Nov 14, 1991 - Rather, it is transformed to analogous dead-end metabolites, (2-methylbenzyl)-succinic acid and (2-methyl- benzyl)-fumaric acid. o-Xylene ...
Vol. 58, No. 2
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1992, p. 496-501
0099-2240/92/020496-06$02.00/0 Copyright C) 1992, American Society for Microbiology
Metabolites Formed during Anaerobic Transformation of Toluene and o-Xylene and Their Proposed Relationship to the Initial Steps of Toluene Mineralization PATRICK J. EVANS,'t WILLIAM LING,' BERNARD GOLDSCHMIDT,2 EDWARD R. RITTER,3 AND L. Y. YOUNG'.2* Departments of Microbiology' and Environmental Medicine,2 New York University Medical Center, 550 First Avenue, New York, New York 10016, and Hazardous Substance Management Research Center, New Jersey Institute of Technology, Newark, New Jersey 071023 Received 18 July 1991/Accepted 14 November 1991
Strain Tl is a facultative bacterium that is capable of anaerobic toluene degradation under denitrifying conditions. While 80% of the carbon from toluene is either oxidized to carbon dioxide or assimilated into cellular carbon, a significant portion of the remainder is transformed into two dead-end metabolites. These metabolites were produced simultaneous to the mineralization of toluene and were identified as benzylsuccinic acid and benzylfumaric acid. Identification was based on comparison of mass spectra of the methyl esters of the metabolites and authentic compounds that were chemically synthesized. Strain Tl is also capable of o-xylene transformation during growth on toluene. o-Xylene does not serve as a source of carbon and is not mineralized. Rather, it is transformed to analogous dead-end metabolites, (2-methylbenzyl)-succinic acid and (2-methylbenzyl)-fumaric acid. o-Xylene transformation also occurred during growth on succinic acid, which suggests that attack of the methyl group by succinyl-coenzyme A is a key reaction in this transformation. We reason that the main pathway for toluene oxidation to carbon dioxide involves a mechanism similar to that for the formation of the metabolites and involves an attack of the methyl group of toluene by acetyl-coenzyme A.
Strain Ti is a denitrifying bacterium that has been shown to oxidize toluene under anaerobic conditions primarily to carbon dioxide and biomass (8). This organism is also capable of o-xylene transformation during growth on toluene; however, o-xylene cannot serve as a sole source of energy or cellular carbon. Metabolites were formed from both toluene and o-xylene during their transformation and were not further metabolized. These dead-end metabolites were determined not to be benzyl alcohol, benzaldehyde, benzoic acid, cresol, hydroxybenzyl alcohol, hydroxybenzaldehyde, hydroxybenzoic acid, methylbenzyl alcohol, tolualdehyde, or toluic acid (8). Since the metabolites were not further degraded, it was reasoned that they were not intermediates in the pathway of toluene oxidation to carbon dioxide. However, since a significant portion of toluene carbon was transformed to these metabolites, investigation of their formation was warranted. The toluene-dependent transformation of o-xylene is significant because o-xylene is relatively recalcitrant to biodegradation under both aerobic (2) and anaerobic (8) conditions. However, incomplete transformations have a potentially negative side since the metabolites formed may be more toxic than the original chemical that was the source of contamination. Therefore, identification of these metabolites can provide information on their toxicity, their formation, and how to initiate their mineralization.
