Biotransformation of Benzothiophene by Isopropylbenzene- Degrading Bacteriat RICHARD W

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Biotransformation of Benzothiophene by Isopropylbenzene- Degrading Bacteriat RICHARD W. EATON'* AN JAMES D. NIT1IERAUER2f Environmental Research Laboratory, U.S. Environmental Protection Agency, Gulf Breeze, Florida 32561,1 and Technical Resources, Inc., GulfBreeze, Florida 32561, and University ofArkansas for Medical Sciences, Little Rock, Arkansas 722052 Received 28 January 1994/Accepted 19 April 1994

Isopropylbenzene-degrading bacteria, including Pseudomonas putida RE204, transform benzothiophene to a mixture of compounds. Induced strain RE204 and a number of its TnS mutant derivatives were used to accumulate these compounds and their precursors from benzothiophene. These metabolites were subsequently identified by 1H and 13C nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry. When strain RE204 was incubated with benzothiophene, it produced a bright yellow compound, identified as trans-4-[3-hydroxy-2-thienylJ-2-oxobut-3-enoate, formed by the rearrangement of cis-4-(3-keto-2,3-dihydrothienyl)-2-hydroxybuta-2,4-dienoate, the product of 3-isopropylcatechol-2,3-dioxygenase-catalyzed ring cleavage of 4,5-dihydroxybenzothiophene, as well as 2-mercaptophenylglyoxalate and 2'-mercaptomandelaldehyde. A dihydrodiol dehydrogenase-deficient mutant, strain RE213, converted benzothiophene to cis-4,5- dihydroxy4,5-dihydrobenzothiophene and 2'-mercaptomandelaldehyde; neither trans-4-[3-hydroxy-2-thienyl]- 2-oxobut-3-enoate nor 2-mercaptophenylglyoxalate was detected. Cell extracts of strain RE204 catabzed the conversion of cis-4,5-dihydroxy-4,5-dihydrobenzothiophene to trans-4-[3-hydroxy-2-thienyl]-2-oxobut-3-enoate in the presence of NAD+. Under the same conditions, extracts of the 3-isopropylcatechol-2,3-dioxygenase- deficient mutant RE215 acted on cis-4,5-dihydroxy-4,5-dihydrobenzothiophene, forming 4,5-dihydroxybenzothiophene. These data indicate that oxidation of benzothiophene by strain RE204 is initiated at either ring. Transformation initiated at the 4,5 position on the benzene ring proceeds by three enzyme-catalyzed reactions through ring cleavage. The sequence of events that occurs following attack at the 2,3 position of the thiophene ring is less clear, but it is proposed that 2,3 dioxygenation yields a product that is both a cis-dihydrodiol and a thiohemiacetal, which as a result of this structure undergoes two competing reactions: either spontaneous opening of the ring, yielding 2'-mercaptomandelaldehyde, or oxidation by the dihydrodiol dehydrogenase to another thiohemiacetal, 2-hydroxy-3-oxo-2,3-dihydrobenzothiophene, which is not a substrate for the ring cleavage dioxygenase but which spontaneously opens to form 2-mercaptophenylglyoxaldehyde and subsequently 2-mercaptophenylglyoxalate. The yellow product, trans4[3-hydroxy-2-thienylJ-2-oxobut-3-enoate, is a structural analog of nins-o-hydroxybenzylidenepyruvate, an intermediate of the naphthalene catabolic pathway; extracts of recombinant bacteria containing zns-o-hydroxybenzylidenepyruvate hydratase-aldolase catalyzed the conversion of ans-4-[3-hydroxy-2-thienyl]-2-oxobut-3-enoate to 3-hydroxythiophene-2-carboxaldehyde, which could then be further acted on, in the presence of NAD+, by extracts of recombinant bacteria containing the subsequent enzyme of the naphthalene pathway, salicylaldehyde dehydrogenase.

Sulfur-containing heterocyclic chemicals, including benzo- dihydrobenzothiophene and cis- and trans-2,3-dihydroxy-2,3- thiophene (benzo-[b]-thiophene, thianaphthene), comprise a dihydrobenzothiophene; it was suggested that the trans-2,3- relatively small but significant fraction of petroleum, coal, and dihydrodiol was formed by the spontaneous isomerization of shale (5, 9, 26, 30, 42). the cis-2,3-dihydrodiol through an undetected aldehyde inter- Although benzothiophene appears to readily undergo bio- mediate (3). A naphthalene-degrading enrichment culture transformation by environmental microorganisms (2, 3, 16, 18, produced a combination of three chemicals, benzothiophene- 19, 23, 25, 31-33, 38), metabolic intermediates or products 2,3-dione, benzothiophene sulfoxide, and benzothiophene-2,3- have been identified in only a few of these studies. A 1-meth- dihydrodiols (2), while a methanogenic consortium produced ylnaphthalene-degrading strain, Pseudomonas strain BT1, was several intermediates, including 2-hydroxybenzothiophene, shown to transform benzothiophene to benzothiophene-2,3- 7-hydroxybenzothiophene, and 2-hydroxythiophene, as well as dione and 3-methylbenzothiophene to the sulfoxide and sul- other aromatic and aliphatic acids and alcohols (23). Aside fone (19). Pseudomonasputida UV4, a dihydrodiol-accumulating from the work of Fedorak and Grbic-Galic (19), few data are mutant derivative of a toluene-degrading strain, transformed provided in support of these structural assignments. benzothiophene to three dihydrodiols: cis-4,5-dihydroxy-4,5- As part of a program to study the metabolism of heterocyclic compounds, various aromatic hydrocarbon-degrading bacteria were examined for the ability to catalyze biotransformations of * Corresponding author. Mailing address: Environmental Research benzothiophene. All 36 isopropylbenzene-utilizing bacteria Laboratory, U.S. Environmental Protection Agency, 1 Sabine Island Dr., Gulf Breeze, FL 32561. Phone: (904) 934-9345. Fax: (904) tested transformed benzothiophene to a bright yellow product 934-9201. Electronic mail address: [email protected]. (Xmax = 435 nm) after growth with isopropylbenzene. This t Contribution 880 from the Environmental Research Laboratory, report describes the identification of this and other products of U.S. Environmental Protection Agency, Gulf Breeze, Fla. the biotransformation of benzothiophene by one of these t Present address: Avanti Corporation, Gulf Breeze, FL 32561. strains, P. putida RE204, and TnS mutant derivatives blocked 3992 VOL. 176, 1994 BENZOTHIOPHENE BIOTRANSFORMATION 3993

