(Received for publication, April 17, 1995; and in revised form, June 30, 1995)
From the
Chorismate synthase catalyzes the conversion of 5-enolpyruvylshikimate-3-phosphate to chorismate. It is the seventh enzyme of the shikimate pathway, which is responsible for the biosynthesis of aromatic metabolites from glucose. The chorismate synthase reaction involves a 1,4-elimination with unusual anti-stereochemistry and requires a reduced flavin cofactor. The substrate analogue (6S)-6-fluoro-5-enolpyruvylshikimate-3-phosphate is a competitive inhibitor of Neurospora crassa chorismate synthase (Balasubramanian, S., Davies, G. M., Coggins, J. R., and Abell, C.(1991) J. Am. Chem. Soc. 113, 8945-8946). We have shown that this analogue is converted to 6-fluorochorismate by Escherichia coli chorismate synthase at a rate 2 orders of magnitude slower than the normal substrate. The decreased rate of reaction is consistent with the destabilization of an allylic cationic intermediate. The formation of chorismate and 6-fluorochorismate involves a common protein-bound flavin intermediate although the fluoro substituent does influence the spectral characteristics of this intermediate. The fluoro substituent also decreased the rate of decay of the flavin intermediate by 280 times. These results are consistent with the antimicrobial activity of (6S)-6-fluoroshikimate not being mediated by the inhibition of chorismate synthase but by the inhibition of 4-aminobenzoic acid synthesis as previously proposed (Davies, G. M., Barrett-Bee, K. J., Jude, D. A., Lehan, M., Nichols, W. W., Pinder, P. E., Thain, J. L., Watkins, W. J., and Wilson, R. G. (1994) Antimicrobial Agents and Chemotherapy 38, 403-406).
Chorismate synthase (EC 4.6.1.4) is the seventh enzyme of the
shikimate pathway (1, 2) and catalyzes the conversion
of 5-enolpyruvylshikimate-3-phosphate 1 (EPSP) ()to
chorismate 2 (Fig. S1). Chorismate is a common
intermediate in the biosynthesis of aromatic metabolites such as
aromatic amino acids and folate cofactors. The chorismate synthase
reaction involves the 1,4-elimination of phosphate and the
C-(6proR) hydrogen with overall anti-stereochemistry(3, 4, 5) . A
concerted elimination appears to be unlikely since studies with model
systems (6, 7) and molecular orbital considerations (8, 9) suggest that concerted 1,4-eliminations with syn-stereochemistry are favored. For these reasons, several
non-concerted mechanisms have been suggested for the chorismate
synthase reaction(10) . Proposed mechanisms include an X-group
mechanism, involving the nucleophilic attack of the C-1 position of the
substrate (5) (Fig. S2d), and a cationic
mechanism, involving the step-wise loss of phosphate and a proton (11, 12) (Fig. S2a). An allylic
rearrangement of phosphate followed by a 1,2-elimination (13) has been discounted because the intermediate iso-EPSP is a competitive inhibitor rather than a substrate of
the Neurospora crassa enzyme (14) .
Figure S1: Scheme 1.
Figure S2: Scheme 2.
Reduced FMN is required for enzyme activity despite there being no overall reduction or oxidation in the conversion of EPSP to chorismate (15, 16, 17, 18, 19) . The role of flavin is not yet clear but it appears to be directly involved in catalysis. A transient flavin intermediate has been characterized by uv/visible spectroscopy during single and multiple turnover experiments using the enzyme from Escherichia coli(20, 21) . It has been suggested that this spectrum is consistent with a charge transfer complex or a C-4a-flavin adduct(20) . However, such a perturbation of the reduced flavin spectrum could, at least in part, be due to noncovalent interactions between the substrate and the flavin. The absence of detectable activity of the N. crassa(22) and E. coli(23) enzymes reconstituted with reduced 5-deaza-FMN provides additional evidence that reduced flavin is chemically, and not just structurally, involved in turnover. In the presence of the inhibitor (6R)-6-fluoro-EPSP the E. coli enzyme forms a protein-bound flavin semiquinone, suggesting the possibility of radical intermediates during normal turnover(24) . A radical mechanism involving the initial abstraction of a hydrogen atom from the C-6 position of the substrate (11, 22, 25) seems unlikely since there are no obvious candidates for a catalytic center that can accept single electrons, such as transition metal ions(24) , particularly given the requirement for fully reduced flavin for activity.
