differential isoenzyme expression (60,62, 81,95,223).
Developmental or environmental stimuli like light, mechanical wounding, or elicitation by microorganisms all
influence DAHP synthase isoenzyme expression (40,60,81,124,207). Gibberellic acid and jasmonate induce the
enzyme in Coptis japonica and Nicotiana tabacum, respectively (72, 199). Although cis-- acting elements have
been identified in the regulatory region of one potato gene encoding DAHP synthase (213), to date none of the
plant DAHP synthase promoters has been studied in detail.
In eukaryotic microorganisms, amino acid biosynthesis is subject to general control (87). For example, in yeast

(109, 147) and the pathogen Candida albicans (150), the general control activator protein GCN4 regulates the
expression of the two DAHP synthase genes. There is some evidence for such regulation in higher plants as
well (69).
3-Dehydroquinate Synthase
In bacteria, reactions two to six of the shikimate pathway are catalyzed by five separate enzymes, but in fungi, a
single polypeptide called the multifunctional AROM complex serves the same purpose. Space constraints
prevent us from dealing in detail with this highly interesting system that was discovered three decades ago by
NH Giles and his associates. AROM complexes have been studied from Neurospora crassa, Aspergillus
nidulans, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pneumocystis carinii. In these
complexes, the enzymes do not appear in the order of the pathway reactions. Protein domains for DHQ
synthase and EPSP synthase form the amino terminal part, domains for shikimate kinase, DHQ dehydratase,
and shikimate dedydrogenase the carboxy terminal part (77). It appears that the arom locus evolved by gene
fusion. DNA encoding the entire AROM complex has been cloned and sequenced from A. nidulans (29), yeast
(39), and P carinii (9), the pathogen that is the principle cause of death for patients with AIDS. A very interesting
evolutionary aspect of fungal aromatic metabolism is a gene duplication of arom that resulted in genes encoding
regulatory proteins for quinate degradation (see section on 3-dehydroquinate and 3-dehydroshikimate). In
higher plants, reactions three and four are catalyzed by a bifunctional enzyme, the remaining three reactions of
the shikimate pathway by separate enzymes that are structurally rather similar to their prokaryotic homologues.
The second reaction of the shikimate pathway is the elimination of phosphate from DAHP to generate 3-
dehydroquinate (DHQ). The reaction is catalyzed by DHQ synthase (Figure 2), a monomeric enzyme with a
molecular weight of 39,000. The enzyme from E. coli (47) requires divalent cations for activity; Co is the most
active metal but Zn may be the ion used in vivo (13). There is evidence for two functionally distinct metal binding
sites per polypeptide chain (185). DHQ synthase is activated by inorganic phosphate, one of the reaction
products, and the enzyme also requires catalytic amounts of NAD for activity, even though the enzyme
catalyzed reaction is redox neutral.
Conversion of DAHP to DHQ proceeds by way of an intramolecular exchange of the DAHP ring oxygen with
carbon 7, driven by the cleavage of the phosphoester. The reaction involves an oxidation, a -elimination of
inorganic phosphate, a reduction, a ring opening, and an intramolecular aldol condensation. The mechanism of
this complicated reaction, already outlined by Sprinson and coworkers, was elucidated by PA Bartlett &JR
Knowles and associates. The true substrate for the enzyme is apparently the pyran form of DAHP (49). The
Beta-elimination of phosphate proceeds with syn stereochemistry (11, 14, 221) and there is compelling
evidence that the enzyme is a simple oxidoreductase (11). The phosphate monoester may either mediate its
own elimination (132) or may be aided by the enzyme (131). The remaining partial reactions proceed
spontaneously (221). The enzyme itself provides a potential conformational template to prevent formation of
undesirable side products (10,149). In these studies, DAHP analogs were used that can only undergo part of
the overall reaction. Among these analogs is a carbacyclic phosphonate that inhibits the enzyme with a Ki of 0.8
nM (220). A similar enzyme mechanism was later found for the 2-deoxy-scyllo-inosose synthase of
Streptomyces fradiae, an enzyme involved in antibiotic biosynthesis (226).