TABLE 1. Bacterial strains and plasihids used Strain or plasmid Description Reference E. coli JM109 recA endAl gyrA96 thi hsdR17 supE44 reLAl A(lac-proAB) (F' traD36 proAB lacIPZAM15) 43 P. putida RE204 Grows with isopropylbenzene, ethylbenzene, or toluene as sole source of carbon and energy 15 RE213 TnS mutant derivative of strain RE204, defective in the metabolism of isopropylbenzene; accumulates 15 2,3-dihydroxy-2,3-dihydroisopropylbenzene from isopropylbenzene RE215 Tn5 mutant derivative of strain RE204, defective in the metabolism of isopropylbenzene; accumulates 15 3-isopropylcatechol from isopropylbenzene RE225 Tn5 mutant derivative of strain RE204, defective in the metabolism of isopropylbenzene; accumulates 15 2-hydroxy-6-oxo-7-methylocta-2,4-dienoate from isopropylbenzene Plasmids pRE701 Encodes trans-o-hydroxybenzylidenepyruvate hydratase-aldolase from NAH7, Apr 11 pRE672 Encodes salicylaldehyde and 1,2-dihydroxy-1,2-dihydronaphthalene dehydrogenases from NAH7, Cmr 11 in the isopropylbenzene metabolic pathway. Transformation of previously described and cell suspensions were passed twice this yellow product by enzymes derived from the NAH7- through a French pressure cell at between 14,000 and 20,000 encoded naphthalene catabolic pathway is also described. lb/in2 at 4°C. Cell debris was removed by centrifugation at A part of this work was presented previously (12). 47,800 x g for 40 min. Protein concentrations were determined by using the bicinchoninic acid protein assay reagent (Pierce MATERIALS AND METHODS Chemical Co., Rockford, Ill.), with bovine serum albumin as the standard. Bacterial strains and plasmids. Bacterial strains and plas- Biotransformation of benzothiophene by P. putida RE204. mids used in this study are described in Table 1. All isopro- Two 100-ml overnight cultures of P. putida RE204, grown in pylbenzene-degrading bacteria were isolated by selective en- minimal medium supplemented with 0.2% succinate and richment with isopropylbenzene and utilize that compound as 0.05% yeast extract, were used to inoculate two 13.5-liter Pyrex well as ethylbenzene and toluene as sole sources of carbon and carboys, each containing 8 liters of the same medium and a energy. 10-cm Teflon-coated magnetic stir bar. The cultures were Media. Luria-Bertani medium (8) was used for the cultiva- stirred vigorously at 30°C for 8 h on Thermolyne type 25500 tion of bacteria except where otherwise indicated. Minimal Maxi-Stirrer stir plates (Barnstead/Thermolyne, Dubuque, medium was R medium (13) containing trace elements (24) Iowa), at which time isopropylbenzene (1 mmol liter-1) was and a carbon source (0.1 to 0.2% [wt/vol]). Media were added to each and incubation continued for 16 h. The com- solidified with 1.5% agar (Bacto Agar or, for minimal media, bined cultures were harvested by centrifugation, and cells were Noble agar [Difco Laboratories, Detroit, Mich.]). resuspended in 2 liters of minimal medium in a carboy with a Chemicals. Benzo-[b]-thiophene, isopropylbenzene, ethyl- magnetic stir bar to which benzothiophene (1 g in 6.5 ml benzene, oxalyl chloride, aluminum chloride, and thiophenol ethanol [95%]) was added in four aliquots at 1-h intervals. were obtained from Aldrich Chemical Company, Milwaukee, Incubation was continued for a total of 22 h, after which no Wis. further change in the UV-visible spectrum of the supernatant Benzothiophene-2,3-dione was synthesized essentially as de- could be observed and at which time cells were removed by scribed by Clark and McKinnon (4; see also references 35 and centrifugation. The pH of the supernatant was then lowered to 40) except that in the initial step of the synthesis, a solution of 2.0 with HCl before extraction three times with diethyl ether (2 thiophenol was added dropwise to a solution of oxalyl chloride, liters) which was dried over sodium sulfate before it was instead of the reverse, to minimize the formation of dithiophe- removed under reduced pressure in the rotary evaporator. nyloxalate instead of the desired product, thiophenyloxalyl Biotransformation of benzothiophene by P. putida RE213. chloride. The thiophenyloxalyl chloride was subsequently cy- Conditions were similar to those for strain RE204 except that clized by using the Lewis acid, AlC13, to catalyze Friedel-Crafts two 1-liter overnight cultures of P. putida RE213 with kana- acylation. The product, benzothiophene-2,3-dione, was recrys- mycin at 100 ,ug ml-' were used as inocula, the medium used tallized from methanol, and its identity was confirmed by 'H in the transformation of benzothiophene was supplemented and 13C nuclear magnetic resonance (NMR) spectroscopy and with 0.2% succinate and adjusted to pH 7.2, and the pH of the gas chromatography-mass spectrometry (GC-MS). NMR spec- supernatant was not adjusted prior to extraction with diethyl tra were in good agreement with those published for this ether. compound (21, 22), as were mass spectra (19). Sodium 2-mer- Purification of products by chromatography on Sephadex captophenylglyoxalate was prepared by dissolving 50 mg of LH-20. Ether-extracted products were redissolved in about 20 benzothiophene-2,3-dione in 5 ml of 6 N NaOH to hydrolyze ml of 95% ethanol. The ethanol solution was filtered through the thioester and, after neutralization with HCl, removing the a Whatman no. 1 filter and applied to a column of Sephadex water by lyophilization. Its identity was confirmed by 'H NMR LH-20 (55 by 5 cm, volume = 1,080 ml), which was developed spectroscopy in deuterated dimethylsulfoxide (DMSO-d6) and with 95% ethanol. The column effluent was monitored by GC-MS of its dipentafluorobenzyl (di-PFB) derivative. recording UV-visible spectra (200 to 500 nm) of fractions (12.9 Preparation of cell extracts. To prepare extracts of Esche- ml) diluted in 50 mM K-Na phosphate buffer (pH 7). Fractions richia coli JM109(pRE701) containing trans-o-hydroxybenzyli- having similar spectra were pooled, and the solvent was denepyruvate hydratase-aldolase, of E. coli JM109(pRE672) evaporated under reduced pressure in the rotary evaporator. containing salicylaldehyde dehydrogenase (11), and of P. Biotransformation of benzothiophene by P. putida RE204 putida RE204 and RE215 (15), bacteria were cultivated as with alternate purification of products by Sephadex G-25 3994 EATON AND NITlERAUER J. BAC-IERIOL. chromatography. Four 100-ml overnight cultures of P. putida carrier gas at an average linear velocity of 37 cm s-' and a RE204, grown in minimal medium supplemented with 0.2% temperature program which retained the oven temperature at glutamate and 0.05% yeast extract, were used to inoculate four 50°C for 1 min and then increased the temperature to 95°C 2,800-ml Fernbach flasks containing 1 liter of the same me- over 2 min and finally to 290°C over 17.5 min. For analyses dium. After the cultures were incubated at 30°C for 3 h, using instrument B, samples were injected