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(Received for publication, September 5, 1995; and in revised form, October 2, 1995) From the
Chloroaromatics, a major class of industrial pollutants, may be
oxidatively metabolized to chlorocatechols by soil and water
microorganisms that have evolved catabolic activities toward these
xenobiotics. We show here that 4-chlorocatechol can be further
transformed by enzymes of the ubiquitous 3-oxoadipate pathway. However,
whereas chloromuconate cycloisomerases catalyze the dechlorination of
3-chloro-cis,cis-muconate to form cis-dienelactone,
muconate cycloisomerases catalyze a novel reaction, i.e. the
dechlorination and concomitant decarboxylation to form
4-methylenebut-2-en-4-olide (protoanemonin), an ordinarily
plant-derived antibiotic that is toxic to microorganisms.
Industrially produced halogenated aromatic compounds constitute
a major class of environmental pollutants. Whereas some compounds are
recalcitrant to degradation and accumulate in the environment, others
disappear rapidly. Microorganisms exhibit an exceptional range of
metabolic versatility, evolutionary potential and opportunism, which
enables them to colonize a variety of habitats too hostile for higher
organisms and to metabolize, and thereby grow, at the expense of a wide
spectrum of noxious compounds, including some that are highly toxic. The aerobic degradation of a wide range of aromatic compounds
involves their successive activation and modification such that they
are channeled toward one of a few key dihydroxylated intermediates such
as catechol, gentisate, or protocatechuate, which are the substrates
for cleavage of the aromatic ring. In the case of the haloaromatics,
although halide elimination during an early step in the catabolic
sequence has been documented in a few
instances(1, 2, 3, 4) , the major
degradative route involves conversion to corresponding halocatechols,
intradiol (ortho) cleavage of the aromatic ring, and halide
elimination during a subsequent reaction ((5) ; Fig. 1).
This so-called ``modified'' ortho-cleavage pathway
is thought to essentially parallel the classical 3-oxoadipate pathway,
and enzymes of the classical 3-oxoadipate pathway have been assumed to
carry out reactions identical to those of their counterparts in the
modified ortho pathway, albeit with much lower activities (6, 7, 8) . In both pathways, catechols are ortho-cleaved by (chloro-) catechol 1,2-dioxygenases, to form
the corresponding cis,cis-muconates, which are then
cycloisomerized by muconate cycloisomerase or chloromuconate
cycloisomerase(6, 7, 8) . In analogy to
muconolactone (4-carboxymethylbut-2-en-4-olide) as product of cis,cis-muconate cycloisomerization, halosubstituted
muconolactones were believed to be intermediates of cycloisomerization
of halosubstituted cis,cis-muconates, and spontaneous
dehalogenation of those intermediates was postulated to give rise to
4-carboxymethylenebut-2-en-4-olides (dienelactones; (8) ).
Those are subsequently hydrolyzed to maleylacetate by dienelactone
hydrolase(8) , followed by the enzymatic reduction of
maleylacetate to 3-oxoadipate, the first common intermediate of the
classical and the modified ortho-cleavage pathways ((9) ; Fig. 1B).
Figure 1:
Aerobic degradation of haloaromatic
compounds. A, haloaromatic compounds are generally metabolized
via catechols as central intermediates(3) . B, whereas
catechol is metabolized by enzymes of the classical 3-oxoadipate
pathway, specialized enzymes are responsible for the metabolism of
chlorocatechols. The pathway shows the previously proposed
intermediates (3, 4, 5, 6, 7) and
protoanemonin. Reactions are indicated as follows: &cjs3587;, enzymes
for the metabolism of bicyclic aromatics; &cjs3800;, enzymes for the
metabolism of mononuclear aromatics to catechol; &cjs3613;, reactions
catalyzed by enzymes of the 3-oxoadipate pathway (type I enzymes);
Based on the models of Ngai
and Kallen (10) and Chari et al.(11) of syn addition to a cis double bond of muconate
proceeding via a carbanionic intermediate,
Schlömann et al.(12) suggested
that the cycloisomerization reaction of
3-chloro-cis,cis-muconate is completed by the elimination of
chloride rather than the protonation of the carbanionic intermediate,
excluding 4-chloromuconolactone as an intermediate. An analogous
elimination of chloride from the carbanion is not possible during
cycloisomerization of 2-chloro-cis,cis-muconate. Recently, it
has been shown that 2- and 5-chloromuconolactone were formed by
muconate cycloisomerase(13) , proving that this class of
enzymes is not able to cause dechlorination during conversion of
2-chloro-cis,cis-muconate and that dechlorination by
chloromuconate cycloisomerases is actually an enzyme-catalyzed process.