Plant DHQ synthases have been purified from Phaseolus mungo (225) and Pisum sativum (154). The primary
structures of the bacterial (128) and plant (17) enzymes were deduced by translation of the corresponding DNA
sequences. Interestingly, DHQ synthase sequences from E. coli and tomato are 52.5% identical, whereas
bacterial and plant DAHP synthases are only 20% identical. Plant DHQ synthase is more closely related to the
bacterial than to the fungal enzyme, unlike the case for DAHP synthase.
Like the plant cDNA encoding DAHP synthases, the tomato cDNA encodes a DHQ synthase with an amino
terminal signal sequence for plastid import (17). The cDNA complements E. coli mutants lacking DHQ synthase.
RNA blots show that gene expression of DHQ synthase is organ specific and follows the pattern of gene

expression for one of the DAHP synthase isoenzymes (17). Furthermore, DHQ synthase mRNA is induced
when tomato cells grown in suspension culture are elicited with extracts from Phytophthora megasperma (17).
In contrast, the DHQ synthase enzyme activity does not change when potato cells are exposed to glyphosate
(152).
3-Dedydroquinate Dehydratase-Shikimate Dehydrogenase The third step of the shikimate pathway, dehydration
of DHQ to give 3dehydroshikimate (DHS), is catalyzed by DHQ dehydratase (Figure 3) that exists in two forms:
type I and II. Some bacteria, like E. coli (32) or S. typhi (133), have type I enzyme, whereas others, like
Streptomyces coelicor (219), Mycobacterium tuberculosis (48,134), or Helicobacterpylori (22), have type II.
Type I DHQ dehydratase catalyzes syn elimination and type II anti-elimination of water (177). The mechanistic
differences are reflected in the structures of these proteins; there is no sequence similarity between the type I
and II enzymes, a rare example of convergent evolution (102). Both enzymes have been crystallized (23,63).
Analysis of diffraction data should reveal some fundamental differences in the reaction mechanisms of enzyme-
catalyzed syn and anti-eliminations.
The best-studied type I DHQ dehydratase is from E. coli (38). The enzyme is a dimer with a subunit molecular
weight of 27,000. The reaction proceeds by way of a Schiff base mechanism in which Lys-170 serves as the
amino donor (30). The imine intermediate has been directly observed by electrospray mass spectrometry (178).
In addition to Lys-170, other active site residues are His-143 (32,115), Met-205 (101), and Arg-213 (106). Since
the amino terminus shows sequence similarity to a region within DHQ synthase, it is thought to be part of the
substrate binding site (101). There appears to be a VDL sequence motif among enzymes of the shikimate
pathway (26).
The S. coelicor type II enzyme is a dodecamer with a subunit molecular weight of 16,000. The active site
contains Arg-23 (107), Tyr-28 (106), a His (102), and a Trp (19) but no Lys residue. No imine intermediate has
been identified, ruling out formation of a Schiff base. Arg-23 of S. coelicor corresponds to Arg-213 in type I
enzymes, Tyr-28 of type II to Phe-219 in type I enzymes. Arg-23 and Tyr-28 of type II are in a nine residue motif
that corresponds to a similar motif in type I containing residues Arg-213 and Phe-219. This motif is positionally

not conserved but is considered part of the substrate binding sites for the two types of enzymes, a situation that
is very similar to the positioning of the Cys-X-X-His motifs in pro- and eukaryotic DAHP synthases.
Some fungi have both type I and II DHQ dehydratases. In such organisms, the type I enzyme is considered the
anabolic form in the main trunk of the shikimate pathway and the type II enzyme the catabolic form in the
pathway that soil microorganisms use to degrade the abundant plant metabolite quinate. In contrast, the
bacterium Acinetobacter calcoaceticus has a catabolic type I and an anabolic type II DHQ dehydratase (43).
Finally, in the bacterium Amycolatopsis (formerly Nocardia) methanolica, DHQ dehydratase II serves both an
anabolic and a catabolic function (44).
