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Biochemistry |
Mikrobiologie, Institut für Biologie II, Universität Freiburg, Schänzlestr. 1, 79104 Freiburg, Germany1
Author for correspondence: Johann Heider. Tel: +49 761 203 2774. Fax: +49 761 203 2626. e-mail: heiderj{at}ruf.uni-freiburg.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Keywords: anaerobic toluene catabolism, benzylsuccinate, CoA-transferase, stereochemistry, ß-oxidation
Abbreviations: DCPIP, dichlorophenolindophenol; PMS, phenazine methosulfate
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). The simplest alkylbenzene, toluene, appears to be one of the most readily degradable hydrocarbons under anaerobic conditions: it is preferentially catabolized from crude oil in anaerobic enrichment cultures (Rabus et al., 1996 , 1999 ). The pathway involved in anaerobic toluene degradation has been identified in toluene-grown denitrifying and sulfate-reducing bacteria (Biegert et al., 1996 ; Beller & Spormann 1997a , b , 1998 ; Rabus & Heider, 1998 ). The first step of anaerobic toluene catabolism is the addition of the methyl group of toluene to the double bond of fumarate to yield the first intermediate, benzylsuccinate (Biegert et al., 1996 ; Fig. 1). This reaction is unprecedented in biochemistry and is catalysed by benzylsuccinate synthase, a novel glycyl-radical enzyme (Leuthner et al., 1998 ). The reaction mechanism of benzylsuccinate synthase most probably proceeds analogously to chemical free-radical addition reactions in chemical synthesis (‘Giese-reactions’), which proceed under relatively mild conditions and therefore are particularly useful with substituted or chiral reactants (Giese & Meister, 1977 ). Further catabolism of benzylsuccinate proceeds via oxidation to benzoyl-CoA, the common and the last aromatic intermediate in the anaerobic mineralization pathways of most aromatic compounds. This pathway has been proposed to proceed via ß-oxidation, in analogy to mineralization of fatty acids (Biegert et al., 1996 ; Fig. 1), but no detailed information has been obtained so far.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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) was grown at 30 °C under denitrifying conditions in mineral salt medium with toluene or benzoate as sole substrate, as described previously (Tschech & Fuchs, 1987 ; Biegert et al., 1996 ). Cultures were harvested by flow-through centrifugation in the exponential growth phase; cells were stored frozen in liquid N2. Frozen cells were suspended in the same volume of basal buffer (10 mM triethanolamine hydrochloride/NaOH pH 7·5; 10%, v/v, glycerol) containing 0·05 mg DNase I (g cells)-1. All procedures were performed at 4 °C under N2 or N2/H2 (95:5, v/v) atmosphere. Cells were broken by a passage through a French pressure cell at 137 MPa; the lysate was centrifuged at 100000 g for 60 min. The supernatant was kept frozen at -70 °C until use. Membrane proteins were prepared from washed membranes by differential centrifugation steps, as follows. The cell lysate was centrifuged for 1 h at 20000 g to remove cell debris and unbroken cells, then the supernatant was centrifuged at 100000 g for 1 h. The resulting pellet was washed with the original volume of basal buffer and resuspended in basal buffer containing 0·5% (w/v) Triton X-100. The suspension was centrifuged for 1 h at 100000 g, and the supernatant containing solubilized membrane proteins was stored at -70 °C.
Chemical preparations
Synthesis of succinyl-CoA and benzylsuccinyl-CoA.