The
enzymes of the shikimate pathway are potential targets for antibiotics
and herbicides because they are present in bacteria, fungi, and plants
but not in mammals. Chorismate synthase is a prime candidate because it
has been identified as one of the rate-limiting enzymes of this
pathway(26) . The 6R- and 6S-isomers of
6-fluoroshikimate are antibacterial agents displaying minimum
inhibitory concentrations of 64 and 0.5 µg ml against E. coli K12, respectively(27) . Both
isomers of 6-fluoroshikimate are converted in vitro to
6-fluoro-EPSP by E. coli shikimate kinase and EPSP
synthase(28) . Since (6R)-6-fluoro-EPSP lacks the
(6proR)-hydrogen, it is, as expected, not a substrate of
chorismate synthase. This compound promotes a one-electron oxidation of
the E. coli enzyme (24) and is a competitive inhibitor
of the N. crassa enzyme (K
= 3.0 µM(28) ; K
= 2.2
µM(29) ). It is therefore possible that the
antimicrobial action of (6R)-6-fluoroshikimate is mediated, at
least in part, by the inhibition of chorismate synthase.
The
6S-isomer of 6-fluoro-EPSP (3; Fig. S1) is also
a competitive inhibitor of the N. crassa enzyme (K = 0.2
µM(28) ). The conversion of this analogue to
6-fluorochorismate was not detected with the enzyme from this
source(28) . The antimicrobial action of
(6S)-6-fluoroshikimate is, however, unlikely to be mediated by
the inhibition of chorismate synthase by (6S)-6-fluoro-EPSP.
This is because the susceptibility of E. coli to the
antimicrobial agent (6S)-6-fluoroshikimate is overcome by the
addition of 4-aminobenzoate, a folate cofactor precursor, and not by
the aromatic amino acids phenylalanine, tyrosine, and
tryptophan(27) . These observations suggest that
(6S)-6-fluoro-EPSP is a substrate of E. coli chorismate synthase and that the product, 6-fluorochorismate,
inhibits 4-aminobenzoate synthase.
In this paper we report that the substrate analogue (6S)-6-fluoro-EPSP 3 is converted to the new compound 6-fluorochorismate by E. coli chorismate synthase. The enzyme mechanism and the mode of action of the microbial inhibitor (6S)-6-fluoroshikimate are discussed in the light of this observation.
All chemicals and biochemicals were of the highest grade available and unless otherwise stated were purchased from Sigma (Poole, Dorset, United Kingdom). Sodium dithionite was purchased from B. D. H. Chemicals (Poole, Dorset, U.K.). All spectrophotometric measurements were obtained with a 1-cm path length.
Fig. 1shows the HPLC of an anaerobic reaction mixture
containing (6S)-6-fluoro-EPSP 3 and reduced flavin
before and after the addition of E. coli chorismate synthase.
The fluorinated substrate analogue was converted to a single product
that had a retention time (20.6 min) different from that of chorismate
(25.0 min). A stoichiometric liberation of inorganic phosphate and an
increase in absorbance at 275 nm accompanied this reaction. The
concentration of the substrate analogue was estimated using the
assumption that the product had the same extinction coefficient at 275
nm as the diene chorismate (2630 M cm
). On the basis of this estimation, the
yield of free phosphate was 96 ± 13%. These results are
consistent with the conversion of the (6S)-6-fluoro-EPSP 3 to 6-fluorochorismate 4.