The fourth step in the shikimate pathway is the reduction of DHS to shikimate. In E. coli, the reaction is
catalyzed by an NADP-dependent shikimate dehydrogenase (Figure 4) of molecular weight of 29,000 (5). Some
microorganisms have a shikimate dehydrogenase that is pyrrolo-quinoline quinone dependent (42).
In plants, step three and four of the shikimate pathway are catalyzed by the bifunctional DHQ dehydratase-
shikimate dehydrogenase. A 59-kD enzyme from Pisum sativum has been purified to electrophoretic
homogeneity (135). The dehydratase activity resides in the amino terminal half of the polypeptide. A partial
cDNA has been obtained that encodes a type I dehydratase with all the identified active site residues and that is
inhibited by borohydride as well (31). A longer, although also incomplete, cDNA has been obtained for the
Nicotiana tabacum homologue (20). This sequence translates also into a protein with both activities, the
dehydratase in the amino terminal and the dehydrogenase in the carboxy terminal half. The ratio of the turnover
numbers for these two plant enzymes is about 1:10, meaning that DHS never accumulates (171).
The sequences for both plant enzymes are more similar to bacterial than to lower eukaryotic homologues. In
addition, the 5'-end of the tobacco cDNA translates into a putative signal sequence for plastid import (20). This
sequence is rich in hydroxylated amino acid residues but has a net negative charge, a feature that has not been
seen previously for plastid transit peptides. There is evidence for isoenzymes but no isoenzyme-encoding DNA
has been cloned yet.
Shikimate dehydrogenase enzyme activity increases about 20% within six days after mechanical wounding of
Capsicum annum L. leaves (36). If this increase is significant, the dehydrogenase would join DAHP synthase
(40) as the second enzyme of the shikimate pathway that is wound-inducible.
Although the plant bifunctional DHQ dehydratase-shikimate dehydrogenase has not been fully characterized at
the molecular level, shikimate dehydrogenase has been widely used as a marker in genetics and in crop
breeding for many years. Because this enzyme can be detected in starch or isoelectric focusing gels by activity
staining, it is one of a handful of enzymes used for studies of heritable variation. The pIs of DHQ dehydratase-
shikimate dehydrogenase isozymes differ sufficiently to create characteristic and unique gel patterns when the
isoenzymes are separated by charge and stained for activity. The activity patterns, or zymograms, can serve as
fingerprints for plant genotypes used to identify parents, clones, and seed lots (203).

While the analysis of random, amplified, polymorphic DNA (RAPD) has begun to supplant the use of
zymograms, comparison of zymogram to RAPD technology has shown good correlation in some applications
(88, 208). Since zymogram analysis uses readily available reagents and a simple apparatus, the use of
shikimate dehydrogenase activity as a genetic marker is still prevalent. Recently, shikimate dehydrogenase
zymograms have been used to determine the validity or extent of outcrossing (27), to evaluate genetic variation
within a population (71, 90), and to derive evolutionary relationships between cultivars, ecotypes, and species
(12, 164). In a few cases, a particular pattern of shikimate dehydrogenase isozymes has been linked to a
phenotypic trait (110, 222).
Shikimate Kinase
In the fifth step of the shikimate pathway, shikimate kinase (Figure 5) catalyzes the phosphorylation of shikimate
to yield shikimate 3-phosphate (S3P). E. coli has two shikimate kinases; isoenzymes I and II are monomers of
molecular weight 19,500 but share only 30% sequence identity (66,217). Both enzymes can function in vitro in
aromatic amino acid biosynthesis with isoenzyme II having a K^sub m^ for shikimate of 200 (mu)M; the
corresponding K^sub m^ for I is more than 100 times larger. Thus in vivo, isoenzyme I may not be an enzyme of
the shikimate pathway at all; it may have a function in cell division (209). The expression of the gene encoding
shikimate kinase II, but not I, is controlled by a synergism between the trp- and tyr-repressors (78) and possibly
also by integration host factor (114). The three-dimensional structure of a type II shikimate kinase from Erwinia
chrysanthemi has been solved to high resolution for two different enzyme-substrate complexes (105). The
enzyme seems to undergo large conformational changes during catalysis.