at 270°C onto a type isopropylbenzene was added to 1 mM and the incubations HP-5 capillary column (25 m by 0.32 mm, with a film thickness were continued for 4 h. Cells were harvested by centrifugation, of 0.52 p,m), using helium as the carrier gas at an average linear pooled, washed, and resuspended in 1 liter (total) of minimal velocity of 22 cm s-1 and a temperature program which medium (pH 7.2) supplemented with 0.2% glutamate and to increased the oven temperature from 50 to 150°C over 20 min which benzothiophene (510 mg dissolved in 2 ml of acetone) and then to 290°C over 14 min. was added. Following 16 h of incubation at 30°C, bacterial cells Proton and 13C NMR spectra were obtained with a General were removed by centrifugation and the supernatant was Electric model QE Plus spectrometer at 300 and 75 MHz, concentrated by lyophilization. Water was added to about 60 respectively. Assignment of chemical shifts was aided by homo- ml, and undissolved material was subsequently removed by nuclear automated chemical shift-correlated spectroscopy centrifugation. Approximately 20 ml of the aqueous solution (H,H-COSY), heteronuclear COSY (H,C-COSY), and at- was applied to a column (90 by 5 cm, volume = 1,760 ml) of tached proton test (APT) analyses where indicated. Sephadex G-25, which was developed with water. The column UV-visible spectra were recorded with an HP8452A diode effluent was monitored by recording the UV-visible spectra of array spectrophotometer when Sephadex column fractions fractions (14.4 ml) diluted in 50 mM K-Na phosphate buffer were monitored. Otherwise, a Perkin-Elmer Lambda 6 double- (pH 7). Fractions having similar spectra were pooled, and beam spectrophotometer was employed; spectra obtained with water was removed by lyophilization. this instrument and other figures were prepared for publication Small-scale biotransformations of benzothiophene by wild- by using CorelDRAW 3.0 (Corel Corp., Ottawa, Ontario, type isopropylbenzene-degrading bacteria. To produce small Canada). quantities of benzothiophene metabolites for analysis, isolated colonies of each bacterial strain were spread on carbon-free RESULTS minimal agar plates, which were then exposed to ethylbenzene vapor under a bell jar for 4 days. Each plate was then flooded Thirty-six bacterial strains, previously isolated for the ability with carbon-free minimal medium and scraped to remove the to grow with isopropylbenzene (cumene) and also shown to cells from the agar surface. Each cell suspension was made up grow with toluene and ethylbenzene, were tested for the ability to 5 ml and placed into a 25-ml screw-cap vial (75 by 20 mm), to transform various aromatic and heterocyclic chemicals. and a crystal (1 to 2 mg) of benzothiophene was added. Bottles Patches of these strains grown on minimal agar plates with were incubated with vigorous aeration at 30°C for 15 to 60 min isopropylbenzene or ethylbenzene as the sole carbon source for most strains, and overnight for less active strains, until the became intensely yellow within 15 min when crystals of ben- medium became yellow. Cells were removed by centrifugation zothiophene were added to the lid of the plate. This yellow in a microcentrifuge, and the supernatant was filtered through color-producing ability was inducible; bacteria grown on suc- an Acrodisc 13CR filter (Gelman Sciences, Ann Arbor, Mich.) cinate did not turn yellow immediately when exposed to into a gas chromatography (GC) vial, which was crimp sealed benzothiophene, although almost all became slightly yellow with a Teflon-coated seal before high-pressure liquid chroma- after overnight incubation with benzothiophene. tography (HPLC) analysis. When trans-4-[3-hydroxy-2-thi- Transformation of benzothiophene by one of these strains, enyl]-2-oxobut-3-enoate was apparent in the supernatants, its P. putida RE204 (15), was studied in detail. This strain was identity was confirmed by testing it as substrate for cell extracts used because its pathway for isopropylbenzene degradation of E. coli JM109(pRE701) containing trans-o-hydroxybenzyli- had been described previously (Fig. 1) and TnS-generated denepyruvate hydratase-aldolase as previously described (11). mutant strains RE213, RE215, and RE225, defective in 2,3- Identification of products. HPLC analyses were performed dihydroxy-2,3-dihydroisopropylbenzene dehydrogenase (Fig. with a 5-pum Hypersil ODS reverse-phase column (100 by 2.1 1, enzyme B), 3-isopropylcatechol dioxygenase (Fig. 1, enzyme mm) (Hewlett-Packard [HP]) in an HP model 1090 chromato- C), and 2-hydroxy-6-oxo-7-methylocta-2,4-dienoate hydrolase graph. The solvent, which flowed at a rate of 0.5 ml min-1, was (Fig. 1, enzyme D), respectively, were available (15). a gradient of CH3CN-50 mM phosphate (pH 3.5) in which the Products of the biotransformation of benzothiophene by P. CH3CN concentration increased from 10 to 80% at the rate of putida RE204. The yellow product (BT204A) was purified by 3%/min. The column was monitored with a diode array chromatography on Sephadex LH-20 with 95% ethanol as the detector which allowed the recording of the UV-visible spectra solvent. It emerged from the column as a peak with highest (190 to 400 nm) of chromatographic peaks. concentration in fraction 92; fractions 88 to 97 were pooled, For GC-MS analyses, trimethylsilyl (TMS) derivatives of and the solvent was removed to yield 138 mg of BT204A. acids were prepared by using N,O-bis(trimethylsilyl)-tri- Minor products appeared in low concentrations over a large fluoroacetamide (BSTFA) containing 1% trimethylchlorosi- number of fractions both before and after this peak, and those lane (TMCS) as instructed by the manufacturer (Pierce). formed in this transformation were not studied further (but see Alternatively, pentafluorobenzyl (PFB) derivatives were pre- below). pared by using pentafluorobenzylbromide (7). Product BT204A. Product BT204A (Fig. 2, chemical VII) Mass spectra were obtained with either an HP model 5988A was identified as trans-4-(3-hydroxy-2-thienyl)-2-oxobut-3-eno- mass spectrometer coupled to an HP model 5890 series II gas ate. The UV-visible spectrum of BT204A at pH 7 had a chromatograph (instrument A) or an HP model 5971A mass maximum at 435 nm (£ = 15,340 M` cm-'), while at pH 3.5 = spectrometer coupled to an HP model 5890 series II gas the absorbance maximum shifted to 360 nm (£ 13,900 M-1 chromatograph (instrument B). For analyses using instrument cm-l). The 1H NMR spectrum of BT204A in DMSO-d6 (with A, samples were injected at 290°C onto a type HP-5 capillary H,H-COSY analysis) showed four protons with chemical shifts column (cross-linked 5% phenylmethylsilicone, 25 m by 0.2 of 6.69 (d, H-d), 6.78 (d, H-a), 7.75 (d, H-b), and 7.93 (d, H-c) mm, with a film thickness of 0.11 pum), using helium as the ppm with coupling constants Jab = 5.4 Hz and Jcd = 15.6 Hz. VOL. 176, 1994 BENZOTHIOPHENE BIOTRANSFORMATION 3995

