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Nonaromatic Products from Anoxic Conversion of Benzoyl-CoA with Benzoyl-CoA Reductase and Cyclohexa-1,5-diene-1-carbonyl-CoA Hydratase

2000; Elsevier BV; Volume: 275; Issue: 29 Linguagem: Inglês

10.1074/jbc.m001833200

ISSN

1083-351X

Autores

Matthias Boll, Diana Laempe, Wolfgang Eisenreich, Adelbert Bacher, Thomas Mittelberger, Jürgen Heınze, Georg Fuchs,

Tópico(s)

Enzyme Structure and Function

Resumo

The enzymes benzoyl-CoA reductase and cyclohex-1,5-diene-1-carbonyl-CoA hydratase catalyzing the first steps of benzoyl-CoA conversion under anoxic conditions were purified from the denitrifying bacterium, Thauera aromatica. Reaction products obtained with [ring-13C6]benzoyl-CoA and [ring-14C]benzoyl-CoA as substrates were analyzed by high pressure liquid chromatography and by NMR spectroscopy. The main product obtained with titanium(III) citrate or with reduced [8Fe-8S]-ferredoxin from T. aromatica as electron donors was identified as cyclohexa-1,5-diene-1-carbonyl-CoA. The cyclic diene was converted into 6-hydroxycyclohex-1-ene-1-carbonyl-CoA by the hydratase. Assay mixtures containing reductase, hydratase, and sodium dithionite or a mixture of sulfite and titanium(III) citrate as reducing agent afforded cyclohex-2-ene-1-carbonyl-CoA and 6-hydroxycylohex-2-ene-1-carbonyl-CoA. The potential required for the first electron transfer to the model compoundS-ethyl-thiobenzoate yielding a radical anion was determined by cyclic voltammetry as −1.9 V versus a standard hydrogen electrode. The energetics of enzymatic ring reduction of benzoyl-CoA are discussed. The enzymes benzoyl-CoA reductase and cyclohex-1,5-diene-1-carbonyl-CoA hydratase catalyzing the first steps of benzoyl-CoA conversion under anoxic conditions were purified from the denitrifying bacterium, Thauera aromatica. Reaction products obtained with [ring-13C6]benzoyl-CoA and [ring-14C]benzoyl-CoA as substrates were analyzed by high pressure liquid chromatography and by NMR spectroscopy. The main product obtained with titanium(III) citrate or with reduced [8Fe-8S]-ferredoxin from T. aromatica as electron donors was identified as cyclohexa-1,5-diene-1-carbonyl-CoA. The cyclic diene was converted into 6-hydroxycyclohex-1-ene-1-carbonyl-CoA by the hydratase. Assay mixtures containing reductase, hydratase, and sodium dithionite or a mixture of sulfite and titanium(III) citrate as reducing agent afforded cyclohex-2-ene-1-carbonyl-CoA and 6-hydroxycylohex-2-ene-1-carbonyl-CoA. The potential required for the first electron transfer to the model compoundS-ethyl-thiobenzoate yielding a radical anion was determined by cyclic voltammetry as −1.9 V versus a standard hydrogen electrode. The energetics of enzymatic ring reduction of benzoyl-CoA are discussed. high pressure liquid chromatography 4-morpholinepropanesulfonic acid heteronuclear multiple quantum correlation total correlation spectroscopy incredible natural abundance double quantum transfer spectroscopy titanium(III) Traditionally, the biodegradation of aromatic compounds has been considered as a domain of aerobic metabolism. More recently, an increasing number of bacteria has been shown to metabolize aromatic compounds under anoxic conditions. Benzoyl-CoA (Fig.1, compound 1) has been identified as a central intermediate in anoxic degradation of many aromatic substrates (for review see Refs. 1.Heider J. Fuchs G. Eur. J. Biochem. 1997; 243: 577-596Crossref PubMed Scopus (256) Google Scholar, 2.Heider J. Fuchs G. Anaerobe. 1997; 3: 1-22Crossref PubMed Scopus (108) Google Scholar, 3.Harwood C.S. Gibson J. J. Bacteriol. 1997; 179: 301-309Crossref PubMed Google Scholar, 4.Harwood C.S. Burchhardt G. Herrmann H. Fuchs G. FEMS Microbiol. Rev. 1999; 22: 439-458Crossref Google Scholar). Benzoyl-CoA reductase has been purified from the denitrifying bacteriumThauera aromatica (5.Boll M. Fuchs G. Eur. J. Biochem. 1995; 234: 921-933Crossref PubMed Scopus (187) Google Scholar). The 160-kDa protein consists of four different subunits specified by the bcrCBAD genes. The enzyme contains a minimum of two Fe-S clusters (5.Boll M. Fuchs G. Eur. J. Biochem. 1995; 234: 921-933Crossref PubMed Scopus (187) Google Scholar, 6.Boll M. Albracht S.J.P. Fuchs G. Eur. J. Biochem. 1997; 244: 840-851Crossref PubMed Scopus (73) Google Scholar) and is only active under strictly anoxic conditions (5.Boll M. Fuchs G. Eur. J. Biochem. 1995; 234: 921-933Crossref PubMed Scopus (187) Google Scholar). The reduction of benzoyl-CoA is coupled to the hydrolysis of two molecules of ATP/molecule of benzoyl-CoA. The natural electron donor for the enzyme-catalyzed reduction of benzoyl-CoA is a [8Fe-8S]-ferredoxin with a midpoint potential of −450 mV (versus a standard hydrogen electrode) (7.Boll M. Fuchs G. Eur. J. Biochem. 1998; 251: 946-954Crossref PubMed Scopus (56) Google Scholar). Alternatively, titanium(III) citrate, dithionite, or reduced methyl viologen can serve as artificial reductants. Some CoA-thioesters of fluoro-, amino-, and hydroxybenzoate can be reduced by benzoyl-CoA reductase albeit at low rates. Moreover, low molecular mass compounds such as hydroxylamine and azide are also reduced. All these reactions strictly depend on the hydrolysis of ATP (5.Boll M. Fuchs G. Eur. J. Biochem. 