MATERIALS AND METHODS Microorganism and culture conditions. Strain Ti was isolated and anaerobically grown on toluene and other substrates at a pH of 7.5 in a phosphate-buffered mineral salts medium with KNO3 (5 to 20 mM), as described previously (8, 9). Cultures grown on various substrates in the presence of o-xylene (see Tables 1 and 2) were incubated for 4 days. Syntheses, extraction of metabolites, and derivatizations. Benzylsuccinic acid was synthesized by the method of Cohen and Milovanovic (4). Benzylmaleic acid was synthesized by the method of Doebner and Kersten (6). The metabolites were extracted from the culture fluid by acidification to pH 2.0 (approximately) with H3PO4 and extraction with diethyl ether. The ether extract was dried over anhydrous sodium sulfate, and then the ether was evaporated under a stream of argon. Benzylsuccinic acid, benzylmaleic acid, and the metabolites of toluene and o-xylene were derivatized by three different methods, all yielding identical results. All of the methods converted the carboxylic acids into methyl esters. The first method involved the addition of 2 ml of BC13 in methanol (Supelco, Bellefonte, Pa.) to the solid, heating of the reactants in a sealed tube in a boiling water bath for 3 min, addition of 1 ml of water, and then extraction with 1 ml of pentane. The second method entailed the addition of 2 ml of methanol and 0.4 ml of 50% (vol/vol) concentrated sulfuric acid to the solid, heating of the reactants at 55°C in a sealed tube for 30 min, and then addition of 1 ml of water and extraction with 1 ml of pentane. The third method entailed the addition of 2 ml of methanol and 1 drop of concentrated sulfuric acid to the solid, heating of the reactants at 55°C in a sealed test tube for 30 min, and then addition of 1 ml of water and extraction with 1 ml of pentane. Mass spectra. Electron impact mass spectra (70 eV) were obtained for the methyl esters for a mass range of 35 to 400 atomic mass units, using a model 5988A mass spectrometer
Corresponding author. t Present address: Celgene Corp., P.O. Box 4914, Warren, N.J. *
07059. t Present address: Department of Chemical Engineering, Villanova University, Villanova, PA 19085. 496
VOL. 58, 1992
METABOLITES OF ANAEROBIC TOLUENE DEGRADATION
equipped with a model 5890 gas chromatograph (HewlettPackard Corp., Paramus, N.J.). Spectra were obtained at a rate of 1.32 scans per s. The mass spectrometer ion source was held at 250°C while the spectra were obtained. Sample mixtures, prepared in pentane, were injected onto an HP-5 fused silica capillary column (12.5 m by 0.22-mm inside diameter), using a split/splitless injector held at 225°C. Injection volumes ranged from 1 to 20 p.l, depending on concentration, and were injected in the splitless mode. The column was held at 65°C for 2 min and was ramped to 100°C at a rate of 30°C/min, held at 100°C for 2 min, and then ramped to 275°C at a rate of 15°C/min. The mass spectrometer ion source was turned on 2 min after initiation of the temperature program. The interface line to the ion source was held at 250°C throughout the run. The mass spectrometer was calibrated and tuned by using perfluoro-tributylamine (FC-43) as the calibration compound immediately prior to running samples. Liquid chromatography and metabolite quantitation. Coelution of underivatized metabolites and synthetic products was monitored by high-performance liquid chromatography (HPLC) with UV detection at 254 nm, as described previously (8). Quantitation of the toluene metabolites was accomplished by HPLC. Synthetic benzylsuccinic and benzylmaleic acids were used as standards for quantitation of metabolically produced benzylsuccinic and benzylfumaric acids, respectively. Measurement of oxygen. Oxygen concentration in the headspace of the serum bottles was measured with a model 1200 gas partitioner (Fisher Scientific, Pittsburgh, Pa.). Instead of argon, which coelutes with oxygen, a nitrogencarbon dioxide mixture (30% carbon dioxide) was used to prepare the bottles and medium anaerobically, as described previously (8). The sensitivity limit was 0.04% oxygen. The dissolved oxygen in the medium was measured colorimetrically with an OX-LEV assay (Hach Co., Loveland, Colo.). The sensitivity of this assay was 1 mg/liter. Statistical analysis. Statistical analysis of the two-level factorial experiment was completed as described by Box et al. (3). The interaction between succinic and oxalacetic acids was calculated from the data in Table 2 as [(A4 - A3) - (A2 - A1)]/2, where Ai is the area of (2-methylbenzyl)-fumaric acid produced under condition i. The standard error was calculated as described previously (3). RESULTS Metabolite production. Figure 1 shows normalized profiles of carbon dioxide and the toluene metabolite during growth on 1 mM toluene and 5 mM nitrate. Previously, this metabolite was referred to as the 6.4-min metabolite (8). As illustrated in Fig. 1, the mineralization of toluene, as represented by carbon dioxide formation, and the transformation of toluene, as indicated by the formation of the metabolite, are simultaneous. Metabolites of toluene. The existence of metabolites of toluene and o-xylene, one metabolite from each, was initially observed by HPLC (8). They were previously referred to as the 6.4- and 9.1-min metabolites, respectively (8). They were extractable into diethyl ether under acidic conditions, indicating an acidic property. Initial gas chromatography-mass spectrometry (GC-MS) investigations of the acids and of the methyl esters of the acids indicated that the metabolites were probably dicarboxylic acids. This conclusion was substantiated by the observation of two strong absorption peaks adjacent to each other in the carbonyl region of an infrared
497
1.0 0.9 6 0.8 C o 0.7
0
0.6
0.5 o 0.4 1f 0.3 z 0.2 0.1 0.0 °
0
5 10 15 20 25 30 35 Time (h)
FIG. 1. Normalized concentrations of carbon dioxide (O) and the 6.4-min (8) toluene metabolite (V) during batch growth of strain Ti on 1 mM toluene. Since the concentrations are normalized, their magnitudes cannot be compared.