OHH A HB H 1V VI Bo H III IV FIG. 1. Pathway for the metabolism of isopropylbenzene by P. putida RE204. Chemical designations: I, isopropylbenzene; II, cis-2,3-dihydroxy- 2,3-dihydroisopropylbenzene; III, 2,3-dihydroxyisopropylbenzene; IV, 2-hydroxy-6-oxo-7-methylocta-2,4-dienoate; V, 2-hydroxypenta-2,4-dienoate; VI, isobutyrate. Enzymes: A, isopropylbenzene-2,3-dioxygenase; B, 2,3-dihydroxy-2,3-dihydroisopropylbenzene dehydrogenase; C, 3-isopropylcatechol 2,3-dioxygenase; D, 2-hydroxy-6-oxo-7-methylocta-2,4-dienoate hydrolase.

The 13C NMR spectrum (with APT and H,C-COSY analyses) G-25 with water as the solvent (Fig. 3). Although Sephadex is showed signals at 113.92 (C-2), 114.64 (C-7), 120.65 (C-4), normally used to separate molecules according to size, it also 131.94 (C-5), 137.63 (C-6), 160.83 (C-3 or C-9), 164.65 (C-3 or has an affinity for compounds with aromatic structures (27), C-9), and 184.37 (C-8) ppm. A difference in chemical shift and both Sephadex G-25 and Sephadex LH-20 have been used between carbons 4 and 5 of 11.29 ppm suggests that the previously for the separation of intermediates and products of hydroxyl group is attached to carbon 3, since deshielding due the bacterial metabolism of aromatic compounds (e.g., refer- to the hydroxyl group would be predicted to cause a large ences 10, 11, and 14). Three products were observed. The first decrease in the chemical shift of the neighboring carbon (12.7 product (BT204A) eluted with a maximum in fraction 108 and ppm). If the hydroxyl group were attached to C-2, a decrease was identical to trans-4-(3-hydroxy-2-thienyl)-2-oxobut-3-eno- of approximately 2.5 ppm in the chemical shift of C-4 would be ate previously isolated. The second product, BT204B, eluted predicted (36). with the highest concentration in fraction 116; fractions 114 to The TMS derivative of BT204A emerged from the GC 120 were pooled, and water was removed to yield 30 mg of (instrument B) at 31.32 min with major ions at mlz (percentage BT204B. The third product, BT204C, eluted with the highest of intensity, proposed composition of ions) 342 (5, [M]+), 327 concentration in fraction 134; fractions 128 to 141 were pooled, (8, [M - CH3]X), 225 (100, [M - TMS - CO2]+), 167 (5, [M and the water was removed to yield 60 mg of BT204C (or - TMS - CO2 - C2H2S]+), 147 (6), 73 (57, [TMS]+), and 45 BT204Ca; see below). Because inorganic salts were not previ- (9) indicative of a difunctionalized (di-TMS) product of a ously removed, they may have been present in some of these compound of molecular weight 198. fractions; thus, yields may be lower than they appear to be. A minor contaminant, not observed in the NMR spectra of These salts did not affect subsequent NMR and GC-MS BT204A, which emerged from the GC at 13.80 min had major analyses. ions at mlz (percentage of intensity, proposed composition of Product BT204B. Product BT204B was identified as 2-mer- ions) 164 (5, [M]+), 136 (100, [M - CO]+), 108 (45, [M - CO captophenylglyoxalate (Fig. 2, chemical XIV). The 'H NMR - CO]+), 92 (5), 82 (11), 76 (6), and 69 (23). This mass spectrum of BT204B showed signals at 7.32 (t, H-b), 7.48 (t, spectrum is identical to that published for benzothiophene-2,3- H-c), 7.62 (d, H-a), and 7.95 (d, H-d) ppm with coupling dione (19) and that of the compound synthesized in this work constants Jab = 8 Hz, Jbc = 7.4 Hz, Jcd = 7 Hz, Jac = 1.2 Hz, (Fig. 2, chemical X). and Jbd = 0.6 Hz and was identical to that of 2-mercaptophe- In a second experiment, products of the biotransformation nylglyoxalate synthesized in this work. The 13C NMR spectrum of benzothiophene by strain RE204 were concentrated by (with APT analysis) showed signals at 125.20 (C-5), 125.63 lyophilization and purified by chromatography on Sephadex (C-3), 131.96 (C-2), 133.04 (C-6), 134.15 (C-4), 139.01 (C-1),

Hc OH

Vll VliH Hf VII Vill IX X

OH Hd 0 Ha\OH H°s[X HbxCHcO H xl Xll XiII Ha XIV QH H OH OH X

XV XVI XVII FIG. 2. Structures of chemicals identified in this study. Chemical designations: VII, trans-4-[3-hydroxy-2-thienyl]-2-oxobut-3-enoate; VIII, cis-4,5-dihydroxy-4,5-dihydrobenzothiophene; IX, trans-2,3-dihydroxy-2,3-dihydrobenzothiophene; X, benzothiophene-2,3-dione; XI, 3-hydroxythiophene-2-carboxaldehyde; XII, 5-hydroxybenzothiophene; XIII, 3-hydroxybenzothiophene; XIV, 2-mercaptophenylglyoxalate; XV, 4-hydroxybenzothiophene; XVI, 2-hydroxybenzothiophene; XVII, 2'-mercaptomandelaldehyde. 3996 EATON AND NITTERAUER J. BACTERIOL.

(Fig. 2, chemical XIII) or their keto tautomers. These data, including the coupling constant, Jab' of 4.8 Hz for protons a and b, could indicate either cis or trans stereochemistry (29). E The trans configuration for BT204Ca is assigned on the basis of E40C additional analyses (see below). Compound BT204Ca is not present in neutral culture supematants; it is formed from E BT204C. The most likely chemical precursor to BT204Ca is 30- 1 ~~~224 2'-mercaptomandelaldehyde (Fig. 2, chemical XVII) which,

c 12-,d 234 purified and dried, would spontaneously cyclize to form the

0 thiohemiacetal, trans-2,3-dihydroxy-2,3-dihydrobenzothiophene. Products of biotransformation of benzothiophene by P.