1995; 234: 921-933Crossref PubMed Scopus (187) Google Scholar). In analogy to the Birch reduction of aromatic compounds by metallic sodium in liquid ammonia, the enzyme-catalyzed reduction of benzoyl-CoA has been proposed to proceed by two one-electron transfer reactions, each of which is followed by a protonation step (8.Koch J. Eisenreich W. Bacher A. Fuchs G. Eur. J. Biochem. 1993; 211: 649-661Crossref PubMed Scopus (75) Google Scholar, 9.Buckel W. Keese R. Angew. Chem. 1995; 107: 1595-1598Crossref Google Scholar). The crucial step is the first electron transfer to the aromatic ring yielding a radical anion. EPR studies indicated that the hydrolysis of ATP is responsible for conformational changes in the vicinity of [4Fe-4S] clusters, most probably resulting in a lowered reduction potential of the clusters (6.Boll M. Albracht S.J.P. Fuchs G. Eur. J. Biochem. 1997; 244: 840-851Crossref PubMed Scopus (73) Google Scholar). [ring-13C6]Cyclohexa-1,5-diene-1-carbonyl-CoA was identified in a product mixture obtained after incubation of a crude extract preparation from T. aromatica containing [ring-13C6]benzoate, coenzyme A, ATP, Mg2+, and titanium(III) citrate (8.Koch J. Eisenreich W. Bacher A. Fuchs G. Eur. J. Biochem. 1993; 211: 649-661Crossref PubMed Scopus (75) Google Scholar). Cyclohexa-1,5-diene-1-carbonyl-CoA (Fig. 1, compound2) was proposed tentatively to be the committed product of benzoyl-CoA reductase. Furthermore, an enzyme (Dch protein; subsequently designated as 1,5-dienoyl-CoA hydratase) catalyzing the formation of 6-hydroxycyclohex-1-ene-1-carbonyl-CoA (compound3) by addition of water to the cyclic 1,5-diene (compound 2) has been purified and characterized (10.Laempe D. Eisenreich W. Bacher A Fuchs G. Eur. J. Biochem. 1998; 255: 618-627Crossref PubMed Scopus (36) Google Scholar). In contrast to the results obtained with T. aromatica,cyclohexa-2,5-diene-1-carboxylate (Fig.2, compound 4) and cyclohexa-1,4-diene-1-carboxylate (compound 5) were reported as products of benzoic acid reduction in cell suspensions of the phototrophic bacterium Rhodopseudomonas palustris. The structure assignments were based on gas chromatography and mass spectrometry after hydrolytic treatment of the reaction mixture (11.Gibson K.J. Gibson J. Appl. Env. Microbiol. 1992; 58: 696-698Crossref PubMed Google Scholar). The present study was initiated to establish the product of benzoyl-CoA reductase from T. aromatica. For this purpose, benzoyl-CoA labeled with 13C or 14C was incubated with purified benzoyl-CoA reductase using various electron donors. Enyzme products were analyzed by HPLC1 and two-dimensional homonuclear and heteronuclear NMR spectroscopy. Furthermore, the effect of a thioester residue on the reduction potential of the aromatic moiety was determined by cyclic voltammetry usingS-ethyl-thiobenzoate as a model compound. Reagents were obtained from Aldrich, Fluka, Merck, Sigma, Roth, Roche Molecular Biochemicals, and Gerbu. Chromatography materials were obtained from Amersham Pharmacia Biotech, Bio-Rad, and Merck, respectively. [ring-13C6]Benzoic acid was purchased from MS Isotopes, and [ring-14C]benzoate was obtained from American Radiolabeled Chemicals Inc. and Biotrend Chemikalien GmbH. T. aromatica strain K172 (DSM 6984) was isolated in our laboratory (12.Tschech A. Fuchs G. Arch. Microbiol. 1987; 148: 213-217Crossref PubMed Scopus (234) Google Scholar, 13.Anders H.-J. Kaetzke A. Kämpfer P. Ludwig W. Fuchs G. Int. J. Syst. Bacteriol. 1995; 45: 327-333Crossref PubMed Scopus (254) Google Scholar) and has been deposited in Deutsche Sammlung von Mikroorganismen (Braunschweig, Germany).Clostridium pasteurianum (ATCC 6013) was obtained from the American Type Culture Collection. [ring-13C6]Benzoyl-CoA was obtained via esterification of benzoic acid withN-hydroxysuccinimide (14.Gross G.G. Zenk M.H. Z. Naturforschg. 1966; 21b: 683-690Google Scholar). [ring-14C]Benzoyl-CoA and cyclohex-1-ene-1-carbonyl-CoA were prepared enzymatically from [ring-14C]benzoate (specific radioactivity, 2.025 GBq mmol−1) and cyclohex-1-ene-1-carboxylate, respectively, using coenzyme A and enriched benzoate-CoA ligase fromT. aromatica as described earlier (15.Ziegler K. Braun K. Böckler A. Fuchs G. Arch. Microbiol. 1987; 149: 62-69Crossref Scopus (70) Google Scholar). [ring-13C6]benzoyl-CoA and [ring-14C]benzoyl-CoA were mixed (600 kBq mg−1) and co-purified by semi-preparative HPLC as described (10.Laempe D. Eisenreich W. Bacher A Fuchs G. Eur. J. Biochem. 1998; 255: 618-627Crossref PubMed Scopus (36) Google Scholar). The yield was 50–80%. Treatment of benzoic acid phenyl ester with sodium ethyl mercaptide affordedS-ethyl-thiobenzoate (16.Seifert A. J. Prakt. Chem. 1885; 31: 471-473Crossref Google Scholar). An aliquot (80 μl) of the reaction mixture was dissolved in 920 μl of toluene and purified by preparative thin layer chromatography using silica gel 60 plates (Merck, 2 mm, 20 × 20 cm) with toluene as solvent. A strip of the plate was sprayed with 0.01% fluorescein (m/v) in ethanol, and the bands were visualized under ultraviolet light (254 nm). The product band was extracted with toluene and dried under reduced pressure (1H NMR using CDCl3 as solvent: δ (ppm); 1.37 (3H, t, CH3), 3.10 (2H, q, CH2), 7.33–8.00 (5H, m, C6H5). T. aromatica was grown anoxically at 28 °C in a mineral salt medium containing a mixture of 4-hydroxybenzoate and nitrate (ratio 1:3.5) as sole source of carbon and energy (12.Tschech A. Fuchs G. Arch. Microbiol. 