We report here, that both muconate and chloromuconate cycloisomerases
cause dehalogenation during conversion of
3-chloro-cis,cis-muconate but that different reactions were
catalyzed.
Muconate cycloisomerase (EC 5.5.1.1) was measured
following the consumption of cis,cis-muconate at 260 nm by a
modification of the method of Sistrom and Stanier(22) . The
cuvettes initially contained 50 mM, 2 mM
MnSO Chlorocatechol 1,2-dioxygenase, chloromuconate
cycloisomerase (EC 5.1.1.7) and dienelactone hydrolase (EC 3.1.1.45)
were determined according to published
methods(4, 5, 6) . The protein
concentration was estimated as described by Bradford (23) .
The catechol 1,2-dioxygenase type I from B13 was purified
from the same cell-free extract by a similar procedure, but the heat
treatment and the last anion-exchange step were avoided. For the first
anion-exchange chromatography, a 20-ml linear gradient of NaCl
(0-500 mM) was applied. Muconate cycloisomerase form P. putida strain RW10 was purified by similar procedures. In order to obtain catechol 1,2-dioxygenase I or II preparations
free of the responsible isoenzyme and free of any cycloisomerizing
activities, those proteins were partially purified. The
ultracentrifugated cell free extract was applied to a Mono Q HR 5/5
column, and the proteins were eluted with 5 ml of 50 mM Tris,
pH 7.5, followed by a 20-ml linear gradient of NaCl (0-500
mM) of the same buffer. This procedure allows for the
separation of the enzymes and isoenzymes of the classical and modified ortho-pathway.
For high
resolution gas chromatography-mass spectroscopy, the sample was
analyzed by using a Carlo Erba/mega series gas chromatograph equipped
with a 30-m DB1 column. The gas chromatograph was linked to a Kratos MS
50 mass spectrometer. Helium was used as the carrier gas, and a
potential of 70 eV was used for electron ionization. Dynamic high
resolution MS of the molecular ion and the major fragment ions was
performed using perfluorokerosene as internal reference. High
resolution
The syntheses of cis-dienelactone
((E)-4-carboxymethylenebut-2-en-4-olide), trans-dienelactone
((Z)-4-carboxymethylenebut-2-en-4-olide) and 3-chlorocatechol
have been described previously(25, 26) .
4-Chlorocatechol was purchased from Aldrich. All other chemicals
were of analytical grade and obtained from Fluka AG, Merck AG, and
Aldrich Chemie GmbH.
In order to analyze whether this new metabolite was formed from
4-chlorocatechol in a single catalytic step or in a coupled reaction
and whether enzymes of the 3-oxoadipate pathway were responsible for
its formation, both the catechol 1,2-dioxygenase type I and the
muconate cycloisomerase from Pseudomonas sp. B13 were purified
to homogeneity. Specific activity of catechol 1,2-dioxygenase type I
(29 units/mg of protein) agreed well with literature data (20 units/mg
of protein; (6) ). Muconate cycloisomerase was purified
91-fold, giving a preparation with a specific activity of 137 units/mg
of protein. The protein showed a single band in SDS-polyacrylamide gel
electrophoresis corresponding to a molecular mass of 40 kDa. Spectrophotometric analysis of 4-chlorocatechol (100
µM) turnover by purified muconate cycloisomerase of B13
revealed an increase in absorption at 260 nm as predicted for
3-chloro-cis,cis-muconate formation(7) . Turnover of
4-chlorocatechol by catechol 1,2-dioxygenase type II gave the same
spectroscopic changes. In both cases, increase in absorption of 1.2
± 0.05 is indicative for quantitative
3-chloro-cis,cis-muconate formation. In both cases, the
quantitative consumption of 4-chlorocatechol was confirmed by HPLC
analysis. The appearance of two products was observed. One of those
(retention volume, 6.6 ml) co-chromatographed with authentic cis-dienelactone and was reported to be spontaneously formed
from 3-chloro-cis,cis-muconate under the acidic conditions
used for HPLC analysis(28, 29) . Consequently,
catechol 1,2-dioxygenase types I and II carry out the same reaction
with 4-chlorocatechol as substrate, forming
3-chloro-cis,cis-muconate (retention volume, 9.5 ml) as the
product. When a 3-chloro-cis,cis-muconate containing
reaction mixture was incubated with muconate cycloisomerase (2
mM Mn
UV-visible spectroscopic data for protoanemonin