Succinyl-CoA and benzylsuccinyl-CoA were synthesized from the internal anhydrides. Benzylsuccinic anhydride is not commercially available and was synthesized from benzylsuccinate and acetic anhydride, as follows. Benzylsuccinate (384 µmol) was dissolved in 800 µl acetic acid at 80 °C, and an equimolar amount of acetic anhydride was added. The assay was then incubated at 120 °C, until the solvent acetic acid and acetic acid formed from acetic anhydride were completely evaporated. Quantitative formation of yellow-brown benzylsuccinic anhydride was obtained. The preparation of CoA-thioesters from the anhydrides was as described by Schachter & Taggart (1976) ; the compounds were freeze-dried and stored at -20 °C. 13C-NMR analysis of the synthesized benzylsuccinyl-CoA revealed that the preparation consisted of the two isomeric mono-CoA thioesters (60% 2-carboxymethyl-3-phenylpropionyl-CoA and 40% 3-carboxy-4-phenylbutyryl-CoA; data not shown). The two chemically synthesized isomers co-migrated in HPLC analysis under the conditions described, as did biologically formed benzylsuccinyl-CoA.
Enantiomer separation of racemic benzylsuccinate.
The (R)-(+)- and (S)-(-)-enantiomers of benzylsuccinate were prepared from the racemate according to Byers & Wolfenden (1973) by stereoselective precipitation with (S)- and (R)-1-phenylethylamine, respectively. The (R)- and (S)-benzylsuccinate fractions obtained were checked for enantiomer purity by HPLC analysis of the 1-phenylethylamine diamides, as described below [values obtained: 99% for (R)-(+)-benzylsuccinate, 90% for (S)-(-)-benzylsuccinate].
Derivatizion of benzylsuccinate with 1-phenylethylamine and HPLC analysis.
Benzylsuccinate (9·6 µmol) was suspended in 50 µl dimethylformamide. To this assay, 76·8 µmol ethyldiisopropylamine and 48 µmol O-(7-azobenzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate were added. After 1 min, 48 µmol (S)-(-)-1-phenylethylamine was added. The mixture was incubated for 1 h at room temperature. To determine the stereospecifity of benzylsuccinate synthase, ether-extracted [phenyl-14C]benzylsuccinate (220 MBq) from an enzymic conversion assay with [phenyl-14C]toluene and fumarate was mixed with racemic benzylsuccinate and derivatized to the diamide. HPLC analysis was performed at room temperature with UV detection at 214 nm and simultaneous radioactivity detection by a flow-through detector (Ramona, Raytest). The assay was diluted 1:100 with dimethylformamide, and 15 µl of the resulting solution was applied to a LiChrospher 100 RPC-18 column. The column was eluted with a gradient of 30–74% acetonitrile in 0·1% (v/v) trifluoracetate over 30 min with a flow rate of 1 ml min-1. If necessary, the peak fractions were collected and dried in a vacuum concentrator.
Enzymic synthesis of [phenyl–14C]benzylsuccinate.
Cell extract (2 ml, containing 50 mg protein ml-1) of T. aromatica cells grown anaerobically on toluene was incubated anaerobically at 30 °C with 200 µM [phenyl-14C]toluene (ARC/Biotrend; specific radioactivity 2 GBq mmol-1) and 10 mM fumarate. The reaction was stopped after 30 min by adding sulfuric acid (5%, w/v, final concentration), and 14C-labelled benzylsuccinic acid was extracted with diethyl ether from the assays, as described by Biegert et al. (1996) .
Enzymic assays and analysis of the substrates and the products
CoA-transferase.
In a photometric enzyme assay, succinyl-CoA:benzylsuccinate CoA-transferase activity was coupled to the reaction of endogenous succinate dehydrogenase of T. aromatica, which was supplied by a solubilized membrane preparation. The assay was performed at 30 °C in 100 mM Tris/HCl pH 7·5 under anaerobic conditions. The reaction mixture contained 0·1 mM dichlorophenolindophenol (DCPIP), 0·05 mM phenazine methosulfate (PMS), 0·03 U succinate dehydrogenase (equivalent to 40 µg solubilized membrane protein), 10–50 µl 100000 g supernatant (0·5–2·3 mg protein) and 50 µM succinyl-CoA. The reaction was started by adding benzylsuccinate to a final concentration of 600 µM. The reduction of DCPIP was followed at 600 nm [
=22000 M-1 cm-1 (Dawson et al., 1986 )]. The assay required strictly anaerobic conditions because the reoxidation rate of the electron acceptors by traces of introduced oxygen was faster than the enzyme activity to be measured.