1987; 148: 213-217Crossref PubMed Scopus (234) Google Scholar). Hydroxybenzoate and nitrate were continuously fed at a molar ratio of 1:3.5. Cells were harvested by centrifugation under anaerobic conditions and were stored in liquid nitrogen. Cell extract was prepared as described (17.Brackmann R. Fuchs G. Eur. J. Biochem. 1993; 213: 563-571Crossref PubMed Scopus (92) Google Scholar). Benzoyl-CoA reductase was partially purified from T. aromatica (wet cell mass, 200 g) under strictly anaerobic conditions in a glove box containing an atmosphere of N2/H2 (95:5, v/v) as described earlier (5.Boll M. Fuchs G. Eur. J. Biochem. 1995; 234: 921-933Crossref PubMed Scopus (187) Google Scholar). Benzoyl-CoA reductase was further purified by chromatography on a column of Blue Sepharose CL 6B (Amersham Pharmacia Biotech, 4.2 × 12.5 cm) that had been equilibrated with 20 mm Tris-HCl, pH 7.5. The column was developed with three bed volumes of equilibration buffer and was then developed with 20 mm Tris-HCl, pH 7.5, containing 500 mm KCl (flow rate, 2 ml min−1). Fractions were assayed for benzoyl-CoA reductase (5.Boll M. Fuchs G. Eur. J. Biochem. 1995; 234: 921-933Crossref PubMed Scopus (187) Google Scholar) and cyclohexa-1,5-diene-1-carbonyl-CoA hydratase activity (10.Laempe D. Eisenreich W. Bacher A Fuchs G. Eur. J. Biochem. 1998; 255: 618-627Crossref PubMed Scopus (36) Google Scholar). Ferredoxin was purified fromT. aromatica (wet cell mass, 200 g) as described earlier (7.Boll M. Fuchs G. Eur. J. Biochem. 1998; 251: 946-954Crossref PubMed Scopus (56) Google Scholar). The yield was 60 mg of pure protein. Benzoyl-CoA reductase was assayed spectrophotometrically using reduced methyl viologen as electron donor (5.Boll M. Fuchs G. Eur. J. Biochem. 1995; 234: 921-933Crossref PubMed Scopus (187) Google Scholar) or by HPLC as described earlier (6.Boll M. Albracht S.J.P. Fuchs G. Eur. J. Biochem. 1997; 244: 840-851Crossref PubMed Scopus (73) Google Scholar). The reaction mixture for HPLC analysis contained 150 mm MOPS/KOH, pH 7.3, 10 mm MgCl2, 7.5 mm ATP, 10 mm phosphoenolpyruvate, 0.2 mm[ring-14C]benzoyl-CoA, 80 nkat of pyruvate kinase, and purified benzoyl-CoA reductase from T. aromatica(0.1–0.2 unit) in a total volume of 1 ml. Titanium(III) citrate or sodium dithionite were added to a final concentration of 5 mm. Assays using reduced ferredoxin (37 μm) from T. aromatica as reductant were performed under an atmosphere of 100% hydrogen. Reduction of T. aromaticaferredoxin was catalyzed by a ferredoxin-free crude preparation of hydrogenase from C. pasteurianum (1.5 units mg−1) that had been prepared as described previously (7.Boll M. Fuchs G. Eur. J. Biochem. 1998; 251: 946-954Crossref PubMed Scopus (56) Google Scholar). When ferredoxin was used, other reductants were omitted. Unless otherwise stated, the reaction was started by adding 50 μl of a purified benzoyl-CoA reductase solution. The enzymatic hydration of cyclohex-1,5-diene-1-carbonyl-CoA and the reverse reaction, dehydration of 6-hydroxycyclohex-1-ene-1-carbonyl-CoA, were determined by a continuous spectrophotometric assay (λ = 310 nm) at 37 °C as described (10.Laempe D. Eisenreich W. Bacher A Fuchs G. Eur. J. Biochem. 1998; 255: 618-627Crossref PubMed Scopus (36) Google Scholar). Additionally, dienoyl-CoA hydratase was determined by HPLC analysis of substrate consumption and product formation. The assay mixture for HPLC analysis contained 150 mm MOPS/KOH, pH 7.3, 0.1 mm 6-hydroxycyclohex-1-ene-1-carbonyl-CoA, and approximately 0.1 unit of dienoyl-CoA hydratase in a volume of 0.25 ml. The mixture was incubated at 37 °C. After 10 min, equilibrium was reached, and approximately one-half of 6-hydroxycyclohex-1-ene-1-carbonyl CoA was converted into the cyclic 1,5-diene. Subsequently, sodium dithionite, sodium sulfite, or a mixture of sodium sulfite and titanium(III) citrate were added to a final concentration of 1 mm each. Aliquots were retrieved at intervals and applied to a Lichrosphere 100 RP C-18 HPLC column (Merck; bed volume, 1 ml; flow rate, 1 ml min−1) that had been equilibrated with 50 mm potassium phosphate, pH 6.7, containing 10% acetonitrile (v/v). The column was developed with the equilibration buffer. The effluent was monitored by scintillation counting (Ramona, Raytest, Straubenhardt, Germany) or photometrically at 260 nm. Retention volumes were 11.1 ml for benzoyl-CoA, 13.9 ml for cyclohexa-1,5-diene-1-carbonyl CoA, 4.4 ml for 6-hydroxycyclohex-1-ene-1-carbonyl CoA, 2.5 ml for 6-hydroxycyclohex-2-ene-1-carbonyl-CoA, and 16.5 ml for cyclohex-2-ene-1-carbonyl-CoA. The assignment of the HPLC peaks to individual CoA-thioesters was performed by NMR analysis of13C-labeled compounds (see below). Reaction mixtures (5 ml) contained 150 mm MOPS/KOH, pH 7.3, 10 mmMgCl2, 7.5 mm ATP, 10 mmphosphoenol pyruvate, and 400 nkat pyruvate kinase, and 10 mm titanium(III) citrate or 10 mm sodium dithionite. The reaction