spectrum of the toluene metabolite. Further detailed GC-MS analyses demonstrated the existence of two metabolites from toluene. The parent ions of the methyl esters of these metabolites were 234 and 236 Da. The difference of 2 Da suggested that the difference between them was an additional carbon-carbon bond. Chemical ionization MS indicated that the 234- and 236-Da ions were indeed parent ions and not fragments of higher-molecular-weight compounds. From these collective results, it was hypothesized that the metabolites were products of a four-carbon dicarboxylic acid addition to the methyl group of toluene. The 234-Da metabolite was hypothesized to be benzylfumaric or benzylmaleic acid, and the 236-Da metabolite was hypothesized to be benzylsuccinic acid. These compounds were chemically synthesized for comparison. The first metabolite was identical to synthetic benzylmaleic acid, as shown in Fig. 2. The underivatized benzylmaleic acid also coeluted on HPLC with the first metabolite, which further supports the identification. However, as further elaborated in the Discussion, from a biological point of view, this toluene metabolite is most likely benzylfumaric acid (i.e., trans) and not benzylmaleic acid (i.e., cis). Our analyses could not distinguish between the two since mass spectra of cis and trans isomers are quite similar (14), and the chromatographic conditions were not able to resolve cis and trans isomers. On the other hand, the differences in relative abundance between Fig. 2A and B are consistent with differences observed for cis and trans molecules. The appearance of a 91-Da fragment ruled out benzylidenesuccinic acid (benzylitaconic acid) as a possible metabolite; it is unlikely that the mass spectrum of this compound would contain a fragment at 91 Da. The 91-Da fragment is normally associated with benzyl or tropylium ion (16). The second metabolite observed by GC-MS was found to be identical to synthetic benzylsuccinic acid, as shown in Fig. 3. This is also supported by the observation that the underivatized benzylsuccinic acid coeluted with a small peak on HPLC that eluted prior to benzylmaleic acid. Benzylsuccinic acid absorption is weak at 254 nm; thus, the small peak on HPLC seen previously was not deemed to be significant and was not reported (8).
498
APPL. ENVIRON. MICROBIOL.
EVANS ET AL.
A
115
A
176
117
4)
43
U a
131
a
0
.0 cx a
91
43
163
I
43
a
91
.1.1,. X, .S ,., ,. 1 i .4.J8 60 c
~~~~~~174202 ,v.
a
23
2iii 205236
103
l, . ,1,. . ,..,.
80 100 120 140 160 180 200 220 240 m/e
60
n 40
80
a) c
100 120 140 160 180 200 220 24C m/e
>
M a
B
176
117
43
91
131
a C
.0
4)
a
163
43
a 43
103
80 100 120 140 160 180 200 220 240 m/e FIG. 2. Mass spectra of the dimethyl esters of (A) the first metabolite recovered from the culture fluid after growth on toluene and (B) synthetic benzylmaleic acid [C6H5CH2C(CO2)CH(CO2H)]. 60
40
60
80
204
236'
100 120 140 160 180 200 220 240
m/e FIG. 3. Mass spectra of the dimethyl esters of (A) the second metabolite recovered from the culture fluid after growth on toluene and (B) synthetic benzylsuccinic acid [C6H5CH2CH(CO2)CH2 (CO2H)].