Figure 1: Conversion of (6S)-6-fluoro-EPSP by chorismate synthase. An anaerobic solution containing (6S)-6-fluoro-EPSP (6.4 mM), chorismate synthase (9 µM), FMN (10 µM), dithionite (1 mM), and buffer was incubated at 22 °C for 5 h. HPLC of samples taken (a) before the addition of enzyme and (b) after the reaction shows the conversion of the substrate analogue to a single product.
NMR spectroscopy of a reaction
mixture showed the loss of the signals associated with the starting
material and the formation of new signals consistent with
6-fluorochorismate. The 2-CH, 3-CH, 4-CH, and (proE)-8-CH H signals of the product were well resolved (Fig. 2). The HOD peak obscured the 5-CH resonance partially and
the (proZ)-8-CH resonance completely. The product clearly
retained the enolpyruvyl side chain as the (proE)-8-CH signal
remained essentially unchanged. Homonuclear
H decoupling
experiments confirmed the connectivities between the resonances
associated with positions 2-CH through to 5-CH. The double doublet
F resonance of (6S)-6-fluoro-EPSP at
-174.35 ppm disappeared (Fig. 3, inset) giving
rise to a new quartet at -111.39 ppm (Fig. 3). Finally,
the
P singlet resonance of (6S)-6-fluoro-EPSP at
-135.8 ppm was replaced by one associated with inorganic
phosphate at -136.9 ppm (data not shown).
Figure 2:
H NMR spectrum (270.05 MHz) of
the product of the reaction of (6S)-6-fluoro-EPSP with
chorismate synthase. The product 6-fluorochorismate (0.5 ml) was
prepared using the conditions described in the legend to Fig. 1without further purification. The bulk of the
H
O was exchanged by three cycles of lyophilization and
reconstitution with D
O.
Figure 3:
F NMR spectrum (254.05 MHz)
of the product of the reaction of (6S)-6-fluoro-EPSP with
chorismate synthase. See Fig. 2. The inset shows the
spectrum of the substrate before the addition of the
enzyme.
The above data clearly establish that (6S)-6-fluoro-EPSP is an alternative substrate of E. coli chorismate synthase that yields 6-fluorochorismate as the only detectable product. (6S)-6-Fluoro-EPSP is therefore not just a competitive inhibitor of this enzyme, as has been reported for the N. crassa chorismate synthase(28) .
A time course monitoring the
formation of 6-fluorochorismate by an increase in absorbance at 275 nm
showed that the initial rate of reaction was 270 ± 20 times
lower than that observed with the normal substrate (data not shown).
There was curvature in the time course such that toward the end of the
reaction the rate slowed to 370 ± 30 times lower than with EPSP.
Finally, the reaction stopped abruptly as the substrate analogue became
exhausted. The sensitivity of the assay did not allow the determination
of the K, suggesting its value was at least 5-fold
lower than that for EPSP (1.3 µM(33) ).
When the enzyme was incubated with reduced flavin and either 6-fluorochorismate or chorismate, the slow oxidation of a proportion of the enzyme-bound flavin to the semiquinone was observed over a period of minutes. This reaction may be responsible for the curvature in the time course of the conversion of (6S)-6-fluoro-EPSP. A detailed investigation of this curious oxidative reaction, which is similar to that seen with (6R)-6-fluoro-EPSP(24) , is beyond the scope of the present paper. It is clear, however, that the oxidative reaction is slower than turnover with (6S)-6-fluoro-EPSP and that these processes are independent.
Single turnover experiments with a 1.5-fold molar excess of enzyme
over (6S)-6-fluoro-EPSP gave spectroscopic changes associated
with the formation and decay of a flavin intermediate (Fig. 4).
The difference spectrum of the intermediate was similar but not
identical to that observed using EPSP (Fig. 5). The
was somewhat decreased and shifted from 395 to
385 nm. The shoulder at 445 nm became a more discrete peak at 450 nm
and the isosbestic point at 355 nm was shifted slightly to 352 nm. The
fluoro substituent clearly has some influence on the spectral
characteristics of the intermediate. With either substrate the spectrum
changed only in intensity and not in shape throughout the course of the
reaction.