was started by the addition of a mixture of [ring-14C]benzoyl-CoA (44.4 kBq) and 5 mg of [ring-13C6]benzoyl-CoA. The mixtures were incubated under anoxic conditions at 37 °C for 20–40 min. The reaction was stopped by stirring for 15 min at 0 °C under an aerobic atmosphere. The solution was lyophilized, and the powder was dissolved in 1 ml of 20 mm ammonium formate, pH 5. The solution was applied to a Lichrosorb RP-C18 column (25 × 2 cm; Merck; flow rate, 8 ml min−1), which had been equilibrated with 20 mm ammonium formate, pH 5. After washing the column with two bed volumes of 10% methanol (v/v) in the same buffer, the CoA thioesters were eluted with 98% methanol (v/v) in the same buffer. Methanol was evaporated under reduced pressure at 30 °C, and the residue was lyophilized. Product recovery was determined by liquid scintillation counting and was 60–80% of the initial amount of14C added to the assay. When reduced ferredoxin was used as reductant, the reaction was performed in a 100-ml stoppered bottle containing 9.6 ml of the standard assay mixture. The bottle was flushed with hydrogen for 20 min under continuous stirring. A ferredoxin-free hydrogenase preparation (H2 reduction activity: 5–6 μmol/min) from C. pasteurianum (7.Boll M. Fuchs G. Eur. J. Biochem. 1998; 251: 946-954Crossref PubMed Scopus (56) Google Scholar), [ring-13C6]benzoyl-CoA (7 mg), 85 kBq [ring-14C]benzoyl-CoA, and 3.5 mg ferredoxin from T. aromatica (final concentration, 37.5 μm) were added. The mixture was flushed with hydrogen for 20 min under continuous stirring. The assay was started by the addition of benzoyl-CoA reductase (0.8 unit) and was terminated after 15 min by adding 250 μl of 1% formic acid (final pH, 3–4). The mixture was then lyophilized. The lyophilized samples were dissolved in D2O at pH 6 (uncorrected glass electrode reading).1H and 13C NMR spectra were measured at 17 °C using a four-channel Bruker DRX500 spectrometer equipped with pulsed field gradient accessory, a lock switch unit, and an ASPECT station. One-dimensional and two-dimensional HMQC, HMQC-TOCSY, and INADEQUATE experiments were performed as described earlier (8.Koch J. Eisenreich W. Bacher A. Fuchs G. Eur. J. Biochem. 1993; 211: 649-661Crossref PubMed Scopus (75) Google Scholar). The duration of the 1H spin-lock was 60 ms in the HMQC-TOCSY experiment. The midpoint potential ofS-ethyl-thiobenzoate was determined at 25 °C by cyclic voltammetry. All measurements were carried out in super-dry acetonitrile with tetrabutyl ammonium hexafluorophosphate as supporting electrolyte. A three-electrode cell compartment was used throughout. For the electrochemical measurements, a potentiostat model 173 and a programmer model 175 (EG&G, Burlington, Vermont) were used. All potentials were calibrated against a ferrocene/ferrocenium couple. Polyacrylamide gel electrophoresis and SDS-polyacrylamide gel electrophoresis were performed as described by Schägger and von Jagow (18.Schägger J. van Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10480) Google Scholar). Proteins were visualized by Coomassie Blue staining (19.Zehr B.D. Savin T.J. Hall R.E. Anal. Biochem. 1989; 182: 157-159Crossref PubMed Scopus (129) Google Scholar). Protein was determined by the methods of Bradford (20.Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216440) Google Scholar) or Lowry et al. (21.Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) using bovine serum albumin as standard. Benzoyl-CoA reductase was purified from T. aromatica as described earlier (5.Boll M. Fuchs G. Eur. J. Biochem. 1995; 234: 921-933Crossref PubMed Scopus (187) Google Scholar). The enzyme was enriched 40-fold by four chromatographic steps with a yield of 25% to a specific activity of 0.48 μmol min−1 mg−1 (measured with reduced methyl viologen as electron donor (5.Boll M. Fuchs G. Eur. J. Biochem. 1995; 234: 921-933Crossref PubMed Scopus (187) Google Scholar)) and >95% purity as estimated by SDS gel electrophoresis. This protein fraction contained benzoyl-CoA reductase (7 μmol min−1) and cyclohexa-1,5-diene-1-carbonyl-CoA hydratase (86 μmol min−1) activity. Further purification of benzoyl-CoA reductase was achieved by affinity chromatography on Cibachron Blue (Amersham Pharmacia Biotech). The effluent of the column contained benzoyl-CoA reductase activity (4.5 μmol min−1) and 7% of the initial hydratase activity (6.0 μmol min−1). Using buffer containing 0.5 m KCl, 70% of the initial dienoyl-CoA hydratase activity could be eluted from the column. A mixture of benzoyl-CoA reductase (7 μmol min−1) and 1,5-dienoyl-CoA hydratase (86 μmol min−1) was incubated with [ring-14C]benzoyl-CoA in the presence of reduced ferredoxin or titanium(III) citrate. Reversed phase HPLC of the reaction mixture showed four peaks (1, 2, 3, and F) reflecting at least three radioactive products (Fig.3 A). The compound reflecting peak 3 was apparently more polar than benzoyl-CoA (peak 1, compound 1), product 2 was less polar than benzoyl-CoA, and product F had the same retention volume as free aromatic carboxylic acids and was probably formed by hydrolytic cleavage of the thioester. Subsequent experiments were performed with [ring-13C6]benzoyl-CoA as substrate in an attempt to optimize