Plant shikimate kinase has been purified to near electrophoretic homogeneity from spinach (170) and pepper
(35). The enzyme is inhibited by 2,4-D; the herbicidal activity of 2,4-D may be due in part to its ability to inhibit
shikimate kinase. The chloroplast-localized enzyme is regulated by energy charge but not by light. Plant cDNA
encoding shikimate kinase has been cloned from tomato; this plant has only one gene encoding this protein
(169). The cDNA-deduced amino acid sequence is quite similar to the sequences of microbial homologues but
contains in addition an amino terminal extension that resembles a transit sequence for chloroplast import. In
vitro synthesized tomato shikimate kinase precursor is processed and taken up by isolated chloroplasts (169).
These results are consistent with the assumption of a single shikimate kinase in tomato that is localized
exclusively in chloroplasts.
5-Enolpyruvylshikimate 3-Phosphate Synthase
In the sixth step of the shikimate pathway, a second PEP enters the pathway. It is condensed with S3P to yield
5-enolpyruvylshikimate 3-phosphate (EPSP) and inorganic phosphate. This reversible reaction is catalyzed by
EPSP synthase (Figure 6), a monomeric enzyme of molecular weight 48,000. The enzyme has been purified
from prokaryotes and eukaryotes and the E. coli EPSP synthase has been crystallized. X-ray structure analysis
indicates two domains with the active site near the interdomain crossover segment (189). Enzyme-substrate
complexes were also characterized by NMR spectroscopy (194).

Separate studies proposed an ordered (4) and a random (68) kinetic mechanism for the enzyme-catalyzed
reaction that proceeds through a tetrahedral intermediate. This tetrahedral intermediate had already been
suggested by Sprinson and coworkers and was verified through extensive physicochemical investigations
(174,183). It has been detected directly by interfacing a rapid mixing apparatus with an electrospray mass
spectrometer (145). Reaction intermediates were also demonstrated by solid-state NMR spectroscopy (91,195)
and the stereochemical course of the reaction was detailed in studies with fluoro derivatives of PEP (99).
EPSP synthase is the only cellular target for the herbicide glyphosate (190). Glyphosate does not bind to the
naked enzyme but rather to the enzyme-S3P complex. Glyphosate binding is competitive with PEP. For a long
time, the ternary complex enzyme-S3P-glyphosate has been considered a transition state analog in which
glyphosate takes the place of PEP. Surprisingly, other enzymes that have PEP as substrates are not inhibited
by glyphosate. Recently, enzyme-- ligand distances within the glyphosate-containing ternary complex were
measured by NMR (127), and now reports from several laboratories indicate that the complex may not be a
transition state analog (165), because PEP and glyphosate binding are apparently not identical (126). In support
of these findings, it has been noticed for some time that enzymes from different organisms show great
variations in their sensitivities to the herbicide. Inhibitor constants may vary by as much as three orders of
magnitude when some plant and bacterial enzymes are compared, while variations in the K^sub m^'s for PEP
are much less. Thus, in spite of the competitive nature, glyphosate and PEP binding is not totally equivalent.
This nonequivalency is corroborated through mutant enzyme studies (120,144,180,181) that show no direct
correlation between glyphosate inhibitor potency and the loss of catalytic efficiency. Since glyphosate is a
competitive inhibitor with respect to PEP but does not bind in the same fashion as PEP, Sikorski &Gruys now
call the inhibition an "adventitious allosteric interaction" (183). Under specific in vitro conditions, the EPSP
synthase from Bacillus subtilis is an oligomeric protein with two non-equivalent PEP binding sites. Glyphosate
binding is competitive with respect to one site only; thus this enzyme has been considered a classical allosteric
protein (120).
Glyphosate is the only commercial herbicide that acts on EPSP synthase despite tremendous efforts to find
equally effective inhibitory compounds (2, 211). However, recently discovered transition state analogs in which
the shikimate ring is replaced by a benzene ring are very effective inhibitors of EPSP synthase (129,130,175).