isolated from Anemona pulsatilla have been reported by Shaw (30) (
Formation of
protoanemonin (always greater than 70% of the theoretical yield) was
also observed when cell extracts from cultures of different bacteria
producing enzymes of the 3-oxoadipate pathway, i.e. benzoate-grown Pseudomonas sp. B13, P. putida KT2442 and Sphingomonas sp. RW1, and salicylate-grown P. putida RW10, were incubated with 10.2 mM 4-chlorocatechol. This is indicative of a general reaction
mechanism for muconate cycloisomerizing enzymes. The specific activity
for protoanemonin production by cell extracts was always >50 units/g
protein, and further metabolism was negligible (<1 unit/g of
protein), indicating that protoanemonin is probably a nonmetabolizable,
dead-end product of the classical 3-oxoadipate pathway. In contrast
to the situation with bacteria induced for type I ortho-pathway enzymes (3-oxoadipate pathway) enzymes only, no
formation of protoanemonin from 4-chlorocatechol was detected when an
extract of a 3-chlorobenzoate-grown culture of strain B13, i.e. an extract containing type II-modified ortho-pathway
enzymes, was used. In this case, maleylacetate resulting from the
concerted action of chloromuconate cycloisomerase and dienelactone
hydrolase, was the only product observed. It should be noted at this
juncture that 3-chlorobenzoate-grown B13 cells are induced for both the
modified and the classical pathways and that the cell extract contained
both muconate cycloisomerase and chloromuconate cycloisomerase. These
two enzymes were partially purified from the extract by anion-exchange
chromatography; individually they catalyzed the expected reactions, i.e. protoanemonin formation by muconate cycloisomerase and cis-dienelactone formation by chloromuconate cycloisomerase,
but combined fractions quantitatively transformed
3-chloro-cis,cis-muconate into cis-dienelactone,
indicative for the reported high activity of chloromuconate
cycloisomerase with this substrate(8) .
Figure 2:
In vivo production and effect of
protoanemonin. Acetate (
In order to study the effect of
protoanemonin on various xenobiotic-degrading bacteria, we synthesized
this compound from 4-chlorocatechol in a coupled reaction (catechol
1,2-dioxygenase type I and muconate cycloisomerase) catalyzed by
cell-free extracts of Pseudomonas sp. B13 bacteria grown on
benzoate. Pure preparations of protoanemonin were obtained after
extraction with diethyl ether with a yield higher than 90%. In all
cases, protoanemonin inhibited the growth of microorganisms,
concentrations between 15 and 150 ppm being bacteriostatic (Table 3).
Figure 3:
Models for the mechanisms of
cycloisomerization of muconate and 3-chloromuconate. For details, see
text. H
Despite their high
degree of homology, it is evident that muconate cycloisomerase and
chloromuconate cycloisomerase are not merely isoenzymes with distinct
substrate specificities. Different mechanisms were observed not only
with 3- but also with 2-chloro-cis,cis-muconate(13) ,
indicating different active site structures. It is interesting to
speculate that evolution of the chloromuconate cycloisomerase active
site structure may have been selected by the need to prevent formation
of the antibiotic protoanemonin. The three-dimensional structure of
both muconate cycloisomerase from P. putida(37) and
of chloromuconate cycloisomerase from Alcaligenes eutrophus JMP 134 (38) have now been elucidated, and conformational
differences in the active site as well as differences in the polarity
and size of the channel leading to the active site were reported.
Site-directed mutagenesis will give insights in amino acids responsible
for variation in substrate specificity and transformation mechanisms.
Volume 270,
Number 49,
Issue of December 8, 1995 pp. 29229-29235
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, reactions catalyzed by enzymes of the modified ortho-pathway (type II enzymes). Enzymes involved are as
follows: C12O I, catechol 1,2-dioxygenase type I; C12O
II, catechol 1,2-dioxygenase type II; MCI, muconate
cycloisomerase; CMCI, chloromuconate cycloisomerase; MI, muconolactone isomerase; ELH, enollactone
hydrolase; DHL, dienelactone hydrolase; MAR,
maleylacetate reductase.