the sensitivity and selectivity of NMR detection. The reaction mixture was desalted and was analyzed by NMR spectroscopy. Carbon atoms derived from the13C-enriched phenyl ring of benzoyl-CoA substrate gave 12 intense 13C NMR signals (Fig.4) that were attributed to two products (compounds 2 and 3) by two-dimensional NMR experiments. More specifically, two well separated spin systems were gleaned from a HMQC-TOCSY experiment, establishing the connectivities between 13C and 1H atoms as well as between1H atoms of the 13C-labeled ring moieties (Fig.5 and TableI).Figure 5Partial HMQC (A) and HMQC-TOCSY (B) spectrum of the assay mixture containing [ring-13C6]cyclohexa-1,5-diene-1-carbonyl CoA (compound 2) and [ring-13C6]6-hydroxycyclohex-1-ene-1-carbonyl CoA (compound 3). Signals of compound2 and compound 3 are indicated by italic numbers and numbers in regular type, respectively. The duration of 1H spin-lock was 60 ms in the HMQC-TOCSY experiment.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table INMR data of products formed from [phenyl- 13 C]benzoyl-CoA by enzymatic action of benzoyl-CoA reductase and hydratasePositionChemical shift aReferenced to external 3-trimethylsilylpropane 1-sulfonate.Coupling constant ± JCC cDetermined from one-dimensional13C NMR spectra; coupled atoms to the respective index atoms are in parentheses.Correlation experiments13C1HHMQC-TOCSYINADEQUATEppmHzBenzoyl-CoA (1) 1138.3(t) bSignal multiplicities in parentheses (t = pseudotriplet; dd = double doublet).57.8(2/6), 9.12/6 2/6129.2(t)7.85nd2/6, 3/5, 41, 3/5 3/5131.2(t)7.49nd3/5, 2/6, 42/6, 4 4136.5(t)7.6653.5(3/5), 9.54, 3/5, 2/63/56-Hydroxycyclohex-1-ene-1-carbonyl-CoA (3) 1140.7(dd)68.9(2), 47.1(6), 4.22, 6 2147.4(dd)7.2168.9(1), 38.2(3)2, 3′, 3, 41, 3 328.1(t)2.38(3′), 2.2136.1(2, 4)2, 3′, 3, 4, 5′, 52, 4 418.0(t)1.7033.8(5, 3)2, 6, 4, 5′, 5, 3, 3′3, 5 532.2(dd)1.91(5′), 1.6736.3(6), 32.7(4), 2.92(w), 6, 5′, 5, 4, 3, 3′ dw, weak signal intensity.4, 6 664.0(dd)4.6646.8(1), 37.0(5)6, 5′, 55, 1Cyclohex-1,5-diene-1-carbonyl-CoA (2) 1137.4(dd)68.8(2), 53.0(6), 6.62, 6 2140.6(dd)6.9768.4(1), 38.7(3), 7.6, 3.12, 3, 41, 3 324.7(dd)2.3738.7(2), 34.8(4), 6.0, 2.82, 5, 6, 3, 42, 4 422.8(t)2.2037.6(3, 5)2, 6, 5, 4, 33, 5 5131.6(t)6.0268.8(6), 7.05, 6, 4, 34, 6 6121.9(dd)6.2267.8(5), 53.5(1), 6.2, 1.46, 5, 4, 3(w)5, 1Cyclohex-2-ene-1-carbonyl-CoA (9) 151.8(dd)3.1340.2, 32.62, 3, 4, 6, 6′, 5, 5′2, 6 2125.1(dd)5.4769.4, 40.61, 3, 4, 5(w)1, 3 3134.3(dd)5.7967.3, 37.02, 4, 1, 5(w), 5′(w)2, 4 426.3(t)1.8235.02, 3, 1, 5, 5′, 6, 63, 5 522.0(t)1.50, 1.3933.23, 4, 2, 1, 6, 6′4, 6 628.7(t)1.71, 1.5129.41, 2, 5, 5′, 3(w)5, 16-Hydroxycyclohex-2-ene-1-carbonyl-CoA (8) 154.0(t)3.5535.92, 4, 3(w), 5(w), 5′(w)2, 6 2124.2(dd)5.3669.5, 38.03, 1, 41, 3 3132.9(dd)5.8270.0, 36.04, 2, 1(w), 5(w), 5′(w)2, 4 425.3(t)2.0332.93, 2, 1, 6, 5, 5′3, 5 524.4(t)1.93, 1.5333.24, 6, 1, 3(w)4, 6 659.2(t)3.3134.91, 5, 5′, 4, 3(w), 2(w)5, 1a Referenced to external 3-trimethylsilylpropane 1-sulfonate.b Signal multiplicities in parentheses (t = pseudotriplet; dd = double doublet).c Determined from one-dimensional13C NMR spectra; coupled atoms to the respective index atoms are in parentheses.d w, weak signal intensity. Open table in a new tab Each of the 12 13C signals described above was split into a double doublet or a pseudo-triplet by 13C13C coupling constants of 30–70 Hz. Apparently, each of the observed13C atoms was directly bonded to two adjacent13C atoms. This result suggests that both products retained the connectivity of the ring carbon atoms of the benzoyl substrate. Some of the signals showed additional fine splittings (coupling constants of 1–8 Hz; Table I) as a result of13C13C couplings via two or three bonds. Four of the six intense 13C NMR signals of compound2 had chemical shifts values (121.9, 131.6, 137.4, and 140.6 ppm; cf. Fig. 4 and Table I) consistent with sp2-hybridization of the corresponding carbon atoms. Two signals were detected at 27.7 and 22.8 ppm reflecting two sp3-hybridized carbon atoms. This 13C NMR signature in conjunction with the detected13C13C coupling pattern was consistent with a cyclic diene structure. Two-dimensional INADEQUATE, HMQC, and HMQC-TOCSY experiments (Table I and Fig. 5) afforded the entire network of carbon-carbon, carbon-proton, and proton-proton connectivities (28 detected homo- and heterocorrelations between 1H and13C of the carbocyclus). Specifically, HMQC spectroscopy revealed correlations of the 13C NMR signals at 121.9, 131.6, and 140.6 ppm to 1H NMR signals at 6.22, 6.02, and 6.97 ppm, respectively. The 13C signal at 137.4 ppm did not correlate to any 1H NMR signal in the HMQC experiment. This finding suggested a cyclohexadiene-1-carbonyl derivative. Carbon-carbon coupling constants (68.8 and 53.0 Hz) detected for the NMR signal at 137.4 ppm (C-1) as well as INADEQUATE spectroscopy showed that C-1 was adjacent to two sp2-hybridized carbon atoms with13C NMR signals at 140.6 ppm (C-2) and 121.9 ppm (C-6). The signal at 121.9 ppm (C-6) gave an additional carbon-carbon correlation to the signal at 131.6 ppm (C-5), indicating a cyclohexa-1,5-diene-1-carbonyl system. The presence of a CoA residue was revealed by the 1H NMR data (not shown), thus establishing compound 2 as cyclohexa-1,5-diene-1-carbonyl-CoA. The second product in the mixture (compound 3) was identified as 6-hydroxycyclohex-1-ene-1-carbonyl-CoA by the same approach, as described above for compound 2 (Table I). Compound 3 had been identified earlier as the reaction product of 1,5-dienoyl-CoA hydratase from T. aromatica (10.Laempe D. Eisenreich W. Bacher A Fuchs G. Eur. J. Biochem. 