Enzyme assays for HPLC analysis contained 0·9 ml 100 mM Tris/HCl pH 7·5, 600 µM benzylsuccinate and 30 µl dialysed 100000 g supernatant (45 mg protein ml-1). The reaction was started by adding succinyl-CoA (200 µM final concentration) and incubated at 30 °C under aerobic or anaerobic conditions. Samples of 0·3–1 ml were taken at different times. The reaction was stopped by adding formic acid (10%, v/v, final concentration) and the precipitated proteins were removed by centrifugation (20000 g, 4 °C, 10 min). The reverse reaction was performed as mentioned above, except that benzylsuccinate in the assay mixture was substituted by 2 mM succinate and the reaction was started with 600 µM benzylsuccinyl-CoA. The supernatants were analysed by HPLC at room temperature with UV detection at 260 nm. Samples of 50 µl or CoA-thioester standards were applied on the column. The following systems were used: (a) a LiChrospher 100 RPC-18 (5 µm) column (Merck) was eluted with a gradient of 1–20% (v/v) acetonitrile in 50 mM potassium phosphate pH 6·8 over 25 min with a flow rate of 1 ml min-1; (b) a Poros R1 column (10 µm, Perseptive Biosystems) was eluted with a gradient of 0–50% (v/v) acetonitrile in 0·1% (v/v) trifluoroacetate over 20 min with a flow rate of 5 ml min-1. If necessary, the eluates were collected in fractions of 1 ml and freeze-dried.
Oxidation of benzylsuccinyl-CoA to benzoyl-CoA.
The assay mixture contained 0·9 ml 100 mM Tris/HCl pH 7·5, 2 mM NaNO3 or 1 mM PMS and 50–100 µl 100000 g supernatant (equivalent to 2–4 mg protein). The reaction was started by adding benzylsuccinyl-CoA (1 mM final concentration). Sampling and HPLC analysis were performed as described above. The assay was varied by using nitrate-reductase-depleted cell extract or by addition of supplements, e.g. 0·5 mM NAD or NADP. Details are described in Results and Discussion.
Nitrate reductase.
Nitrate reductase was measured under anaerobic conditions at 30 °C using reduced methyl viologen as electron donor. The assay mixture contained 1 mM methyl viologen, 0·5 mM dithionite, 100 mM Tris/HCl pH 7·5, and 10 µl of a 1:10 diluted 100000 g supernatant or solubilized membrane preparation. The reaction was started by adding sodium nitrate (final concentration 2 mM), and the oxidation of methyl viologen was followed at 710 nm [=2400 mM-1 cm-1 (Boll & Fuchs, 1995 )].
Other methods.
Dialysis of 100000 g supernatants was performed in dialysis tubes (Visking; 12–14 kDa exclusion limit) against 2 x 1 litre of basal buffer. Some experiments were performed with 100000 g supernatants (10 ml portions), which were passaged over a DEAE-Sepharose Column (20 ml; Amersham-Pharmacia) in basal buffer (10 mM triethanolamine hydrochloride/NaOH pH 7·5; 10%, v/v, glycerol; flow rate 1 ml min-1). After elution of the flow-through fractions, the bound proteins were eluted with a step gradient of basal buffer containing 500 mM NaCl, collected and directly used for the assays. Protein was determined according to Bradford (1976) using bovine serum albumin as standard. UV/visible spectra of the CoA-thioesters were recorded by a Perkin Elmer UV/Vis spectrometer Lambda 2S. Electrospray-mass spectrometry of benzylsuccinate derivatives was performed with a Finnigan model TSQ 700 tandem quadrupole mass spectrometer. Freeze-dried HPLC fractions of the compounds obtained with solvent system (b) were dissolved in 0·2% (v/v) trifluoroacetic acid, 25% (v/v) acetonitrile and injected into the mass spectrometer.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). Therefore, we assayed for a benzylsuccinate CoA-transferase activity using succinyl-CoA, the most common CoA donor. The benzylsuccinate-dependent conversion of succinyl-CoA to free succinate was monitored in a coupled enzyme assay with succinate dehydrogenase of T. aromatica as auxiliary enzyme. Succinate dehydrogenase activity was assayed with the artificial electron acceptors PMS and DCPIP. The enzyme was exclusively localized in washed membrane fractions, and a specific activity of 130 nmol succinate oxidized min-1 (mg protein)-1 was obtained in supernatants of Triton X-100 extracted membranes (Table 1). This supernatant (40 µg protein per test) was used as auxiliary enzyme source for CoA-transferase assays.