Strains, Media, and Growth
Most of the bacterial
strains used in this study have been described previously: Pseudomonas sp. B13(14) , Pseudomonas putida KT 2442(15) , Sphingomonas sp. RW1(16) , Pseudomonas sp. LB400(17) , P. putida P111(18) , Sphingomonas paucimobilis Q1(19) , Alcaligenes eutrophus H850(20) , Rhodococcus globerulus P6(21) , Escherichia coli DH5
(Life Technologies, Inc.). P. putida RW10 was
isolated based on its capability to mineralize 4- and
5-chlorosalicylate. Cells were grown in M9 medium supplemented with the
indicated carbon source (usually 5 mM) and incubated at 30
°C on a rotary shaker at 140 rpm in baffled Erlenmeyer flasks.Preparation of Cell Extracts
Cells were harvested
by centrifugation at the end of the logarithmic growth phase,
resuspended in the appropriate buffer (50 mM Tris, pH 7.5,
supplemented with 2 mM MnSO
for analysis or
purification of muconate cycloisomerase) and passed once through a
French pressure cell (Aminco) operating at 18,000 p.s.i. The cell
debris was removed by centrifugation at 100,000 
g for
45 min. The clear supernatant is referred to as the cell extract.Enzymatic Assays
Catechol 1,2-dioxygenase (EC
1.13.11.1) was measured by following the formation of cis,cis-muconate at 260 nm by a modification of the method of
Dorn and Knackmuss(6) . The cuvettes initially contained 33
mM Tris, pH 8.0, 2 mM EDTA, and an appropriate amount
of enzyme. After a 5-min incubation, the reaction was started by the
addition of catechol or 4-chlorocatechol to a final concentration of
0.2 mM., pH 8, 80 µMcis,cis-muconate,
and an appropriate amount of enzyme. Transformation of
3-chloro-cis,cis-muconate was followed by HPLC. (
)Enzyme Purifications
Enzyme purifications were
performed on a Pharmacia Biotech Inc. fast protein liquid
chromatography system. For the purification of muconate cycloisomerase
from Pseudomonas sp. B13, the cells were grown with benzoate,
and the cell extract was incubated at 60 °C for 5 min and
centrifuged at 15,000 g for 10 min. The supernatant
fluid was applied to a Mono Q HR 5/5 anion-exchange column. The column
was operated at a flow rate of 1 ml/min. Proteins were eluted with 10
ml of Tris (50 mM, pH 7.5, supplemented with 2 mM MnSO), followed by 5 ml of the same buffer containing
70 mM NaCl and a 5-ml linear gradient of NaCl (70-140
mM). Active fractions were precipitated with 70% ammonium
sulfate, loaded onto a phenyl-Superose HR 5/5 (hydrophobic interaction)
column, and the enzyme activity was eluted with a 35-ml linear gradient
of ammonium sulfate (0.7-0.0 M). The active fractions
were pooled, adjusted to 70% with ammonium sulfate, centrifuged at
10,000 g for 10 min, resuspended in 200 µl of the
same buffer, and loaded onto a Superose 12 HR 10/30 column (gel
filtration). Proteins were eluted with 50 mM Tris, pH 7.5,
supplemented with 2 mM MnSO. The active fractions
were applied to a second anion-exchange chromatography as described
above.SDS-Polyacrylamide Gel Electrophoresis
SDS
electrophoresis was performed according to Laemmli(24) . Low
molecular mass standards (phosphorylase b, 97.4 kDa; bovine
serum albumin, 66.2 kDa; ovalbumin, 45 kDa; carbonic anhydrase, 31 kDa;
soybean trypsin inhibitor, 21.5 kDa; and lysozyme, 14.4 kDa) were
purchased from Bio-Rad. Proteins were stained with an aqueous solution
containing 1% (w/v) Coomassie Brilliant Blue R-250 and 7% (v/v) acetic
acid and destained with an aqueous solution containing 10% (v/v) acetic
acid and 10% (v/v) isopropanol.Analytical Methods
Substrates and products were
quantified by using a Beckman System Gold HPLC equipped with SC columns
(125 4.6 mm) filled with 5-mm particles of Lichrospher 100 RP8
(Bischoff). An aqueous solution of 15% (v/v) methanol and 0.1% (v/v)
H
PO in Milli Q (Millipore) water was used as
the mobile phase (flow rate, 1 ml/min). Usually, samples of 20 µl
were analyzed. The column effluent was monitored simultaneously at 210
and 270 nm by use of a diode array detector, and the scans between 210
and 300 nm of the significant peaks were stored by the system. Typical
net retention volumes (ml) and characteristic absorption maxima
(between 240 and 300 nm) were as follows: maleylacetate, 1 ml; trans-dienelactone, 3 ml, 
= 274 nm;
protoanemonin, 6 ml, = 260 nm; cis-dienelactone, 6.6 ml, = 275 nm;
3-chloro-cis,cis-muconate, 9.5 ml, = 257 nm; 4-chlorocatechol, 23 ml, = 285 nm.Preparative Conversion of 4-Chlorocatechol to
Protoanemonin
The reaction mixture initially contained, in a
final volume of 230 ml, 20 mM Tris, pH 8, 13 units of purified
catechol 1,2-dioxygenase type I, and 60 µmol of 4-chlorocatechol.