Metabolites of o-xylene. The mass spectra of the methyl esters of the two metabolites of o-xylene transformation are shown in Fig. 4 and 5. Figure 4 demonstrates that the peaks at 115, 129, 216, and 248 Da correspond to the respective peaks in Fig. 2 at 91, 115, 202, and 234 Da plus 14 Da. Since +14 Da is equivalent to a methyl group, the specific difference between toluene and o-xylene, the data in Fig. 4 suggest a structure of (2-methylbenzyl)-fumaric acid. The presence of a peak at 184 Da, which corresponds to 174 Da in Fig. 2 plus 10 Da, may be due to differences in fragmentation patterns between benzylfumaric acid and (2-methylbenzyl)-fumaric acid. (2-Methylbenzyl)-fumaric acid was previously referred to as the 9.1-min metabolite (8). All of the major peaks in Fig. 5 are equivalent to the respective peaks in Fig. 3 plus 14 Da. This metabolite is most likely (2-methylbenzyl)-succinic acid. Quantitation of toluene metabolites. The benzylsuccinic acid and benzylfumaric acid metabolites were quantified in a culture of strain Ti grown on 1 mM toluene. The measured concentrations of benzylsuccinic acid and benzylfumaric acid were 0.03 and 0.14 mM, respectively. The concentration of benzylfumaric acid was determined by using synthetic benzylmaleic acid as a standard. This concentration may be an overestimate since the extinction coefficients of cis and trans aromatic acids can be different by a factor of 2, an example being cis- and trans-cinnamic acid (20). Thus, the concentration of benzylfumaric acid may be as low as 0.07 mM. With this in mind, the sum of the concentrations of benzylsuccinic and benzylfumaric acids is estimated to be approximately 0.10 to 0.17 mM.
Transformation of phenylalanine. Phenylalanine transformation by strain Ti was assessed because a strain of Chromobacterium violaceum has been found to transform phenylalanine to benzylfumaric and benzylsuccinic acids (17). L-Phenylalanine (1 mM) was tested for utilization as a carbon source for growth of strain Ti and for transformation to benzylsuccinic acid or benzylfumaric acid. L-Phenylalanine did support the growth of strain Ti. However, benzylsuccinic and benzylfumaric acids were not detected in the culture fluid by HPLC (data not shown).
129 4) ~0 C
.0
184
216
43
115 a
40
60
80 100 120 140 160 180 200 220 240 260
m/e FIG. 4. Mass spectra of the dimethyl ester of (2-methylbenzyl)fumaric acid [C6H5(CH3)CH2C(CO2)CH(CO2H)] recovered from the culture fluid after growth on toluene and o-xylene.
VOL. 58, 1992
~~~METABOLITES
TABLE 2.
145
105
(2-methylbenzyl)-fumaric
Production of
acid in the
pLM o-xylene
presence of succinic and oxalacetic acids and 100
131
0,
499
OF ANAEROBIC TOLUENE DEGRADATION
190 Initial
159
0
.0
9117
a)
(2-Methylbenzyl)-
(mM)
fumaric acid
115
-o
concn
Condition
C
0
0,
Succinate
Oxalacetate
area
units (SD)
1
0
0
12,800 (3,350)
2
2
0
309,000 (47,900)
3
0
2
158,000 (10,600)
4
2
2
633,000 (75,100)
succinic
acid-mediated
25 40
80
60
100 120 140 160 180 200 220 240 260
enhanced
m/e dimethyl ester of (2-methylbenzyl)[C6H5(CH3)CH2CH(C02)CH2(CO2H)] recovered from the culture fluid after growth on toluene and o-xylene. FIG.
Mass spectra of the
5.
transformation
of o-xy-
lene, the production of (2-methylbenzyl)-fumaric acid in the
succinic acid
presence of succinic acid and oxalacetic acid
be greater than the
together should
of their individual effects. Table 2
sum
shows that the effects of succinic and oxalacetic acids
on
(2-methylbenzyl)-fumaric acid formation are synergistic since the (2-methylbenzyl)-fumaric acid area under condition Role of
succini'c
acid in metabolite formation. Table 1 shows
(2-methylbenzyl)-fumaric acid from 100 RM o-xylene after growth on a series of aromatic compounds mM) and nonaromatic compounds (2 mM). The normalized the formation of
concentrations of this metabolite
are
tabulated relative to its
production after growth on pyruvic acid. No enhancement of (2-methylbenzyl)-fumaric acid production was observed relative to pyruvic acid during growth on fumaric acid, L-malic acid, and aromatic compounds other than toluene. In fact, less (2-methylbenzyl)-fumaric acid was formed when the organism was grown on aromatic acids relative to pyruvate. Growth on oxalacetic acid led to a slight enhancement of (2-methylbenzyl)-fumaric acid production, while growth on toluene
or
succinic acid gave the greatest enhancements.