318 putida RE213. Chromatography of the ether-extracted products of benzothiophene transformation by dihydrodiol-accu- 1 110d 1 14 150t mulating mutant strain RE213 on Sephadex LH-20 with 95% fraction ethanol as the solvent separated two compounds. The first, FIG. 3. Chromatographic separation of products of biotransforma- BT213A, eluted with a peak in fraction 88 (Xmax = 292 nm); tion of benzothiophene by strain RE204, using Sephadex G-25. fractions 85 to 89 were pooled, and solvent was removed to Numbers above chromatographic peaks indicate the km.x (nanom- yield 124 mg of BT213A. The second, BT213B, of the eluted with a eters) UV-visible spectra of fractions that make up the peaks. peak in fraction 94 (XmaX = 244 nm); fractions 92 to 96 were pooled, and solvent was removed to yield 41 mg of BT213B. Product BT213A. BT213A was analyzed by 'H and "3C 168.73 (C-8), and 198.20 (C-7) ppm. The di-PFB derivative NMR spectroscopy and GC-MS. These data are consistent analyzed by GC-MS (instrument a) (R, = 24.79 min) gave ions with the identification of BT213A as either cis-4,5-dihydroxy- at m/z (percentage of intensity, proposed composition of ions) 4,5-dihydrobenzothiophene (Fig. 2, chemical VIII) or cis-6,7- 542 (0.6, [M]+), 524 (1.4), 361 (32, [M - PFB]+), 317 (49, [M dihydroxy-6,7-dihydrobenzothiophene. However, BT213A has - PFB - CO2]), 289 (0.8, [M - PFB - CO2 - CO]+), 269 been designated the 4,5-dihydrodiol because the wild-type (2.3), 237 (1.1), 211 (1.0), 181 (100, [PFB]+), 161 (4, [PFB - strain RE204 transforms benzothiophene to trans-4-(3-hy- HF]+), 136 (14, [M - PFB - CO2 - PFB]+), 108 (10, [M - droxy-2-thienyl)-2-oxobut-3-enoate, whose formation can be PFB - CO2 - CO - PFB]+), and 69 (6). This was identical to accounted for by a route involving the 4,5-dihydrodiol, not the the mass spectrum of a PFB derivative of synthetic 2-mercaptophenylglyoxalate. 6,7-dihydrodiol; also, extracts of strain RE204 convert BT213A Product BT204C. Pooled and dried fractions of material to trans-4-(3-hydroxy-2-thienyl)-2-oxobut-3-enoate (see below). The 'H NMR spectrum of BT213A from the third chromatographic peak, having a UV spectrum in DMSO-d6 with with absorbance maxima at 224 and 318 nm, were left in the H,H-COSY analysis showed protons with chemical shifts of freezer (-20°C) for 1 month before analysis. At that time, the 4.23 (q, H-d), 4.45 (t, H-c), 4.83 (d, H-g), 4.93 (d, H-h), 5.8 (t, UV spectrum had changed to one having maxima at 208, 244, H-e), 6.48 (d, H-f), 7.06 (d, H-b), and 7.34 (d, H-a) ppm with and 286 nm. This product, BT204Ca, formed from BT204C, coupling constants Jab = 5.0 Hz, Jcd = 5.3 Hz, Jde = 3.1 Hz, Jef has been identified as trans-2,3-dihydroxy-2,3-dihydrobenzo- = 9.8 Hz, Jcg = 5.7 Hz,Jdh = 6.2 Hz, and Jfd = 2.0 Hz. The 13C thiophene. NMR spectrum with APT analysis showed signals at 66.97 (C-4 The 'H NMR spectrum of BT204Ca in CDC13 showed or C-5), 69.52 (C-4 or C-5), 120.03 (C-7), 124.02 (C-2), 128.10 protons with chemical shifts of 5.14 (d, H-a), 5.58 (d, H-b), 7.19 (C-3), 130.54 (C-6), 134.00 (C-7a), and 138.67 (C-3a) ppm. (t, H-d), 7.20 (d, H-c), 7.26 (t, H-e), and 7.39 (d, H-f) ppm with Protons c and d, with coupling constant Jcd = 5.3 Hz, are coupling constants Jab = 4.8 Hz, Jef = 7.4 Hz, and Jcd and Jde assigned a cis relative stereochemistry by comparison to the = -7 Hz. The 13C NMR spectrum showed eight signals at similar dihydrodiols, cis- and trans-1,2-dihydroxy-1,2-dihy- 78.46 (C-2 or C-3), 82.61 (C-2 or C-3), 123.28, 125.21, 125.38, dronaphthalenes. The coupling constant for the protons at- and 129.42 (C-4, C-5, C-6, and C-7), 136.3 (C-3a or C-7a), and tached to carbons 1 and 2 in the cis isomer is 5.1 Hz, while that 138.3 (C-3a or C-7a) ppm. Analysis of BT204Ca by GC-MS for the trans isomer is 10.1 Hz (28). (instrument A) gave three chromatographic peaks. The major BT213A gave three GC-MS (instrument A) total ion chro- peak (90%, Rt = 14.91 min) possessed a mass spectrum matographic peaks of approximately equal areas. The first consistent with the identification of BT204Ca as 2,3-dihydroxy- peak (Rt = 13.96 min) had major ions of m/z (percentage of 2,3-dihydrobenzothiophene and had ions of m/z (percentage of of 168 150 intensity, proposed composition of ions) 168 (79, [M]+), 150 intensity, proposed composition ions) (37, [M]+]), (100, [M - H2O]+), 139 (41, [M - CHO]+), 122 (53, [M - (14, [M - H20]5, 139 (100, [M - CHO]+, 137 (35), 122 (29, H20- CO]+), 121 (52, [M - H20- CHO]+, 111 (15), 96 [M - H20 - CO]+), 121 (56, [M - H20 - CHO]+), 107 (60), (23), and 85 (7). This mass spectrum is consistent with a 77 (93), and 69 (19). The first minor peak (5%, Rt = 12.48) had ions of m/z (percentage of intensity, proposed composition of dihydrodiol structure. The mass spectrum of the second chromatographic peak (R, = 14.07 min) had ions of m/z (percent- ions) 150 (94, [M]+), 122 (74, [M - CO]'), 121 (100, [M - CHO]'), 78 (37), and 69 (15). The second minor peak (5%, Rt age of intensity, proposed composition of ions) 150 (100, = 12.78 min) had ions of m/z (percentage of intensity, pro- [M]+), 122 (33, [M - CO]+), 121 (50, [M - CHO]+), and 96 posed composition of ions) 150 (100, [M]+), 122 (30, [M - (9) and resembled that of the third chromatographic peak (Rt CO]+), 121 (99, [M - CHO]+), 105 (10), 78 (27), 76 (21), and = 14.26 min), which had ions of mlz (percentage of intensity, 69 (12). These two mass spectra are those that would be proposed composition of ions) 150 (100, [M]+), 122 (14 [M - expected for the two products of the dehydration of 2,3- CO]+), 121 (30, [M - CHO]'), and 96 (3). The latter two mass dihydroxy-2,3-dihydroxybenzothiophene, 2-hydroxybenzothio- spectra are those expected for 4-hydroxybenzothiophene (Fig. phene (Fig. 2, chemical XVI), and 3-hydroxybenzothiophene 2, chemical XV) and 5-hydroxybenzothiophene (Fig. 2, chem- VOL. 176, 1994 BENZOTHIOPHENE BIOTRANSFORMATION 3997

FIG. 4. Analysis of culture supernatants by HPLC. Strains RE213 and RE204 were incubated with benzothiophene, and the supernatants were analyzed by HPLC as described in Materials and Methods. (A) HPLC chromatogram of RE213 supernatant recorded at 207 nm; (B) HPLC chromatogram of RE204 supematant recorded at 207 nm; (C) spectrum of purified cis-4,5-dihydroxy-4,5-dihydrobenzothiophene (R, = 2.70 min); (D) spectrum of the 2.82-min peak from RE213 supematant; (E) spectrum of the 2.81-min peak from RE204 supernatant; (F) spectrum of synthetic benzothiophene-2,3-dione (R, 8.54 min); (G) spectrum of purified trans-2,3-dihydroxy-2,3-dihydrobenzothiophene (R, = 2.85 min); (H) spectrum of purified trans-4-[3-dihydroxy-2-thienyl]-2-oxobut-3-enoate (RJ = 0.99 min); (I) spectrum of synthetic 2-mercaptophenylglyoxalate (RI = 1.15 min); (J) spectrum of the 1.10-min peak from RE204 supernatant; (K) spectrum of the 1.06-min peak from RE204 supematant (data in panels J and K were recorded at different points in the same chromatographic peak). An additional relevant spectrum not shown is that of benzothiophene (R, = 14.80 min; Xm,, = 226, 256, 288, and 296 nm). ical XII) formed by dehydration of cis-4,5-dihydroxy-4,5-dihy- Analysis of culture supernatants by HPLC. Culture super- drobenzothiophene in the 290°C injector. natants of P. putida RE204, its mutant derivatives, and other Identification of product BT213B. BT213B was analyzed by isopropylbenzene-degrading bacteria incubated with benzo- 1H and 13C NMR spectroscopy and GC-MS. The results thiophene were analyzed by HPLC (Fig. 4). Besides the void obtained from these analyses are identical to those obtained volume peak at about 0.6 min and the peak due to benzothio- with compound BT204Ca and thus indicate that BT213B is phene at 14.80 min, the supernatant from strain RE204 trans-2,3-dihydroxy-2,3-dihydrobenzothiophene. contained chemicals emerging at 1.10 and 2.81 min (Fig. 4B). The UV spectra of compounds BT204Ca and BT213B The 1.10-min peak from strain RE204 contains a mixture of dissolved in 50 mM K-Na phosphate (pH 7) remained un- two chemicals (Fig. 4J). The major component of the peak is changed for at least 24 h. This finding suggests that if these trans-4-(3-hydroxy-2-thienyl)-2-oxobut-3-enoate (Fig. 4H), products were formed and accumulated during incubation of which can be distinguished from the second component by strain RE204 or RE213 with benzothiophene, they should have spectral analysis across the peak (Fig. 4K). The second com- been detected in the HPLC analysis of the culture superna- ponent appears to be 2-mercaptophenylglyoxalate (Fig. 41), tants (below). which has the same retention time and absorbance maxima 3998 EATON AND NITITERAUER J. BAC=ERIOL.