Figure 4:
The
formation and decay of the flavin intermediate during a single turnover
stopped-flow experiment with (6S)-6-fluoro-EPSP. After mixing,
the anaerobic reaction mixture contained (6S)-6-fluoro-EPSP
(13.5 µM), enzyme (20 µM), FMN (20
µM), dithionite (1 mM), and buffer. The smooth
overlaid curve was fitted to the observed data with two first-order
rate constants describing the formation (210 s) and
subsequent decay (0.19 s
) of the
intermediate.
Figure 5: Difference spectra of the transient flavin intermediate relative to reduced flavoenzyme. The difference spectra of the transient flavin intermediates with (6S)-6-fluoro-EPSP (-) and EPSP(- -) were those obtained when their amplitudes were maximal after 300 and 5 ms, respectively. After mixing, the anaerobic reaction mixtures contained substrate (20 µM), enzyme (15 µM), photoreduced FMN (40 µM), oxalate (1 mM), and buffer. The blank spectrum was obtained from a control experiment without substrate.
Fitting the kinetic data obtained with
(6S)-6-fluoro-EPSP using two single exponentials gave values
of 210 ± 10 and 0.186 ± 0.004 s for
the formation and decay of the intermediate, respectively (Fig. 4). Values of 160 and 52 s
,
respectively, have been obtained using data with the normal substrate,
EPSP (see Fig. 2in (32) ). The formation of the
intermediate is therefore 1.3-fold more rapid with the substrate
analogue under these conditions. Such a rapid rise in absorbance
results in much of the change occurring within the 4-ms dead time of
the stopped-flow spectrophotometer. The rate of decay of the
intermediate was 280 ± 10 times slower than that observed for
EPSP.
We have shown that (6S)-6-fluoro-EPSP is converted
to 6-fluorochorismate by E. coli chorismate synthase at a rate
between 270 and 370 times slower than EPSP. This is in contrast to the
observation that this analogue is a competitive inhibitor of the N.
crassa enzyme(28) . If it is a substrate of the N.
crassa enzyme it is turned over at least 500 times slower than
EPSP(28) . This is one of several differences between the
enzymes from these sources. For example,
carbon-(6proR)-hydrogen bond breaking does not contribute
significantly to rate limitation with the E. coli enzyme (V = 1.13 ±
0.03(32) ) but is partially rate-limiting with the N.
crassa enzyme (
V = 2.7
± 0.2(29) , 2.64 ± 0.02(22) ). The most
striking difference is that the E. coli enzyme is
monofunctional (17) while the N. crassa enzyme is
bifunctional, having an additional NADPH-dependent flavin reductase
activity(16, 17) .
The formation of chorismate and 6-fluorochorismate appear to share a common flavin intermediate although the fluoro substituent does influence its spectral characteristics. No other intermediates, such as a flavin semiquinone radical, were detected during the single turnover experiment. With EPSP, the rate-limiting step occurs after the formation of the flavin intermediate and could be phosphate cleavage (12) , the decay of the flavin intermediate, or the release of either product. Carbon-(6proR)-hydrogen bond breaking occurs after the formation of the flavin intermediate but this is not the rate-limiting step with EPSP(32) . Although the rate-limiting step is not necessarily the same with the fluorinated substrate analogue, it clearly still occurs after the formation of the flavin intermediate.
The decreased rate of decay of the flavin intermediate with the fluorinated substrate did not result in an obvious increase in its transient concentration. This may indicate that essentially all of the enzyme-bound FMN is in the form of the flavin intermediate during a single turnover with either substrate. However, it is difficult to compare precisely the transient concentrations because their difference spectra are not identical. It appears that the initial rate of turnover during the continuous assay and the rate of decay of the intermediate were decreased by similar amounts. It is therefore likely that steps after the decay of the flavin intermediate, which may include product release, are not significantly rate-limiting with either substrate. A similar conclusion was drawn from deuterium kinetic isotope effect studies(32) .