Transformation of
o-xylene' in the presence of pyruvate
was
4 is greater than the
sum
of the
synergistic effect scribed by Box et al. (3),
3. This
is
areas
under conditions 2 and
statistically significant;
de-
as
the interaction between succinic
was 89,400 (area units) and the standard 20,100. This result suggests that, when oxalacetic
and oxalacetic acids error was
and succinic acids
both present, oxalacetic acid inhibits
are
the oxidation of succinic acid; thus,
succinic acid is available
as
a
of
amount
greater
substrate for
a
o-xylene
trans-
formation relative to when oxalacetic acid is absent. Fur-
thermore,
the
results
suggest
that
acid
oxalacetic
is
an
unlikely substrate for o-xylene transformation since neither fumaric nor L-malic acid enhanced o-xylene transformation. Oxalacetic acid is easily formed from fumaric acid or L-malic acid via the Krebs cycle. To determine whether succinic acid
was
involved in the
previously (8). However, growth on toluene greatly enhanced the transformation of o-xylene relative to pyruvate. We hypothesized that succinic acid was primarily responsible for o-xylene transformation and the effect observed with oxalacetic acid (Table 1) is attributable to inhibition of succinate dehydrogenase by oxalacetic acid (13), thus causing accumulation of succinic acid. This greater amount of succinic acid would then be available for o-xylene transformation. To assess this hypothesis, a two-level factorial design was implemented to quantify the degree of interaction
transformation of benzaldehyde rather than toluene, strain Ti was grown on succinic acid (2 mM) in the presence of
between succinic and oxalacetic acids with respect to their
detection limits for these assays, the maximal amounts Of
not observed
effects
on
as was
the transformation
of
reported (8),
o-xylene. If oxalacetate
benzaldehyde (0.5 mM) and formation of benzylfumaric acid was a ssessed. No benzylfumaric acid was detected in the culture fluid during growth. Role of oxygen. To assess the possible role of molecular oxygen in toluene oxidation by strain Ti, it was grown on 1 mM toluene in 150 ml of medium in
liquid
were
(2-methylbenzyl)-fumaric acid produced by o-xylene transformation during growth on selected substrates Normalized concentration of
were
below detecthe
employed. Using
02 headspace and liquid were calculated to be 0.16 and ~xmol, respectively. A mechanism of toluene degradation
require
0.5
amount
Of
MMOI
an
initial monooxygenase reaction would
02 per 02 required is 75 Of
mmol of toluene. j±mol.
The total
Thus, the amount
of
only 6.5% of that required for an attack and is stoichiometrically insuf-
oxygen available makes up
initial monooxygenase Normalized
Growth substrate
concn
(SD)
Pyruvate ...............................1.00 (0.06) Succinate...............................6.39 (0.18) Fumarate...............................1.19 (0.16) L-Malate ...............................1. 25 (0.04M) Oxalacetate .............................1.94 (0.37) Toluene................................4.32 (0.55) Benzoate ...............................0.094 (0.002) p-Cresol................................0.39 (0.15) Phenylacetate ............................0.30 (0.08)
Phenylpropionate..........................0.47
(0.002)
m-Toluate...............................0.076 (0.11)
a"Concentrations
bottle,
in the
that would involve 1.
were
serum
headspace and in the
measured at the outset. Both
tion limits for the assays that
4.7
TABLE
160-ml
a
and the concentrations of oxygen in the
are
normalized with respect to the data for
pyruvic acid.
ficient to support such
a
mechanism.
DISCUSSION
Anaerobic transformation of toluene and o-xylene shown
to
involve
the
addition
of
identical
was
four-carbon
methyl group of these aromatic hydrocarbons. resulting metabolites, which were not further metabolized, were identified by mass spectra to be benzylsuccinic and benzylfumaric acids from toluene and (2-methylbenzyl)succinic and (2-methylbenzyl)-fumaric acids from o-xylene. The unsaturated metabolites are most likely the trans (e.g., benzylfumaric acid) rather than the cis isomer (e.g., benzyl-
groups to the
The
500
APPL. ENVIRON. MICROBIOL.