At this pH, catechol 1,2-dioxygenase type I exhibited maximal activity,
and 3-chloro-cis,cis-muconate, the assumed reaction product,
was reported to be stable. The reaction was carried out at 25 °C
under continuous agitation, and the progress of the reaction was
followed by monitoring the increase in absorbance at 260 nm. After 2 h,
the absorbance at 260 nm remained constant, and HPLC analysis confirmed
the complete consumption of 4-chlorocatechol. The pH of the reaction
mixture was adjusted to 6.5 by the addition of 60 ml of 500 mM MES/NaOH buffer, pH 6.5, and the reaction was initiated by
addition of MnSO (2 mM) and purified muconate
cycloisomerase (7 units). After 2.5 h protoanemonin was the major
metabolite detected by HPLC (>98%) and was extracted twice with 250
ml of diethyl ether. About 4 mg of a pale yellow oil were obtained
after careful evaporation of the ether.Spectroscopic Methods
UV spectra were either
recorded using a Beckman DU 70 spectrophotometer or were available from
the diode array analysis of the HPLC effluent. In order to estimate the
molecular extinction coefficient of protoanemonin, the solution
obtained after complete enzymatic conversion of 4-chlorocatechol (using
purified catechol 1,2-dioxygenase and muconate cycloisomerase) was
diluted 10 times with water, and the spectrum was recorded between 220
and 320 nm. A solution containing the same amount of enzymes and
buffers but without substrate was used as background.H NMR spectra were recorded on a CXP 300
spectrometer (Bruker) using tetramethylsilane as internal standard and
deuterated methanol as solvent.Chemicals
cis,cis-Muconate was prepared in vitro from 0.2 mM catechol using partially
purified catechol 1,2-dioxygenase from Pseudomonas sp. B13.
This solution was used to follow the muconate cycloisomerase activity
during the purification procedure.
Transformation of 4-Chlorocatechol by Enzymes of the
3-Oxoadipate Pathway of Pseudomonas sp. B13
According to the
currently accepted scheme for the metabolism of chlorinated catechols,
4-chlorocatechol will be converted by catechol 1,2-dioxygenase type I
and muconate cycloisomerase to cis-dienelactone with
reasonable activities(6, 7, 8) .
Theoretically, due to low activity of dienelactone hydrolase (8) maleylacetate will be the final metabolite when cell
extracts of benzoate-grown B13 are incubated with 4-chlorocatechol.
However, we observed the formation of a new absorption maximum at 260
nm when the conversion of 4-chlorocatechol (100 µM) by
such extracts was followed spectrophotometrically. Those spectroscopic
changes neither fit with the formation of maleylacetate ( = 243 nm; (27) ), nor of cis- or trans-dienelactone ( = 275 nm).
Analysis of the reaction mixture by HPLC revealed the formation of a
single metabolite that did not co-chromatograph with any of those
compounds described to be formed during chlorocatechol metabolism via
an ortho-cleavage
pathway(6, 7, 8, 9, 13, 28) . was added to stabilize muconate
cycloisomerase), practically no spectroscopic changes were observed.
Monitoring of the enzyme-catalyzed turnover by HPLC, however, clearly
showed the disappearance of 3-chloro-cis,cis-muconate and the
concomitant production of the new compound, which was shown to be
formed from 4-chlorocatechol by cell extract of benzoate-grown B13
cells. Because this compound was found to be unstable at basic pH (Table 1), muconate cycloisomerase-catalyzed preparative
transformation of 3-chloro-cis, cis-muconate was carried out
under slightly acidic conditions.