(232 and 266 nm) found in the composite spectrum (Fig. 4J). The 2.81-min peak from strain RE204 has a spectrum (Fig. 4E) with max at 224 and 318 nm; this spectrum is identical to that of product RE204C (proposed to be 2'-mercaptomandelaldehyde) after its purification by Sephadex G-25 chromatography. The supernatant from the incubation of strain RE213 with benzothiophene contained a single peak at 2.82 min (Fig. 4A). The spectrum of the peak (Fig. 4D) appears to be a composite of at least two spectra, that of cis-4,5-dihydroxy-4,5-dihydrobenzothiophene (Fig. 4C) and that of the putative 2'-mercaptomandelaldehyde (also formed by strain RE204 [Fig. 4E]). r_= Urij This supernatant did not contain detectable amounts of trans- 2,3-dihydroxy-2,3-dihydrobenzothiophene (Fig. 4G), benzothiophene-2,3-dione (Fig. 4F), or 2-mercaptophenylglyoxalate 0.8 (Fig. 41). Similar HPLC analysis of the supernatant of strain RE225, a mutant defective in 2-hydroxy-6-oxo-7-methylocta-2,4-dienoate hydrolase (Fig. 1D), incubated with benzothiophene, demonstrated the presence of the same products as formed by strain RE204 (data not shown). The hydrolase, therefore, is 0 not required for these transformations. Transformation of cis-4,5-dihydroxy-4,5-dihydrobenzothiophene by extracts of strains RE215 and RE204. The two benzothiophene dihydrodiols were tested together with NAD+ as substrates for the isopropylbenzene dihydrodiol dehydroge- strains nase present in extracts of isopropylbenzene-induced 250 3& 350 460 450 500 RE215 and RE204. Extracts of the wild-type strain RE204 transformed cis-4,5-dihydroxy-4,5-dihydrobenzothiophene wavelength (nanometers) (Xmax = 291 nm) to trans-4-(3-hydroxy-2-thienyl)-2-oxobut-3- FIG. 5. Transformation of cis-4,5-dihydroxy-4,5-dihydrobenzothio- enoate (Fig. 5), while extracts of the mutant strain RE215, phene by cell extracts of P. putida RE204. Sample and reference defective in 3-isopropylcatechol dioxygenase, failed to convert cuvettes contained 50 mM K-Na phosphate buffer (pH 7) and 50 nmol to this product of NAD+ in 1-ml volumes at 30°C. The sample cuvette also contained cis-4,5-dihydroxy-4,5-dihydrobenzothiophene cis-4,5-dihydroxy-4,5-dihydrobenzothiophene. Spectra were recorded but instead accumulated a product with an absorbance maxi- before addition of 1 ,ul of cell extract of strain RE204 containing 24 pLg mum at 338 nm (Fig. 6) presumed to be 4,5-dihydroxybenzo- of protein to both cuvettes, immediately after addition, and after 4, 8, thiophene (chemical XVIII). This intermediate is unstable and 12, 16, 20, 24, 28, and 32 min. changed slowly over several hours to a second compound with an absorbance maximum at 307 nm, possibly benzothiophene- 4,5-quinone (chemical XX). Neither compound was isolated. Dihydrodiol dehydrogenase activity was also measured in below). This reaction proceeded at a rate that was 29% of that phosphate buffer (pH 7.5) by monitoring the rate of formation with the usual substrate, trans-o-hydroxybenzylidenepyruvate. of NADH at 340 nm. cis-4,5-Dihydroxy-4,5-dihydrobenzothio- To identify the product, a large-scale incubation was carried phene was transformed by extracts of strain RE215 at a rate of out in which a dialysis bag holding 5 ml of hydratase-aldolase- 102 nmol min-' mg of protein-, which is 73% of the rate at containing cell extract was floated in 200 ml of a solution of 50 which 2,3-dihydroxy-2,3-dihydroisopropylbenzene was trans- mg of trans-4-(3-hydroxy-2-thienyl)-2-oxobut-3-enoate in 50 formed (139 nmol min-' mg of protein-1). The other dihy- mM K-Na phosphate buffer (pH 6.8). This was slowly stirred at drodiol isolated in this study, trans-2,3-dihydroxy-2,3-dihydro- room temperature, and the progress of the reaction was benzothiophene (Fig. 2, chemical IX), was not acted on by monitored spectrophotometrically. When the reaction was these extracts under identical conditions. complete, the dialysis bag was removed, and the reaction Transformation of trans-4-(3-hydroxy-2-thienyl)-2-oxobut- mixture was adjusted to pH 3 with HCl and extracted three 3-enoate by NAH7-encoded trans-o-hydroxybenzylidenepyru- times with methylene chloride. The solvent was subsequently vate hydratase-aldolase and salicylaldehyde dehydrogenase. dried by passing it through sodium sulfate before removal. It Compound BT204A, trans-4-(3-hydroxy-2-thienyl)-2-oxobut-3- was necessary to acidify the solution to recover the product enoate, has a structure analogous to that of trans-o-hydroxy- because the hydroxyl group of 3-hydroxythiophene-2-carboxal- benzylidenepyruvate, an intermediate of the naphthalene cat- dehyde is relatively acidic (pKa = 5.42) (34). The 1H NMR abolic pathway (11). It was expected that the enzyme that acts spectrum of 3-hydroxythiophene-2-carboxaldehyde (Fig. 2, on trans-o-hydroxybenzylidenepyruvate might also act on this chemical XI) in CDCl3 showed protons at 6.79 (d, H-a), 7.61 substrate analog; if so, this would provide a valuable confir- (d, H-b), and 9.65 (s, H-c) ppm with coupling constants Jab = mation of the proposed structure. Cell extracts of E. coli 5.3 and Jac = 0.63 Hz. This is essentially identical to the JM109(pRE701) containing the enzyme trans-o-hydroxyben- spectrum published by Roques et al. (37) for this compound in zylidenepyruvate hydratase-aldolase were incubated with trans- CC14, which showed protons at 6.73 (d, H-a), 7.51 (d, H-b), 4-(3-hydroxy-2-thienyl)-2-oxobut-3-enoate, and the reaction 9.54 (s, H-c), and 10.26 (s, hydroxyl proton) ppm with coupling was monitored spectrophotometrically (Fig. 7). Upon addition constants Jab = 5.2 and Jac = 0.65. When the compound was of extracts, the spectrum of the substrate (Xmax = 435 and 338 analyzed by GC-MS, the major peak emerged at 7.83 min, nm) was replaced by the spectrum of the product, 3-hydroxy- having ions at m/z (percentage of intensity, proposed compo- thiophene-2-carboxaldehyde (\max = 355 and 283 nm) (see sition of ions) 128 (100, [M]+), 127 (99, [M - H]+), 110 (2.5, VOL. 176, 1994 BENZOTHIOPHENE BIOTRANSFORMATION 3999

20)4 t

wavelength (nanometers) FIG. 7. Transformation of trans-4-[3-hydroxy-2-thienyl]-2-oxobut- 3-enoate to 3-hydroxythiophene-2-carboxaldehyde by cell extracts of E. coli JM109(pRE701). The sample and reference cuvettes contained wavelength (nanometers) 50 mM K-Na phosphate buffer (pH 7.0) in 1-ml volumes at 30°C. The sample cuvette also contained 52.5 nmol of substrate. Spectra were FIG. 6. Transformation of cis-4,5-dihydroxy-4,5-dihydrobenzothio- recorded before the addition of 2 ,ul of extract containing 1.8 ,ug of phene by cell extracts of P. putida RE215. The initial contents osf the protein to both cuvettes and after 0.17, 4, 8, 12, 16, 20, 24, and 28 min. cuvettes were as in Fig. 5. Spectra were recorded before the addition For trans-4-[3-hydroxy-2-thienyl]-2-oxobut-3-enoate, £435 iS 15,340 of 1 ,1l of cell extract of strain RE215 containing 21 F±g of protein to M-1 cm-l and £335 is 4,930 M- cm-l; for the product, 3-hydroxythio- both cuvettes, immediately after addition, and after 5, 10, 15, 20, 25, 30, phene-2-carboxaldehyde, 6435 is 825 M-l cm-', 356 is 8,260 M` 35, 40, 45, 50, 55, and 60 min. cm , and £283 is 5,987 M-l cm-'.