Since nM concentrations of some of these compounds inhibit the enzyme effectively, and since these
compounds are readily accessible through organic synthesis, they may represent the next generation of
commercial herbicides that function through inhibition of the shikimate pathway.
Glyphosate-tolerant cell lines from several different organisms have elevated levels of DAHP synthase
(3,41,57,139,182,186,187). The elevation is due to gene amplification (197). Maintenance of these lines in the
absence of the herbicide causes a time-dependent loss of tolerance (138). Plantlets regenerated from tolerant
lines also show elevated EPSP synthase levels. The degree of herbicide tolerance is directly correlated to the
enzyme levels (92).
Plant DNAs encoding EPSP synthases have been isolated from petunia (176), Arabidopsis (103), tomato (52),
and Brassica napus (51). All these cDNAs encode precursor proteins with amino terminal transit sequences. In

vitro uptake experiments with isolated chloroplasts show that these sequences direct plastid import of the
enzyme (33). In petunia, EPSP synthase expression is tissue specific and developmentally regulated (15). A
transcription activator involved in this regulation is a Cys/His type zinc finger protein (202).
Zea mays has two plastidic EPSP synthase isoforms; one appears to be constitutive, whereas the other may be
subject to regulation (45,46). The single tomato enzyme is induced in elicitor-treated cell cultures (60).
Generally, plant EPSP synthases have lower Ki values for glyphosate than their bacterial homologues. Thus,
glyphosate-tolerant transgenic plants have been obtained carrying mutant alleles that either encode bacterial
enzymes (188) or that cause overproduction of the plant enzyme (57,176,186).
Commercially successful glyphosate-tolerant crop plants carry a naturally occurring Agrobacterium tumefaciens
allele that encodes a glyphosate-insensitive EPSP synthase. Glyphosate-tolerant soybean (143) and cotton
(140) plants have a cauliflower 35S promoter-driven transgene construct that encodes the
Petunia hybrida EPSP synthase signal sequence for plastid import, the abovementioned Agrobacterium
tumefaciens EPSP synthase coding region, and the nopaline synthase terminator. Glyphosate-tolerant sugar
beet plants ( 121 ) carry in addition to this transgene construct a bacterial gene encoding glyphosate oxidase
reductase that degrades glyphosate to nontoxic compounds. These three glyphosate-tolerant crop plants are
the vanguard in weed control systems that should reduce not only production costs, but also the amount of
chemicals in soil and water run-off. We can anticipate that more such crops will appear, particularly when
transformation systems for grasses become easier to manipulate.
Chorismate Synthase
The seventh and final step in the main trunk of the shikimate pathway is the trans- 1,4 elimination of phosphate
from EPSP to yield chorismate (8, 74). In this reaction, the second of the three double bonds of the benzene
ring is introduced. The reaction is catalyzed by chorismate synthase (Figure 7) and requires reduced flavin for
activity even though the overall reaction is redox neutral. In this respect the enzyme is similar to DHQ synthase,
the second enzyme in the shikimate pathway.
In chorismate synthase catalysis, the reduced flavin is apparently directly involved in the mechanism of the
reaction (155, 156). The binary flavin-enzyme and the ternary flavin-enzyme-substrate complexes have been
characterized in detail (118). A careful kinetic analysis (21) shows that the flavin reaction intermediate is formed
after EPSP is bound but before it is consumed. The flavin intermediate decays after EPSP is converted to
chorismate and after phosphate is released from the enzyme. The flavin may serve as an electron donor to
EPSP, thereby initiating C-O bond cleavage. The intermediate may be a radical in what is most likely a non-
concerted reaction.