Structural Identification of 4-Methylenbut-2-en-4-olide
as Cycloisomerization Product of 3-Chloro-cis,cis-muconate
The
product formed from 3-chloro-cis,cis-muconate by purified Pseudomonas sp. B13 muconate cycloisomerase was isolated on a
micropreparative scale as described under ``Materials and
Methods.'' The H NMR spectrum (Table 2) shows
four olefinic protons, centered at three different carbon atoms. A
geminal coupling of 2.8 Hz between H-3 and H-4, a vicinal coupling of
5.6 Hz between H-1 and H-2, as well as long range couplings between H-1
and H-4 (1.9 Hz) and H-1 and H-3 (0.7 Hz) were observed. High
resolution gas chromatography-mass spectroscopy verified the
composition C
HO
: m/e, 96.0222 (M, 100%, base peak); m/e, 68.0264 (M
- CO, 46%); m/e, 54.0115 (M
-
C
HO, 41%); m/e, 42.0103
(M - C
HO, 69%). Those
results confirm the identification as 4-methylenebut-2-en-4-olide
(protoanemonin).
= 260 nm; = 14,000 M
cm
). Enzyme-catalyzed transformation of
4-chlorocatechol into protoanemonin resulted in an increase of
absorbance slightly higher than expected from those data, indicative
for quantitative formation of this product (
= 259.5 nm; = 15,100 M
cm
).
Formation of Protoanemonin by Different Muconate
Cycloisomerases
In order to analyze whether or not the formation
of protoanemonin from 3-chloro-cis,cis-muconate is a unique
characteristic of the muconate cycloisomerase from Pseudomonas sp. B13, the corresponding enzyme from salicylate-grown cells from P. putida RW10 was purified.
3-Chloro-cis,cis-muconate was prepared in vitro from
4-chlorocatechol by partially purified (only by anion-exchange
chromatography) catechol 1,2-dioxygenase from Pseudomonas sp.
B13. Following enzymatic conversion, proteins were eliminated by
passage of the reaction mixture through a 10-kDa cut-off pore filter.
Bioconversion of 3-chloro-cis,cis-muconate was carried out in
a final volume of 1 ml with 20 milliunits of P. putida RW10
muconate cycloisomerase under the conditions described for the
preparative production of protoanemonin with Pseudomonas sp.
B13 enzymes. After 30 min, the reaction was completed by stoichiometric
production of protoanemonin as judged by HPLC analysis.In Vivo Production and Antibiotic Effect of
Protoanemonin
When benzoate-grown cells of Pseudomonas sp. B13 or P. putida RW10 (A =
0.8) were incubated with 1 mM 4-chlorocatechol, they
completely consumed the substrate in less than 1 h. The optical density
remained constant throughout the incubation period of 6 h, however,
there was a drastic decrease (one order of magnitude) in the number of
colony-forming units in the course of the experiment. This effect was
not observed when catechol was added. The toxicity of 4-chlorocatechol
toward Pseudomonas sp. B13 cells has already been
reported(26) . In order to analyze whether this toxic effect is
due to 4-chlorocatechol itself or to protoanemonin formed during the
incubation, washed cells of Pseudomonas sp. B13 grown on
acetate, benzoate, or 3-chlorobenzoate were incubated with
4-chlorocatechol. A significant decrease in colony-forming units was
observed only for benzoate-grown cells (Fig. 2). Whereas
benzoate- and 3-chlorobenzoate-grown cells showed a rapid turnover of
4-chlorocatechol in less than 15 min, acetate-grown cells consumed the
compound within 4 h. 3-Chlorobenzoate- as well as acetate-grown cells
metabolized 4-chlorocatechol without any detectable accumulation of
intermediates. Accumulation of protoanemonin was, however, observed in
the supernatant of benzoate-grown cells. The amount of protoanemonin
produced accumulated up to 30% of that expected for the stoichiometric
transformation of 4-chlorocatechol. We therefore conclude that
4-chlorocatechol itself has no major toxic effect but that cell death
is due too protoanemonin.
), benzoate (
), and
3-chlorobenzoate (
) grown cells of Pseudomonas sp. B13
were incubated with 1 mM 4-chlorocatechol, and the number of
colony-forming units (cfu) on LB plates was analyzed. Data are
representative of three independent
experiments.