[M - H2O]+), 99 (2, [M - CO - H]+), 74 (8), 71(5, [M - CO containing 2-mercaptophenylglyoxalate would result in recov- - H - CO]+), and 45 (10). ery of benzothiophene-2,3-dione. The product of the hydratase-aldolase-catalyzed cleavage of Other isopropylbenzene-degrading bacteria. Supernatants trans-4-(3-hydroxy-2-thienyl)-2-oxobut-3-enoate, 3-hydroxy- of other isopropylbenzene-degrading bacteria incubated with thiophene-2-carboxaldehyde, has a structure analogous to that benzothiophene, analyzed by HPLC, were similar to that of of salicylaldehyde (o-hydroxybenzaldehyde) and thus was a strain RE204, with some variations in the relative quantities of potential substrate for the next enzyme of the naphthalene metabolites. While these variations may have resulted from catabolic pathway, salicylaldehyde dehydrogenase. To test this activities of other metabolic pathways in these organisms, possibility, NAD+ and cell extracts of E. coli JM109(pRE672) definite correlations have not been established. Wherever containing salicylaldehyde dehydrogenase were added to spec- trans-4-(3-hydroxy-2-thienyl)-2-oxobut-3-enoate was evident in trophotometer cuvettes at the end of the reaction shown in Fig. culture supernatants, treatment with trans-o-hydroxybenzylide- 7, and the reaction was monitored spectrophotometrically (Fig. nepyruvate hydratase-aldolase was monitored spectrophoto- 8). The spectrum of 3-hydroxythiophene-2-carboxaldehyde was metrically. In all cases, the transformations proceeded as in converted to a spectrum having an absorbance maximum at Fig. 7 with production of the spectrum of 3-hydroxythiophene- 253 nm with the transient appearance of an intermediate 2-carboxaldehyde. having a spectrum with km.x at 276 nm. It is likely that these spectral changes are due to the formation of 3-hydroxythio- DISCUSSION phene-2-carboxylate (chemical XXII); further spontaneous tautomerization to the keto form would produce a ,-keto acid Biotransformation of benzothiophene (Fig. 9, chemical which could readily decarboxylate to give 3-oxo-2,3-dihydro- XXIV) is accomplished in P. putida RE204 by enzymes that thiophene. However, these products were not characterized. normally catalyze reactions of the isopropylbenzene (alkylben- Although 2-mercaptophenylglyoxalate is rapidly converted zene) catabolic pathway (Fig. 1 and Fig. 9, enzymes A to C). to its thiolactone, benzothiophene-2,3-dione, in acidic solu- This biotransformation can be initiated at either the benzene tions with the reverse, ring opening, occurring in alkaline ring or the thiophene ring by isopropylbenzene-2,3-dioxygen- solutions, both compounds are stable in aqueous solutions at ase (Fig. 9, enzyme A) with different consequences. neutral pH (data not shown). This finding suggests that if one Oxidation of the benzene ring yields cis-4,5-dihydroxy-4,5- of these compounds is detected in culture supernatants, as is dihydrobenzothiophene (Fig. 9, chemical VIII), a substrate the case for 2-mercaptophenylglyoxalate, its presence is not for 2,3-dihydroxy-2,3-dihydroisopropylbenzene dehydrogenase due to its spontaneous formation from the other. On the other (Fig. 9, enzyme B) which converts it in the presence of NAD+ hand, acidification and extraction of culture supernatants to 4,5-dihydroxybenzothiophene (Fig. 9, chemical XVIII). This 4000 EATON AND NITTERAUER J. BAcTERiOL.

lates. This rearrangement is analogous to that which occurs following ring cleavage of 1,2-dihydroxynaphthalene during the metabolism of naphthalene (11). In that case, the ring cleavage product, 2-hydroxy-4-[2'-oxo-3,5-cyclohexadienyl]- buta-2,4-dienoate, rapidly rearranges to form first the more stable, aromatic, cis-o-hydroxybenzylidenepyruvate, which then cyclizes to the hemiketal, 2-hydroxychromene-2-carboxylate. Naphthalene-degrading bacteria are able to grow with naphthalene because they possess enzymes that act on the products of this rearrangement (11). Oxidation of the thiophene ring by isopropylbenzene-2,3- a dioxygenase produces cis-2,3-dihydroxy-2,3-dihydrobenzothiophene (Fig. 9, chemical XXV). As a thiohemiacetal and a cis-dihydrodiol, it appears to undergo two competing transformations. One reaction is the spontaneous opening of the thiohemiacetal to give 2'-mercaptomandelaldehyde (Fig. 9, chemical XVII), a chemical which accumulates. A second, competing reaction appears to be that catalyzed by the enzyme 2,3-dihydroxy-2,3-dihydroisopropylbenzene dehydrogenase (Fig. 9, enzyme B), to give 2-hydroxy-3-oxo-2,3-dihydrobenzothiophene, which is also a thiohemiacetal (Fig. 9, chemical XXVII) and spontaneously opens to give 2-mercaptophenyl- wavelength (nanometers) glyoxaldehyde (Fig. 9, chemical XXVIII). This does not accumulate but is oxidized either spontaneously or by cellular FIG. 8. Transformation of 3-hydroxythiophene-2-carboxaldehyde to 3-hydroxythiophene-2-carboxylate by cell extracts of E. coli enzymes to 2-mercaptophenylglyoxalate (Fig. 9, chemical JM109(pRE672). Following the completion of the reaction illustrated XIV). in Fig. 7, 2 ,ll of 5 mM NAD+ was added to both cuvettes. After the Evidence for these pathways comes from studies using P. spectrum was recorded, 10 ,ul of extract containing 160 t.g of protein putida RE204 and its mutant derivative strains RE213, RE215, was added, and spectra were recorded after 0.17, 10, 20, 30, 40, 50, 60, and RE225. Isopropylbenzene-grown strain RE204 converted 70, 80, 90, 100, 110, 120, 130, 140, 150, and 160 mi. benzothiophene to three products which were isolated and identified: trans-4-(3-hydroxy-2-thienyl)-2-oxobut-3-enoate (BT204A; Fig. 9, chemical VII), 2-mercaptophenylglyoxalate is then cleaved by 3-isopropylcatechol-2,3-dioxygenase (Fig. 9, (BT204B; Fig. 9, chemical XIV), and 2'-mercaptomandelalde- enzyme C), initially producing cis-4-(3-keto-2,3-dihydrothi- hyde (BT204C; Fig. 9, chemical XVII), the latter as an isomer, enyl)-2-hydroxybuta-2,4-dienoate (Fig. 9, chemical XIX), trans-2,3-dihydroxy-2,3-dihydrobenzothiophene (BT204Ca; which immediately rearranges to the more stable aromatic Fig. 9, chemical IX). trans-4-(3-hydroxy-2-thienyl)-2-oxobut-3-enoate (Fig. 9, chem- Two dihydrodiols were obtained following diethyl ether ical VII). This is not a substrate for the next enzyme of the extraction of culture supernatants of the dihydrodiol dehydro- isopropylbenzene catabolic pathway, 2-hydroxy-6-oxo-7-meth- genase-deficient mutant strain RE213 incubated with benzo- ylocta-2,4-dienoate hydrolase (Fig. 1, enzyme D), and accumu- thiophene. One of these dihydrodiols was cis-4,5-dihydroxy-

H - _SH - +s

lX ,XVIIx ii XXIX XIV X

H OH I H0i.