1998; 255: 618-627Crossref PubMed Scopus (36) Google Scholar). Cyclohexa-2,5-diene-1-carbonyl-CoA and cyclohexa-1,4-diene-1-carbonyl-CoA, which had been reported as products of benzoyl-CoA reduction catalyzed by benzoyl-CoA reductase in R. palustris (11.Gibson K.J. Gibson J. Appl. Env. Microbiol. 1992; 58: 696-698Crossref PubMed Google Scholar), were not found in our reaction mixtures. After prolonged incubation of [ring-13C6]benzoyl-CoA with benzoyl-CoA reductase, two minor products, [ring-13C6]cyclohex-1-ene-1-carbonyl-CoA (Fig. 6, compound 6) and [ring-13C6]2-hydroxycyclohexane-1-carbonyl-CoA (7), were identified by NMR (data not shown). The NMR data of these compounds have been described elsewhere (10.Laempe D. Eisenreich W. Bacher A Fuchs G. Eur. J. Biochem. 1998; 255: 618-627Crossref PubMed Scopus (36) Google Scholar). We suggest tentatively that these compounds resulted from reduction of2 by benzoyl-CoA reductase and subsequent hydration catalyzed by cyclohexa-1,5-diene-1-carbonyl-CoA hydratase (Fig. 6). The formation of these compounds was not observed when benzoyl-CoA was still present in the assay mixture as shown in Fig. 3 (Aand B). As reported earlier, dithionite can also act as an artificial electron donor for benzoyl-CoA reductase with rates comparable with other artificial electron donors (5.Boll M. Fuchs G. Eur. J. Biochem. 1995; 234: 921-933Crossref PubMed Scopus (187) Google Scholar). Surprisingly, HPLC analysis of the products formed from incubation of [ring-14C]benzoyl-CoA with dithionite and a protein mixture containing benzoyl-CoA reductase and 1,5-dienoyl-CoA hydratase revealed a product pattern (Fig. 3 B) that was different from that obtained with titanium(III) citrate or reduced ferredoxin (Fig. 3 A). Peaks reflecting the cyclic 1,5-diene (compound 2) or the cyclic 6-hydroxy-1-monoene (compound3) were not observed with dithionite as artificial electron donor. On the other hand, two novel peaks reflecting compounds8 and 9 were detected (Fig. 3 B). Moreover, when dithionite (5 mm) was added to a benzoyl-CoA reductase assay that had been started with titanium(III) citrate as electron donor, the HPLC pattern shown in Fig. 3 A changed to that shown in Fig. 3 C. Compound 8 and compound 9 formed by enzymatic conversion of [ring-13C6]benzoyl-CoA were analyzed by NMR spectroscopy (Table I). The detected 13C NMR chemical shifts (120–140 ppm) and coupling signatures (double doublets or triplets) were consistent with cyclic hexamonoene motifs for both compounds. Notably, each of the 13C atoms correlated to directly bonded 1H atoms in the HMQC experiment, thus indicating that the C-1 positions of both compounds carried a hydrogen atom and were not involved in a double bond. INADEQUATE and HMQC-TOCSY experiments afforded the entire network of carbon-carbon and carbon-proton bonds establishing compound9 as [ring-13C6]cyclohex-2-ene-1-carbonyl-CoA and compound 8 as [ring-13C6]6-hydroxycyclohex-2-ene-1-carbonyl-CoA (Fig. 7). No cyclic diene was detected with dithionite as electron donor. To clarify the effects of electron donors on the product pattern, we analyzed the effect of sodium dithionite on a benzoyl-CoA reductase free preparation of 1,5-dienoyl-CoA hydratase by HPLC. Under equilibrium conditions, similar amounts of cyclohexa-1,5-diene-1-carbonyl CoA (peak 2) and 6-hydroxycyclohex-1-ene-1-carbonyl-CoA (peak 3) were present in assays of the hydratase (Fig.8 A). After addition of sodium dithionite, both compounds were converted into 6-hydroxycyclohex-2-ene-1-carbonyl-CoA (Fig. 8, B andC, peaks 8). Fig. 8 B shows that an additional intermediate apolar product (reflecting peak 10) is formed after 1 min of incubation. After 10 min of incubation this peak disappeared in the chromatogram (Fig. 8 C). It is conceivable that peak 10 represents cyclohexa-2,5-diene-1-carbonyl CoA (Fig. 7,compound 10), which can be converted rapidly into compound 8 by the addition of water. When sodium dithionite was replaced by 1 mm sodium sulfite, the formation of compound 8 could not be detected, whereas a mixture containing 1 mm sodium sulfite and 1 mmtitanium(III) citrate had the same effect as sodium dithionite (not shown). In the absence of 1,5-dienoyl-CoA hydratase, no spontaneous reaction of dithionite with 2 and 3 could be observed. For the energetics of benzoyl-CoA reduction, the transfer of the first electron to the aromatic ring of benzoyl-CoA yielding a radical anion is considered as the crucial step in the reaction catalyzed by benzoyl-CoA reductase (9.Buckel W. Keese R. Angew. Chem. 1995; 107: 1595-1598Crossref Google Scholar). A highly negative redox potential is expected for this reaction. The natural substrate benzoyl-CoA is not suitable for the direct determination of this potential because irreversible follow-up reactions occur in protic solvents. Therefore, the substrate