Depending upon the organism, chorismate synthase is either monofunctional, requiring the addition of reduced
flavin to in vitro enzyme assays, or bifunctional, with an associated NADP-driven flavin reductase within the
same polypeptide chain. The best-studied bifunctional chorismate synthase is from Neurospora crassa. The
enzymes from E. coli and higher plants are monofunctional. The E. coli (218) and the plant enzyme from
Corydalis sempervirens (167) have subunit molecular weights of around 40,000; either two or four polypeptides
form the active enzyme complex. The bifunctional N. crassa subunit homolog is bigger, but shows extensive
sequence identity to the other enzymes. Interestingly, no additional domain is found that would account for the
flavin reductase activity. The active site residues for this second function of the polypeptide are apparently
interspersed with residues that are conserved between mono- and bifunctional chorismate synthases. Thus the
larger size of the bifunctional enzyme is not the consequence of an additional domain encoding a flavin
reductase (80).

The first plant cDNA encoding chorismate synthase was obtained from C. sempervirens (166); it has a 5'
sequence that translates into a typical plastid import signal. Both the full-length cDNA and the truncated form
encoding the presumed mature enzyme have been expressed in E. coli. Only the truncated, mature form of the
enzyme is able to catalyze the synthesis of chorismate in vitro or complement the function in vivo in a
heterologous system (82). Since C. sempervirens contains only one gene encoding chorismate synthase, these
findings indicate that the enzyme activity of plant chorismate synthase is dependent upon plastid import and that
there is no chorismate synthesis outside the plastids of at least this higher plant.
The C. sempervirens cDNA was used to identify two homologs in tomato (61) encoding chorismate synthase.
The corresponding cDNA sequences encode polypeptides with amino terminal signal sequences for plastid
import. A third tomato mRNA encoding chorismate synthase is generated through differential splicing of one of
the two gene products (59). All three encoded proteins were heterologously expressed in E. coli as precursors
and as mature, processed forms. Only the mature forms of two isoenzymes were enzymatically active,
confirming the results from C. sempervirens that plant chorismate synthesis proceeds exclusively in plastids
(24). The two active isoenzymes complement an E. coli mutant devoid of chorismate synthase. The two
isoenzymes differ with respect to their Km values for EPSP and their specific enzyme activities.
Differential expression of chorismate synthase in various parts of the plant shows highest amounts of mRNA in
flowers and roots (61), with only the more active form of the enzyme occurring in higher amounts. Fungal
elicitation further increases the amount of the more active isoenzyme (60). Thus in tomato, apparently just one
of the three isoenzymes supplies the bulk of chorismate synthase activity under all physiological conditions.
BRANCH POINTS IN THE MAIN TRUNK
All intermediates of the main trunk of the shikimate pathway are potential branch points leading to other
metabolic pathways (16). DAHP has long been suspected to be a precursor for an aromatic moiety of certain
antibiotics that consists of seven carbons and a nitrogen (mC7N). DHQ is readily converted to quinate, a
ubiquitous plant building block for phytoalexins and UV protectants. Some organisms can use DHS as a sole
carbon source by converting it to tricarboxylic acid cycle intermediates via protocatechuate. Shikimate is a direct
degradation product of quinate and that reaction may be reversible. S3P and EPSP have been considered as
precursors for cyclohexane carboxylate moieties of antibiotics.
3-Deoxy-D-Arabino-Heptulosonate 7-Phosphate
Amycolatopsis mediterranei produces rifamycin B. Part of the structure of this and other ansamycin antibiotics is
a seven-carbon aromatic moiety derived from 3-amino-5-hydroxy benzoic acid (AHBA). This is the initiator for
the formation of the polyketide chain that is eventually cyclized to form the mature antibiotic. AHBA is
presumably derived from a shikimate pathway intermediate (28, 89). Mutant studies with rifamycin-producing
bacteria seem to point to DAHP as a precursor, since DHQ and DHS could not serve as precursors for AHBA,
but a DHQ synthase lacking mutant was still able to produce rifamycin (70).
Some indirect, supporting evidence for DAHP as a precursor of AHBA came from studies with Streptomyces

hygroscopicus, another antibiotic-producing bacterium, that revealed a large gene cluster for the polyketide
synthase. This cluster was physically in close proximity to an open reading frame encoding a plant-like DAHP
synthase that could potentially catalyze the first step in the biosynthesis of the antibiotic (162). Such a gene was
also found in the phenazine producer Pseudomonas aureofaciens (151). DAHP synthase has been purified to
apparent electrophoretic homogeneity from A. mediterranei. A gene encoding a plant-like DAHP synthase was
found within a cluster of A. mediterranei genes involved in rifamycin biosynthesis (6).