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). The measured specific activity of 15 nmol min-1 (mg protein)-1 is about 40% of that needed to explain the observed growth rate of T. aromatica on toluene (0·08 h-1). This discrepancy may be explained by the artificial assay conditions or by side reactions of the coupled enzyme assay, since a direct activity assay yielded a value corresponding to 80% of the activity required for growth (see below). The enzyme was relatively stable against oxygen: 94% activity was recovered in extracts that were exposed to air for 6 h. The complete absence of enzyme activity in control cells of T. aromatica grown on benzoate suggests that the CoA-transferase is indeed involved in the toluene catabolic pathway. Succinyl-CoA:benzylsuccinate CoA-transferase probably corresponds to one of several strongly substrate-induced proteins that have been observed in toluene-grown T. aromatica cells (Leuthner et al., 1998 ). Specific activities of the reaction in cell extracts are close to the minimum required for growth, indicating that this is a second rate-limiting step of anaerobic toluene catabolism, in addition to the initial reaction of benzylsuccinate synthase. This would explain the accumulation of benzylsuccinate as the only detectable intermediate in previous experiments on the degradation of 14C-labelled toluene in cell extracts (Biegert et al., 1996 ). Further support for the involvement of a succinyl-CoA-dependent CoA-transferase in the pathway can be derived from experiments with permeabilized cells of another toluene-degrading denitrifying bacterium, which showed enhanced rates of benzylsuccinate oxidation to a phenylitaconate derivative in the presence of succinyl-CoA (Beller & Spormann, 1998 ).
Product analysis of the succinyl-CoA:benzylsuccinate CoA-transferase reaction and reversibility of the enzyme
The products formed from benzylsuccinate and succinyl-CoA were analysed by HPLC. Succinyl-CoA disappeared at a rate of 49±21 nmol min-1 (mg protein)-1 in extracts of toluene-grown cells, whereas a new CoA-thioester (judged from the UV spectrum of the compound) was formed at a rate of 31±15 nmol min-1 (mg protein)-1 (Fig. 2a). This CoA-thioester was identified as benzylsuccinyl-CoA by electrospray mass spectrometry, yielding a mass of 958·6 Da (expected mass: 958·2 Da). Control extracts of benzoate-grown cells did not catalyse benzylsuccinyl-CoA formation. In order to assay for the reversibility of the succinyl-CoA:benzylsuccinate CoA-transferase reaction, products formed from benzylsuccinyl-CoA and succinate were assayed by HPLC. Benzylsuccinyl-CoA-dependent formation of succinyl-CoA was recorded with extracts of toluene-grown cells (Fig. 2b), but not with benzoate-grown cells (not shown). The calculated initial rates of disappearance of benzylsuccinyl-CoA and formation of succinyl-CoA were very similar and were about twice as fast as the forward direction [73±21 and 70±5 nmol min-1 (mg protein)-1, respectively]. After 1–2 min, the reactions were affected by the presence of thioesterase in the extracts, converting the CoA-thioesters to the free acids. No formation of benzylsuccinyl-CoA was detected when succinyl-CoA was substituted by acetyl-CoA, malonyl-CoA or glutaryl-CoA (data not shown).