analog S-ethyl-thiobenzoate, which is soluble in aprotic solvents, was synthesized and analyzed by cyclic voltammetry. The thioester was reduced at a midpoint potential of −2.1 Vversus an Ag/AgCl reference electrode. The reaction was reversible; minor waves appeared in the reverse scan at a potential of approximately −0.5 V versus Ag/AgCl indicating, a second slow process (data not shown). This follow-up product probably results from protonation of the radical anion yielding a free radical. Residual traces of water in the solvent could be the proton donor for such species. The structures of six CoA thioesters obtained after incubation of [ring-13C6]benzoyl-CoA with purified benzoyl-CoA reductase (BcrCBAD protein) and 1,5-dienoyl CoA hydratase (Dch protein) from T. aromatica were established by NMR analysis. The use of multiply 13C labeled substrate facilitated signal assignments by two-dimensional INADEQUATE, HMQC, and HMQC-TOCSY spectroscopy and enabled NMR analysis with product mixtures. Specifically, the NMR data established cyclohexa-1,5-diene-1-carbonyl-CoA (compound 2) as the product of benzoyl-CoA reduction with titanium(III) or with reduced [8Fe-8S]-ferredoxin from T. aromatica as electron donors. This is in agreement with the results of Koch et al. (8.Koch J. Eisenreich W. Bacher A. Fuchs G. Eur. J. Biochem. 1993; 211: 649-661Crossref PubMed Scopus (75) Google Scholar), who identified this compound as an early product in experiments with crude cell extracts of T. aromatica. The formation of this cyclic hexadiene implicates a two-electron reduction of benzoyl-CoA and fits the previously determined stoichiometry (6.Boll M. Albracht S.J.P. Fuchs G. Eur. J. Biochem. 1997; 244: 840-851Crossref PubMed Scopus (73) Google Scholar). The identification of 6-hydroxycyclohex-1-ene-1-carbonyl-CoA (compound 3) as next intermediate in the benzoyl-CoA pathway in T. aromaticafurther supports the cyclic 1,5-diene structure, because the 6-hydroxy-1-ene can be formed by the enzymatic addition of water to the conjugated double bonds of the diene (10.Laempe D. Eisenreich W. Bacher A Fuchs G. Eur. J. Biochem. 1998; 255: 618-627Crossref PubMed Scopus (36) Google Scholar), either via a 1,4- or via a 1,2-addition mechanism. The four genes of benzoyl-CoA reductase from T. aromaticaand R. palustris show high similarity (78% sequence identity) (22.Egland P.G. Pelletier D.A. Dispensa M. Gibson J. Harwood C.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6484-6489Crossref PubMed Scopus (142) Google Scholar, 23.Breese K Boll M. Alt-Mörbe J. Schägger H. Fuchs G. Eur. J. Biochem. 1998; 256: 148-154Crossref PubMed Scopus (70) Google Scholar). Interestingly, Gibson and Gibson (11.Gibson K.J. Gibson J. Appl. Env. Microbiol. 1992; 58: 696-698Crossref PubMed Google Scholar) detected cyclohexa-2,5-diene-1-carboxylate (compound 4) and cyclohexa-1,4-diene-1-carboxylate (compound 5) as products of benzoate reduction in cell suspension experiments with R. palustris. These compounds were identified by gas chromatography/mass spectrometry techniques after derivatization of the free acids to the corresponding methyl esters. The discrepancy to our results might have different reasons. Either benzoyl-CoA reduction proceeds via different mechanisms in R. palustris as discussed elsewhere (3.Harwood C.S. Gibson J. J. Bacteriol. 1997; 179: 301-309Crossref PubMed Google Scholar, 22.Egland P.G. Pelletier D.A. Dispensa M. Gibson J. Harwood C.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6484-6489Crossref PubMed Scopus (142) Google Scholar), or the diene isomers detected by GC/MS were artificially formed during derivatization of the products. In the so-called Birch reduction of benzoic acid with elemental sodium or lithium as reductants, 2,5-cyclohexadiene-1-carboxylate (compound4) is formed under kinetic control (24.Krapcho A.P. Bothnerby A.A. J. Am. Chem. Soc. 1959; 81: 3658-3666Crossref Scopus (100) Google Scholar). The electron withdrawing character of the acid group of benzoate stabilizes the anion radical at carbon 1 of the ring; the second electron adds inpara position because of electrostatic repulsion. However, in the presence of a strong base, the thermodynamically more stable conjugated 1,5-diene can be obtained (25.Birch A.J. Rao G.S. Adv. Org. Chem. 1972; 8: 1-65Google Scholar, 26.Birch A.J. Hinde A.L. Radom L. J. Am. Chem. Soc. 1980; 102: 3370-3376Crossref Scopus (90) Google Scholar). This base-catalyzed rearrangement seems to occur in the catalytic cycle of T. aromatica. The formation of the hex-1-ene compound (compound 6) at a slow rate (5.Boll M. Fuchs G. Eur. J. Biochem. 1995; 234: 921-933Crossref PubMed Scopus (187) Google Scholar) explained by a two-electron reduction of the 1,5-diene (compound 2) catalyzed by benzoyl-CoA reductase. Although the hex-1-ene (compound 6) and the 2-hydroxyhexane compound (compound 7) may play an important role in the aromatic metabolism of R. palustris (3.Harwood C.S. Gibson J. J. Bacteriol. 1997; 179: 301-309Crossref PubMed Google Scholar, 22.Egland P.G. Pelletier D.A. Dispensa M. Gibson J. Harwood C.