Studies by Floss and coworkers with A. mediterranei and Streptomyces collinus point to aminoDAHP as the
precursor for AHBA (97). The ability to convert aminoDAHP, but not DAHP, in vitro, into AHBA led to the
proposal of a novel shikimate pathway. In this pathway, PEP and E4P together with a still unknown nitrogen
source are converted to aminoDAHP that serves as a precursor for aminoDHQ, aminoDHS, and eventually
AHBA (97). Potential candidates for genes encoding enzymes for this pathway have been identified in the gene
cluster mentioned above. This cluster contains a number of open reading frames with sequences that are very
similar to genes encoding authentic shikimate pathway enzymes (6). The first step in the novel pathway, the
biosynthesis of aminoDAHP, seems more complicated than originally assumed. While an enzyme catalyzing
this step has not yet been described, the last step in AHBA biosynthesis is catalyzed by an enzyme that has
been purified, its cDNA cloned, and expressed in E. coli (98).
It would be interesting to see if the plant DAHP synthase-like genes of S. hygroscopicus or of A. mediterranei,
expressed in a heterologous system and supplied with the proper nitrogen donor or with additional polypeptides,
could yield aminoDAHP. If Streptomyces and Amycolatopses indeed have only one DAHP synthase, namely a
plant-like enzyme, it would then have to fulfill a dual function. At present, it is not known if DAHP synthase from
the shikimate pathway produces a precursor for antibiotics or if a related but separate enzyme synthesizes
aminoDAHP directly. The identification and characterization of enzymes involved in rifamycin biosynthesis will
certainly be of great value for the generation of genetically engineered microorganisms that can produce
clinically useful antibiotic variants.
3-Dehydroquinate and 3-Dehydroshikimate
DHQ can be converted to DHS or to quinate, the precursor to the ubiquitous plant secondary product
chlorogenate, a condensation product of quinate and caffeate. Thus, chlorogenate is made by combining an
early intermediate of the shikimate pathway with a late intermediate of phenylpropanoid metabolism that is
many steps removed. Because the precursors are made in two different cell organelles, elucidating the mode of
regulation of chlorogenate biosynthesis will be a challenging problem. Chlorogenate protects plants against
fungal attack ( 119) and, through accumulation to substantial levels, serves as a UV protectant in several plants.
Quinate itself also accumulates in some plants and has been considered an alicyclic carbon reservoir for
aromatic compound biosynthesis.
The fully reversible conversion of DHQ to quinate is catalyzed by quinate dehydrogenase, an NAD-dependent
enzyme of 42 kD that is subject to regulation by reversible phosphorylation of a Ser residue (94,157) by a Ca-
calmodulin-- dependent protein kinase. The phospho protein is enzymatically active, whereas dephosphorylated
enzyme is inactive. The quinate dehydrogenase is associated with a Ca-sensing 60-kD regulatory subunit (65).
Ca flux, dependent upon voltage-gated Ca channels (205), can initiate signal transduction in carrot cells. The
cytoskeleton plays an important role in the establishment and stability of such channels (204). How these
channels are activated in whole plants and how their activation regulates the flow of DHQ to DHS or quinate has
yet to be addressed.
Plants also have a quinate hydrolyase that catalyzes the conversion of quinate to shikimate (116). Thus quinate
can reenter the main trunk of the shikimate pathway by being converted either to DHQ or to shikimate. If quinate
hydrolyase catalyzes a freely reversible reaction, plants would also have at least two ways to synthesize
quinate: from DHQ, using the quinate dehydrogenase and from shikimate, using the quinate hydrolyase. Both
enzymes are localized in the plastids. Nothing is known about the regulation of the latter enzyme or about the

relative activities of these two pathways.
