(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);
, 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.
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, pH 8, 80 µMcis,cis-muconate,
and an appropriate amount of enzyme. Transformation of
3-chloro-cis,cis-muconate was followed by HPLC. (
)
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 H NMR spectra were recorded on a CXP 300
spectrometer (Bruker) using tetramethylsilane as internal standard and
deuterated methanol as solvent.
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 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.
UV-visible spectroscopic data for protoanemonin
isolated from Anemona pulsatilla have been reported by Shaw (30) ( = 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 (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 (), 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.
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 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.
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.