XXIV XXV XXVII XXVIII

H H XXIA V4HB l.

XX(IV Vill XVIII XIX VIl FIG. 9. Pathways for the biotransformation of benzothiophene by P. putida RE204. Chemical designations are as in Fig. 2. Additional designations: XXIV, benzothiophene; XVIII, 4,5-dihydroxybenzothiophene; XIX, cis-4-(3-oxo-2,3-dihydrothienyl)-2-hydroxybuta-2,4-dienoate; XXV, cis-2,3-dihydroxy-2,3-dihydrobenzothiophene; XXVII, 2-hydroxy-3-oxo-2,3-dihydrobenzothiophene; XXVIII, 2-mercaptophenylglyoxaldehyde; XXIX, 2'-mercaptomandelate. Enzymes: A, isopropylbenzene-2,3-dioxygenase; B, 2,3-dihydroxy-2,3-dihydroisopropylbenzene dehydrogenase; C, 3-isopropylcatechol 2,3-dioxygenase. Compounds in boxes have been purified and identified; heavy arrows indicate transformations that occur during extraction of products; dashed arrows indicate possible transformations that do not appear to occur. VOL. 176, 1994 BENZOTHIOPHENE BIOTRANSFORMATION 4001

4,5-dihydrobenzothiophene (BT213A; Fig. 9, chemical VIII). and this specificity ensures that the product of one reaction will This dihydrodiol was a substrate for an NAD+-requiring be a substrate for the next enzyme of the pathway. These dehydrogenase present in cell extracts of strains RE204 and enzymes may also act on substrate analogs, compounds that RE215. 4,5-Dihydroxybenzothiophene, accumulated by ex- are accommodated by their active sites and have the appropri- tracts of the 3-isopropylcatechol-2,3-dioxygenase-deficient ate reactivity to serve as substrates. Benzothiophene appears to strain RE215, was presumably spontaneously oxidized to its be accommodated in at least two different orientations at the corresponding quinone, while extracts of the wild-type strain active site of the dioxygenase and to undergo different regio- RE204, which contain 3-isopropylcatechol-2,3-dioxygenase, specific reactions; products resulting from pathways initiated converted the 4,5-dihydrodiol through 4,5-dihydroxybenzo- by dioxygenation of this substrate analog have been identified thiophene to a ring cleavage product not directly observed here and elsewhere (3, 19). Complex mixtures of products such because of its immediate rearrangement to trans-4-(3-hydroxy- as those demonstrated here are to be anticipated whenever 2-thienyl)-2-oxobut-3-enoate. fortuitous metabolism of a chemical occurs. If these products The second dihydrodiol (BT213B) was identical to BT204Ca are more recalcitrant or toxic than the starting chemical, this (Fig. 9, chemical IX), identified as trans-2,3-dihydroxy-2,3- may limit the value of cometabolic reactions in the bioreme- dihydrobenzothiophene. The observed coupling constant of 4.8 diation of this and other environmental pollutants. Hz for protons a and b in the 1H NMR spectra of BT204Ca Reductive (NADH- or NADPH-requiring) dioxygenase re- and BT213B does not distinguish cis and trans stereochemistry actions, such as that catalyzed by isopropylbenzene dioxygen- (29), and the assignment of trans stereochemistry is based on ase, are employed by bacteria as the first step in preparing an additional observations. Although it is stable in aqueous aromatic substrate for subsequent ring cleavage by a second solutions, this dihydrodiol was not detected in supernatants of dioxygenase. It is now evident that the introduction of hydroxyl strain RE204 or RE213 incubated with benzothiophene (Fig. groups can not only prepare heterocyclic substrates for ring 4). Furthermore, it was not a substrate for 2,3-dihydroxy-2,3- opening but also directly effect ring opening by the formation dihydroisopropylbenzene dehydrogenase, a reaction which ap- of unstable hemiacetal or hemiketal products. For example, in pears necessary before 2-mercaptophenylglyoxalate can be the bacterial metabolism of dibenzofuran (20), an initial formed from benzothiophene via the cis-dihydrodiol. It is angular dioxygenation produces a hemiketal which spontane- assumed that trans-2,3-dihydroxy-2,3-dihydrobenzothiophene ously opens, yielding 3-(2'-hydroxyphenyl)-catechol. In the is formed during ether extraction or drying as the more stable transformation of benzothiophene, opening of the thiophene product of recyclization of 2'-mercaptomandelaldehyde, the ring occurs because one of the dihydroxylation reactions ring-open form of cis-2,3-dihydroxy-2,3-dihydrobenzothio- produces a cyclic thiohemiacetal whose ring opening occurs phene. without the requirement for additional enzyme-catalyzed re- In a prior study, Boyd et al. (3) isolated three products of actions. biotransformation of benzothiophene by P. putida UV4, a Experiments in which enzymes of the naphthalene catabolic dihydrodiol-accumulating mutant similar to strain RE213. pathway were used to transform trans-4-(3-hydroxy-2-thienyl)- They identified these as the cis-4,5-, cis-2,3-, and trans-2,3- 2-oxobut-3-enoate indicate that it should possible to extend the dihydrodiols of benzothiophene and proposed that the product pathway for benzothiophene metabolism by combining genes having a coupling constant, Jab, of 4.5 Hz is the trans-2,3- encoding enzymes from the isopropylbenzene and naphthalene isomer, since the compound that they identified as the cis-2,3- pathways into a single strain. Of course, this construction does isomer has a smaller coupling constant, Jab' of 1.6 Hz. How- not prevent formation of products of oxidation of the thio- ever, this 1.6-Hz coupling constant is more typical of values for phene ring or facilitate their removal. It may eventually be coupling of an aldehydic proton and an ao-methylenic proton as possible, however, to construct strains that completely degrade are present in 2'-mercaptomandelaldehyde (Fig. 9, chemical benzothiophene by recruiting additional enzymes from previ- XVII). Boyd et al. (3) showed no NMR spectral data for these ously described bacteria that degrade compounds analogous to compounds other than the two coupling constants; their data the products described here, such as thiophene-2-carboxylate do not distinguish between cis- and trans-2,3-dihydrodiols. (1, 17, 41) and 2-hydroxyphenylglyoxalate (39). HPLC analysis of neutral supernatants from the incubation of wild-type strain RE204 with benzothiophene (Fig. 4) re- ACKNOWLEDGMENTS vealed only three products: trans-4-(3-hydroxy-2-thienyl)-2- oxobut-3-enoate, 2-mercaptophenylglyoxalate, and 2'-mercap- We thank W. Gilliam of the US EPA, Gulf Breeze, Fla., for GC-MS While was analyses; B. Blattmann and M. Downey of Technical Resources, Inc., tomandelaldehyde. 2'-mercaptomandelaldehyde and Avanti Corp., Gulf Breeze, for HPLC and GC-MS analyses; and J. produced by the dihydrodiol-accumulating mutant strain Gurst of the Chemistry Department, University of West Florida, RE213, the other two products were not. This finding indicates Pensacola, for NMR spectroscopy analyses. This work benefited from that 2'-mercaptomandelaldehyde formation requires only the discussions with S. Selifonov, Center for Environmental Diagnostics isopropylbenzene dioxygenase and that the formation of trans- and Bioremediation, University of West Florida, and P. J. Chapman, 4-(3-hydroxy-2-thienyl)-2-oxobut-3-enoate and 2-mercapto- US EPA, Gulf Breeze, and from critical reading of the manuscript by phenylglyoxalate requires the action of dihydrodiol dehydro- P. J. Chapman. genase. 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