Fungi and some bacteria can use quinate as their sole carbon source (56) by degrading it via DHQ and DHS to
protocatechuate and on to succinate and acetyl CoA. This catabolic sequence, described for N. crassa (56), A.
nidulans (64), Rhodococcus rhodochrous (25), and A. calcoaceticus (55), is actually induced by quinate.
Regulation of quinate catabolism is by transcriptional control mediated through an activator and a repressor
(53). The activator is homologous to the amino terminal half and the repressor to the carboxy terminal half of the
biosynthetic AROM complex (75,76,117). Quinate blocks the activity of the repressor (112). These regulatory
molecules are apparently very similar in structure and function when related organisms are compared, because
the activator from N. crassa recognizes promoters of A. nidulans (86).
The first three enzymes of quinate degradation are quinate dehydrogenase, DHQ dehydratase, and DHS
dehydratase. The NAD-dependent quinate dehydrogenase that also oxidizes shikimate to DHS is different from
the NADP-- dependent shikimate dehydrogenase of the main trunk of the shikimate pathway (5). Most quinate-
induced catabolic quinate dehydrogenases are NAD-- dependent; however, the enzyme from A. calcoaceticus
uses pyrrolo quinoline quinone as a cofactor (42).
The pathways of quinate degradation and chorismate biosynthesis have clearly different enzymes, even though
they share at least two intermediates, DHQ and DHS. In A. nidulans, quinate degradation to protocatechuate is
regulated at the level of quinate entry into the fungus that is facilitated by a permease (216). Simultaneous
metabolic flux of quinate to chorismate and protocatechuate has also been studied in this organism (111,112).
Overexpression of the catabolic DHS dehydratase inhibits growth of the organism through interference with
chorismate synthesis (113).
OUTLOOK
A combination of protein and DNA sequencing efforts have yielded primary structures for all the enzymes of the
shikimate pathway from several organisms, including higher plants. Some of the enzymes have been
crystallized. The elucidation of tertiary or quaternary structures by X-ray analysis and other physicochemical
methods is ongoing. Together with molecular biological investigations, in particular site directed mutagenesis,
these studies will refine our knowledge of the reaction mechanisms for enzymes of the shikimate pathway. Such
studies are vital for the design of new inhibitors of shikimate pathway enzymes that are likely to attain
importance as drugs or herbicides.
The coming years will see an increased emphasis on the study of the regulation of this prominent plant
pathway. Since the main regulatory mechanism appears to be transcriptional, a detailed analysis of the
regulatory regions for genes encoding shikimate pathway enzymes would be a good start. Isoenzymes have
been described for some shikimate pathway enzymes. A DNA motif to bind a regulatory protein has been
identified in an EPSP synthase promoter and the regulatory protein binding to this motif was obtained. It is safe
to assume that other regulatory proteins will be identified, among them factors that are responsible for induction
of the expression of specific isoenzymes. Such studies will contribute to the elucidation of signal transduction
pathways involved in responses to mechanical wounding or pathogen attack. Investigations on the genetic
control mechanisms should include mutant hunts, even though it may be very difficult to generate knock-out
mutants in genes of the shikimate pathway, given the many products derived from this metabolic sequence.
A detailed analysis of the signal peptides required for import into plastids should also be undertaken. All
shikimate pathway enzymes, including those of non-green tissues, are synthesized as precursors in the cytosol
and imported into plastids. Such import requires signal sequences. During import these signals are processed
off the protein precursors to give rise to the mature proteins. There are two general characteristics of signal
peptides: They are rich in hydroxyl amino acid residues and positively charged. The large variation in the
primary sequences of these signals seems to indicate a lack of any specific sequence requirement. That can
hardly be the case, since primary structure dictates function. Also, one would expect specific motifs or structural
differences for transport into different plastids.

As one of the most active plant pathways in terms of carbon flow, the shikimate metabolic sequence will
continue to be of interest. New basic insights into plant metabolism and its regulation will be generated through
further studies in this area.
ACKNOWLEDGMENTS
We thank Michael Poling for a critical reading of the manuscript. This is journal paper number 15856 of the
Purdue University Agricultural Experiment Station.
Visit the Annual Reviews home page at http://www.AnnualReviews.org
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