EVANS ET AL.
0
CSCoA I
0 11 HO2CCH2CH2CSCoA
H20
(~~-CH2CHCH2 CO2H
CO2H
CO2H
CH2C 1H2
i-X
HSCoA
L'H2L-=H H2 CO2 H
CO2H
IV
III
11
H2 ~-CH3 0
CH3CSCoA
0
H20
+
HSCoA
0 CoCoA -v Ring Cleavage
CH2CH2CSCoA H2
V
2H2 + CH3CSCoA
VI
FIG. 6. Proposed pathway of toluene transformation and mineralization (initial steps) under anaerobic, denitrifying conditions by strain Ti. The upper pathway illustrates transformation and the lower pathway illustrates mineralization. Structures III and IV have been identified.
maleic acid) which served as the structural standard. Benzylfumaric acid was concluded to have formed from benzylsuccinic acid, since succinic acid was shown to be involved in the transformation rather than fumaric, L-malic, and oxalacetic acids. The biological oxidation of the saturated succinic acid analog is likely to be similar to that for succinic acid. By analogy, benzylfumaric acid should be the oxidation product of benzylsuccinic acid since fumaric acid is the oxidation product of succinic acid in the Krebs cycle. Approximately 10 to 17% of the carbon in toluene was transformed to benzylsuccinic and benzylfumaric acids. Previously reported mass balances on toluene degradation by strain Ti demonstrated that 51% was mineralized to carbon dioxide and 29% was assimilated as cellular carbon (8); the remaining 20%, therefore, can be largely accounted for by the formation of benzylsuccinic and benzylfumaric acids. Benzylsuccinic and benzylfumaric acids were shown also to be inhibitors of carboxypeptidase A (17). The oxidation of toluene, a toxic industrial solvent, by strain Ti resulted in the production of carbon dioxide, biomass, and two metabolites that are potentially toxic themselves. Therefore, a means of further degradation of these metabolites, by other bacteria, for instance, or prevention of their accumulation may be important with respect to the development of processes for bioremediation of toluene-containing wastes. Practical applications of biodegradation must ensure that the pollutant of concern is not transformed to a metabolite that is more toxic than the pollutant itself. Benzylsuccinic and benzylfumaric acids have been observed as intermediates in the biosynthesis of Arphamenine A, a carboxypeptidase A inhibitor, by C. violaceum BMG361-CF4 (17). The proposed biosynthesis pathway of Arphamenine A involves a hydrolytic deamination of L-phenylalanine to form ,-phenylpyruvic acid, which is then attacked by acetyl-coenzyme A (CoA) to form benzylmalic acid (17). This is then reduced sequentially to form benzylfumaric acid and benzylsuccinic acid. This pathway is not utilized by strain Ti since it did not transform L-phenylala-
nine under denitrifying conditions to either benzylsuccinic acid or benzylfumaric acid. On the basis of the identification of the metabolites and the role of succinic acid in o-xylene transformation, the upper pathway in Fig. 6 illustrates a likely route for the transformation of toluene to benzylsuccinic and benzylfumaric acids. The methyl group of toluene (structure I) is slightly electrophilic due to the electron-withdrawing nature of the benzene ring. The methyl group is thus suitable for attack by succinyl-CoA, a strong nucleophile, to form benzylsuccinylCoA (structure II). Succinyl-CoA, a Krebs cycle intermediate, can be replenished as toluene degradation proceeds. Also, succinyl-CoA is readily formed from succinic acid and the latter was shown to enhance o-xylene transformation (Table 1). Benzylsuccinyl-CoA (structure II) is subsequently hydrolyzed to form benzylsuccinic acid (structure III), which is then oxidized to form benzylfumaric acid (structure IV). The transformation of o-xylene, which yielded analogous metabolites with an additional methyl group, would follow an identical pathway. Anaerobic degradation of toluene has also been proposed to proceed through benzyl alcohol (10, 12, 15), which can be subsequently oxidized to benzaldehyde. Nucleophilic attack of the benzaldehyde by succinyl-CoA to form (a-hydroxybenzyl)-succinic acid could in theory also yield a four-carbon addition; however, neither this compound nor its dehydration product, benzylidenesuccinic acid (benzylitaconic acid), was detected. Furthermore, benzylfumaric acid was not formed by strain Ti