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). The rate of benzoyl-CoA formation at the expense of nitrate was 4±2 nmol min-1 (mg protein)-1 in untreated extracts. It increased about fourfold in tests supplemented with 1 mM PMS and 0·5 mM NAD (Table 2). The endogenous nitrate reductase activity in the extracts was removed by passage of the extracts through a DEAE-Sepharose column and elution of the bound protein by a step gradient containing 500 mM NaCl in basal buffer. Nitrate reductase activity was found in the flow-through fractions and the benzylsuccinyl-CoA-oxidizing enzymes were recovered in the eluate with a yield of 92% and an enrichment factor of 1·8. No coupling of benzylsuccinyl-CoA oxidation to reduction of nitrate was retained after this step, even if the two fractions were combined (data not shown). The eluate fractions catalysed oxidation of benzylsuccinyl-CoA to benzoyl-CoA if 1 mM PMS was added as electron acceptor, but not with NAD as sole electron acceptor (Table 2). PMS-dependent benzylsuccinyl-CoA oxidation was accelerated sixfold by addition of 0·5 mM NAD to the assay, whereas NADP did not stimulate the reaction (Table 2). The benzylsuccinyl-CoA oxidation rates obtained in NAD-supplemented experiments (in relation to untreated extract) correspond to 40–50% of the rates needed to explain the growth rate of the bacteria on toluene. Since the measured substrate conversion actually represents a series of four individual reactions, this may be regarded as significant match to the predicted pathway. Some implications can also be drawn from the observed electron acceptor dependence of benzylsuccinyl-CoA oxidation. The exact redox potential for oxidation of benzylsuccinyl-CoA to phenylitaconyl-CoA is unknown, but it should be in the range of the fumarate/succinate (+33 mV) or the crotonate/butyrate couple (-24 mV). Therefore, benzylsuccinyl-CoA dehydrogenase should be able to reduce PMS, but not NAD. This probably accounts for the dependence of the overall reaction on a high-potential electron acceptor, e.g. PMS or nitrate. The next oxidation step of the postulated pathway (enzyme 5 in Fig. 1) is the oxidation of a secondary alcohol to a ketone, which occurs at a sufficiently low redox potential to be coupled to reduction of NAD(P). This reaction may account for the observed stimulation of benzylsuccinyl-CoA oxidation to benzoyl-CoA by NAD supplementation.
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). As shown by HPLC runs with diamides from purified benzylsuccinate enantiomers, the earlier-eluting compound is derived from (R)-(+)-, the latter from (S)-(-)-benzylsuccinate (data not shown). This assay was employed to examine the stereochemistry of benzylsuccinate that had been produced enzymically from 14C-labelled toluene and fumarate in cell extracts of toluene-grown T. aromatica (Biegert et al., 1996 ). As shown in Fig. 3, the 14C-labelled diamide of benzylsuccinate and (S)-1-phenylethylamine co-migrated exclusively with the diamide of the (R)-(+)-enantiomer. If any (S)-(-)-benzylsuccinate was formed by the enzyme, it was below the detection limit of the radioactivity monitor. This corresponds to a minimum enantiomer purity of 99% for (R)-(+)-benzylsuccinate. Stereospecific formation of (R)-(+)-benzylsuccinate was also recently reported in a different toluene-degrading denitrifying bacterium (Beller & Spormann, 1998 ). Thus, the reaction of benzylsuccinate synthase proceeds highly stereospecifically, despite the probable involvement of radical intermediates in the reaction mechanism (Heider et al., 1999 ).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Beller, H. R. & Spormann, A. M. (1997a). Anaerobic activation of toluene and o-xylene by addition to fumarate in denitrifying strain T. J Bacteriol 179, 670-676.
Beller, H. R. & Spormann, A. M. (1997b). Benzylsuccinate formation as a means of anaerobic toluene activation by sulfate-reducing strain PRTOL1. Appl Environ Microbiol 63, 3729-3731.[Abstract]
Beller, H. R. & Spormann, A. M. (1998). Analysis of the novel benzylsuccinate synthase reaction for anaerobic toluene activation based on structural studies of the product. J Bacteriol 180, 5454-5457.