The decreased rate of reaction is consistent with the electron withdrawing fluoro substituent destabilizing an allylic cationic intermediate (12) that would be generated by the loss of phosphate from the substrate (Fig. S2a). In a comparable system, substrate analogues were used to establish the mechanism of farnesylpyrophosphate synthetase(36) . A 57-fold decrease in the rate of turnover using a monofluorinated analogue was observed. The results were interpreted as strong evidence for a stepwise, rather than a concerted condensation reaction, involving an allylic cationic intermediate with the fluoro substituent adjacent to the allylic system. An allylic cationic intermediate in the chorismate synthase reaction would also be destabilized, in part, by the negative hyperconjugative effect of the adjacent fluoro substituent(37) . It is not clear what the nature of the observed flavin intermediate is with such a cationic mechanism, but the electron-rich reduced FMN could have an important role as part of an active site that is capable of stabilizing a cation. The formation and decay of a cationic intermediate would not have to be concomitant with the formation and decay of the observed flavin intermediate. Therefore, interactions between a cationic intermediate and the flavin may not be solely responsible for the transient changes in the flavin spectrum.
The negative hyperconjugative effect of the fluorine could destabilize an allylic radical intermediate that would be formed by a mechanism involving an additional one-electron reduction (Fig. S2b). The overall influence of fluorine on the stability of an adjacent radical is difficult to predict, but such a radical mechanism would provide a role for the fully reduced flavin cofactor. It is possible that a flavin semiquinone does not accumulate to an observable level during the single turnover experiments. The nature of the observed flavin intermediate therefore remains obscure with this mechanism also.
Assuming the withdrawing effect of
the 6-fluoro substituent outweighs its
donating effect, the
reaction is unlikely to involve the initial deprotonation of the C-6
position to form an allylic anionic intermediate (Fig. S2c) because the fluorine would not be expected
to decrease the rate of reaction. There is no obvious requirement for
flavin in this mechanism. The alternative X-group mechanism (5) (Fig. S2d) also appears unlikely since the
electron withdrawing fluorine substituent would facilitate the
rate-limiting (12) nucleophilic attack of the substrate at C-1.
In addition, reduced FMN is unlikely to be the X-group, because the
resultant flavin adduct (flavin C-4a or N-5 adduct, for example) would
be severely sterically disfavored. The reduction of a disulfide by the
flavin to form a thiolate nucleophile has also been
discounted(20) .
(6S)-6-Fluoroshikimate is converted to 6-fluoro-EPSP by E. coli shikimate kinase and EPSP synthase (28) and we have shown 6-fluorochorismate to be produced in the presence of E. coli chorismate synthase. These reactions provide a convenient method for the preparation of the new compound 6-fluorochorismate. In a previous study, the susceptibility of E. coli to the antimicrobial agent (6S)-6-fluoroshikimate was overcome by the addition of 4-aminobenzoic acid and not by aromatic amino acids (27) . It is therefore clear that the antimicrobial activity of (6S)-6-fluoroshikimate is not mediated by the inhibition of chorismate synthase by (6S)-6-fluoro-EPSP. 6-Fluorochorismate most probably inhibits 4-aminobenzoic acid synthase as proposed by Davies et al.(27) . The PabA and PabB proteins of 4-aminobenzoic acid synthase are responsible for the conversion of chorismate to the 4-aminobenzoate precursor 4-amino-4-deoxychorismate(38) . The mechanism of this reaction may involve the nucleophilic attack by an enzyme active site residue at the C-6 position of chorismate, resulting in the loss of the 4-hydroxyl group(1) . This would be followed by amination at the C-4 position, with overall retention of configuration at C-4, and regeneration of the active site nucleophile. It is possible that the reaction with 6-fluorochorismate would result in the loss of hydrogen fluoride before the amination step, leading to an aromatic dead-end complex. Alternatively, a fluoride ion may be lost upon amination rather than the regeneration of the enzyme nucleophile. The covalent modification and irreversible inactivation of enzymes using fluorinated substrates are well known(39) .