during growth on succinic acid in the presence of benzaldehyde. On the basis of the transformation pathway that is responsible for the formation of the dead-end metabolites, the initial steps of a simultaneously operating mineralization pathway are proposed and illustrated in the lower pathway in Fig. 6. The first step involves the nucleophilic attack of the methyl group of toluene (structure I) by acetyl-CoA to form phenylpropionyl-CoA (structure V). Phenylpropionyl-CoA then, via ,-oxidation, yields benzoyl-CoA (structure VI), a known intermediate in the anaerobic degradation of benzoic acid (11), which is then subject to ring cleavage and oxida-
VOL. 58, 1992
METABOLITES OF ANAEROBIC TOLUENE DEGRADATION
tion (21). Dangel et al. (5) and Schnell and Schink (18) have presented evidence of benzoyl-CoA being a central intermediate in the anaerobic degradation of a number of aromatic compounds. Although the attack of a methyl group by acetyl-CoA or succinyl-CoA has not been previously reported, the slightly electrophilic nature of the methyl group of toluene in combination with the highly nucleophilic nature of acetyl-CoA makes the proposed pathway plausible. This proposed pathway in which the first intermediate is a CoA ester provides an explanation of why intermediates of anaerobic toluene oxidation have not been observed in denitrifying and iron-reducing systems (7, 8, 12, 15). CoA esters remain intracellular because of their size. In a methanogenic enrichment culture that was originally grown on ferulic acid, and subsequently on a mixture of toluene and methanol (10), possible toluene intermediates were observed to include p-cresol, benzyl alcohol, methylcyclohexane, and o-cresol. However, it is not known whether denitrifying and methanogenic systems transform toluene similarly. In this proposed pathway (Fig. 6), while o-xylene is not mineralized, its transformation may be the result of low enzyme specificity. The isotope dilution data of Kuhn et al. (12) are consistent with this proposed pathway. Benzoate as a carrier was found to contain 14C provided by toluene in denitrifying aquifer columns. p-Hydroxybenzoate, p-cresol, and benzaldehyde as carriers, on the other hand, contained little or no "4C. Of these four compounds, only benzoate is an intermediate in the pathway proposed in Fig. 6. Recent data of Altenschmidt and Fuchs (1) suggest that benzoyl-CoA is an intermediate in toluene degradation, which is consistent with the pathway proposed in Fig. 6. Furthermore, the preliminary enzyme activity studies of Seyfried (19) indicate that toluene- and p-cresol-methyl hydroxylase activities were not inducible or present in cell extracts of strain K172 grown anaerobically on toluene. These data are not consistent with the previously proposed pathways of anaerobic toluene degradation, namely, methyl group hydroxylation and para-hydroxylation (10, 12, 15). Strain Ti is capable of anaerobically transforming toluene and o-xylene via addition of a four-carbon dicarboxylic acid to the methyl group. This pathway represents a novel biochemical mechanism for transformation of aromatic molecules. On the basis of the formation of the metabolites, we propose that toluene degradation to ring cleavage products proceeds by an analogous mechanism involving attack of the methyl group by acetyl-CoA. Substantiation of this pathway and further work are under way. ACKNOWLEDGMENTS We appreciate the help of Roger A. Lalancette at the Department of Chemistry, Rutgers University, Newark, N.J., for the initial GC-MS investigations, Mark Walters and Yiu Chong Leung at the Department of Chemistry, New York University, New York, N.Y., for the infrared spectroscopy, and Steven L. Cohen at the Mass Spectrometry Resource, Rockefeller University, New York, N.Y., for the chemical ionization MS. This research was partially supported by NIEHS ES04895, EPA R-816483, the New York State Center for Hazardous Waste Management, and the New Jersey Hazardous Substances Management Research Center. REFERENCES 1. Altenschmidt, U., and G. Fuchs. 1991. Anaerobic degradation of toluene in denitrifying Pseudomonas sp.: indication for toluene
2. 3. 4.
5.
6. 7.
8. 9.
10. 11.
12.
13. 14.
15. 16. 17.
18. 19.
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