Mechanistic and Evolutionary Implications of
Protoanemonin Formation
Since several studies have shown that
muconate cycloisomerase and chloromuconate cycloisomerase are
evolutionarly closely
related(31, 32, 33, 34) , a common
reaction mechanism for both types of enzymes has been
assumed(12) . The stereochemistry of the reaction catalyzed by
the muconate cycloisomerase of P. putida with muconate as
substrate has been elucidated (Fig. 3A) and a carbanion has
been postulated as the mechanistic intermediate(10) . The
currently accepted mechanism for the enzymatic reaction involves the
formation of a metal-stabilized enol/enolate intermediate followed by a
vinylogous E2 reaction (1,4-elimination)(35, 36) .
Although this reaction model explains the dechlorination of
4-chloromuconolactone catalyzed by chloromuconate cycloisomerase (Fig. 3B), because of the double bond linking the
carboxylate anion, the proposed intermediate does not explain the
formation of protoanemonin in the reaction catalyzed by muconate
cycloisomerase. Other workers have postulated that
4-chloromuconolactone is formed by protonation of the corresponding
carbanion as intermediate and have assumed that this compound is
chemically unstable, such that cis-dienelactone is generated
spontaneously by anti-elimination of hydrogen chloride or
chloride from the carbanion (6) in a nonenzymatic collateral
reaction (Fig. 3C, part 2). However, if this
were the case, the product of the reaction with 3-chloromuconate should
be always cis-dienelactone, independent of the type of enzyme
catalyzing the reaction. This reasoning, together with our new
findings, has led us to propose 4-chloromuconolactone as the
intermediate from which decarboxylation and dechlorination occur
concomitantly in a single reaction catalyzed by muconate cycloisomerase (Fig. 3C, part 1). For the reaction catalyzed
by chloromuconate cycloisomerase, an active site base must be
postulated, which catalyzes fast elimination of the solvent-derived C-5
proton and the halide, thus generating cis-dienelactone (Fig. 3C, part 2).
indicates protons derived from the
solvent, and M
represents a metal ion and/or
a cationic functional group. The structures in brackets represent hypothetical mechanistic
intermediates.
The Ecological Implication of
Protoanemonin
Protoanemonin has been known since 1945 as an
active constituent of plants of the Ranunculaceae family,
which is antibiotically active against a wide spectrum of
microorganisms(39) . We report here on its bacterial formation
from 4-chlorocatechol, a central intermediate of the catabolism of
chloroaromatic compounds, by enzmes of the wide-spread 3-oxoadipate
pathway. Protoanemonin formation was observed in vitro and in vivo, resulting in cell death. As being released from the
cells, it may subsequently affect the entire ecosystem in which it is
produced. Formation of protoanemonin has to be taken into consideration
when microorganisms harboring incomplete catabolic pathways are
confronted with chloroaromatics. Accordingly, the inhibitory effect of
chlorobenzoates on chlorobiphenyl metabolism and degradation by
coinoculation of chlorobenzoate degraders has been observed. Havel and
Reineke (40) attributed the inhibition of mineralization of
polychlorinated biphenyls by laboratory-selected microorganisms in soil
microcosms to an unknown toxic metabolite produced from
4-chlorobenzoate (the end-product of 4-chlorobiphenyl metabolism by the
biphenyl degraders used in this study) by the native microflora present
in their soil slurries. The positive effect on such survival achieved
by co-inoculation with chlorobenzoate degraders (41, 42) is consistent with this hypothesis. Microcosm
experiments performed in our laboratories have shown protoanemonin
formation by the natural microflora to be responsible for the poor
survival of polychlorinated biphenyls-cometabolizing bacteria in
environmental settings(44) . We assume that the microbial
formation of protoanemonin will occur not only in the presence of
chlorinated aromatic pollutants but also during the catabolism of
fluoroaromatics. The cycloisomerization product of
3-fluoro-cis,cis-muconate was reported to be transformed by
enollactone hydrolase of P. putida strain A 3.12 predominantly
into a new compound that was tentatively identified by its
UV-spectroscopic properties as protoanemonin(43) . If this
result is subsequently confirmed, it seems that protoanemonin can be
formed by two completely different enzyme reactions, namely those
carried out by muconate cycloisomerase and by enollactone hydrolase,
according to the nature of the halosubstituent of cis,cis-muconate.
)
We thank Manfred Nimtz and Victor Wray for performing
gas chromatography-mass spectroscopy and NMR analysis, Michael
Schlömann for fruitful discussions, and Dave McKay
for critical reading of the manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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