Biegert, T., Fuchs, G. & Heider, J. (1996). Evidence that oxidation of toluene in the denitrifying bacterium Thauera aromatica is initiated by formation of benzylsuccinate from toluene and fumarate. Eur J Biochem 238, 661-668.[Medline]
Boll, M. & Fuchs, G. (1995). Benzoyl-CoA reductase (dearomatising), a key enzyme of anaerobic aromatic metabolism. ATP dependence of the reaction, purification and some properties of the enzyme from Thauera aromatica strain K172. Eur J Biochem 234, 921-933.[Medline]
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein-dye binding. Anal Biochem 72, 248-254.[Medline]
Byers, L. D. & Wolfenden, R. (1973). Binding of the by-product analog benzylsuccinic acid by carboxypeptidase A. Biochemistry 12, 2070-2078.[Medline]
Dawson, R. M. C., Elliot, D. C., Elliot, W. H. & Jones, K. M. (1986). Data for Biochemical Research, 3rd edn. Oxford, UK: Clarendon Press.
Giese, B. & Meister, J. (1977). Die Addition von Kohlenwasserstoffen an Olefine: eine neue synthetische Methode. Chem Ber 110, 2588-2600.
Heider, J., Spormann, A. M., Beller, H. R. & Widdel, F. (1999). Anaerobic bacterial metabolism of hydrocarbons. FEMS Microbiol Rev 22, 459-473.
Leuthner, B., Leutwein, C., Schulz, H., Hörth, P., Haehnel, W., Schiltz, E., Schägger, H. & Heider, J. (1998). Biochemical and genetic characterisation of benzylsuccinate synthase from Thauera aromatica: a new glycyl radical enzyme catalysing the first step in anaerobic toluene metabolism. Mol Microbiol 28, 615-628.[Medline]
Rabus, R. & Heider, J. (1998). Initial reactions of anaerobic metabolism of alkylbenzenes in denitrifying and sulfate-reducing bacteria. Arch Microbiol 170, 377-384.
Rabus, R., Fukui, M., Wilkes, H. & Widdel, F. (1996). Degradative capacities and 16S rRNA-targeted whole-cell hybridisation of sulfate-reducing bacteria in an anaerobic enrichment culture utilising alkylbenzenes from crude oil. Appl Environ Microbiol 62, 3605-3613.[Abstract]
Rabus, R., Wilkes, H., Schramm, A., Harms, G., Behrends, A., Amann, R. & Widdel, F. (1999). Anaerobic utilisation of alkylbenzenes and n-alkanes from crude oil in an enrichment culture of denitrifying bacteria affiliating with the ß-subclass of Proteobacteria. Environ Microbiol 1, 145-157.[Medline]
Schachter, D. & Taggart, J. V. (1976). Benzoyl coenzyme A and hippurate synthesis. J Biol Chem 203, 925-933.
Tschech, A. & Fuchs, G. (1987). Anaerobic degradation of phenol by pure cultures of newly isolated denitrifying pseudomonads. Arch Microbiol 148, 213-217.[Medline]
Received 2 June 1999;
revised 22 July 1999;
accepted 28 July 1999.
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, Benzylsuccinyl-CoA; 
, succinyl-CoA; 
, benzoyl-CoA. (a) Benzylsuccinyl-CoA synthesis from benzylsuccinate (2 mM) after starting with succinyl-CoA. (b) Reverse reaction of succinyl-CoA:benzylsuccinate CoA-transferase. The reaction contained 2 mM succinate and was started with benzylsuccinyl-CoA. (c) Oxidation of benzylsuccinyl-CoA to benzoyl-CoA in the presence of 1 mM PMS, 0·5 mM NAD and 2 mM NaNO3 as electron acceptors.