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6484-6489Crossref PubMed Scopus (142) Google Scholar), their formation inT. aromatica appears as an in vitro effect without metabolic relevance. The natural electron donor, reduced ferredoxin, and the artificial reductant titanium(III) citrate yielded the same products. Surprisingly, cyclohex-2-ene-1-carbonyl-CoA (compound 9) and 6-hydroxycyclohex-2-ene-1-carbonyl-CoA (compound 8) were identified as products of benzoyl-CoA reductase and cyclohexa-1,5-diene-1-carbonyl-CoA hydratase when dithionite was used as an artificial electron donor. Our results strongly suggest that the artificial formation of the 6-hydroxy-2-ene (compound 8) was a product of dienoyl-CoA hydratase activity via a set of artificial isomerization and hydration reactions. In this case, cyclohexa-2,5-diene-1-carbonyl-CoA (compound 10) is suggested as an intermediate isomerization product (Fig. 7). Under anaerobic conditions in the presence of benzoyl-CoA reductase, MgATP, and an excess of dithionite, this hypothetical diene is further reduced to cyclohexa-2-ene-1-carbonyl-CoA (compound 9). The following observations support our suggestions: (i) When sodium dithionite was added to a benzoyl-CoA reductase/1,5-dienoyl-CoA hydratase assay that had been started with Ti(III) citrate as electron donor and benzoyl-CoA as substrate, the product pattern shifted immediately from the cyclohexa-1,5-diene/6-hydroxymonene mixture (reflecting peaks 2 and 3 in the HPLC chromatogram; cf. Fig.8 A) to a cylohexa-2-ene/6-hydroxy-2-ene mixture (reflecting peaks 9 and 8 in the HPLC chromatogram; cf. Fig.8 C). (ii) Under aerobic conditions, when the hydratase was fully active and benzoyl-CoA reductase was irreversibly inactivated, addition of dithionite induced the formation of 6-hydroxycyclohex-2-ene-1-carbonyl-CoA (compound 8) as the unique product from a cyclohexa-1,5-diene/6-hydroxymonene mixture. 6-Hydroxycyclohex-2-ene-1-carbonyl-CoA (compound 8) is considered to be a dead end product that is not in equilibrium with the corresponding diene 10 (Fig. 7). A mixture of sulfite and Ti(III) citrate could replace dithionite in the artificial product formation, whereas sulfite alone had no effect on dienoyl-CoA hydratase activity. We assume that a sulfoxide radical anion is formed from sulfite by reduction with Ti(III) citrate as an active species; it is known that anaerobic dithionite solutions contain the same radical anion (27.Lambeth D.O. Palmer G. J. Biol. Chem. 1973; 248: 6095-6103Abstract Full Text PDF PubMed Google Scholar). For energetics of anaerobic enzymatic ring reduction, a Birch mechanism has been proposed for enzymatic reduction of the aromatic ring, which predicts alternating one-electron transfer and protonation steps (8.Koch J. Eisenreich W. Bacher A. Fuchs G. Eur. J. Biochem. 1993; 211: 649-661Crossref PubMed Scopus (75) Google Scholar,9.Buckel W. Keese R. Angew. Chem. 1995; 107: 1595-1598Crossref Google Scholar). A ketyl radical anion at the carbonyl C atom of the thioester group rather than a benzene radical anion was suggested as the first intermediate. In case of benzene reduction, the potential for the first electron transfer step is −3.1 V (versus a normal hydrogen electrode) (28.Mortensen J. Heinze J. Angew. Chem. 1984; 96: 64-65Crossref Google Scholar). A thioester group at the aromatic ring should lower this potential significantly (9.Buckel W. Keese R. Angew. Chem. 1995; 107: 1595-1598Crossref Google Scholar). Indeed, usingS-ethyl-thiobenzoate the potential is lowered to −1.9 V (versus a normal hydrogen electrode). This drastic effect illustrates the importance of the thioester group in enzymatic reduction of benzoyl-CoA. However, the energetics of enzymatic ring reduction are still not clear. The two electron reduction of the aromatic ring is driven by the stoichiometric hydrolysis of 2 ATP to 2 ADP + 2 Pi. If the free hydrolysis energy of both ATP molecules (∼−100 kJ) is coupled to a single electron transfer the redox potential can maximally be lowered by 1 V. Assuming this situation, the potential of the first electron transferred from the natural electron donor ferredoxin withE′° = −0.45 (versus normal hydrogen electrode) could be lowered to −1.45 V in enzymatic aromatic ring reduction. In conjunction with the determined potential of the model compoundS-ethyl-thiobenzoate (−1.9 V), the hydrolysis of ATP appears to be insufficient to enable ring reduction. However, partial protonation of the electron accepting carbonyl function by a protonated amino acid residue or a protonated Fe-S cluster could result in a more positive redox potential for the first electron transfer yielding the ketyl radical anions 11 and 12 (Fig.9) as proposed by Buckel and Keese (9.Buckel W. Keese R. Angew. Chem. 1995; 107: 1595-1598Crossref Google Scholar). The subsequent protonation of the radical anion seems to be highly exergonic, thus driving the endergonic first electron transfer step. The transfer of the second electron to the protonated free radical (compound 13) should require less energy than the formation of the radical anion, and protonation of compound 14 to the diene product (compound 2) is considered a highly exergonic reaction. We thank A. Werner and F. Wendling for expert help with the preparation of the manuscript.

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