APPLIED AND ENVIRONMENTAL MICROBIOLOGY, JUIY 1986, p. 98-100 Vol. 52, No. 1 0099-2240/86/070098-03$02.00/0 Copyright ©D 1986, American Society for Microbiology Demethylation of Veratrole by Cytochrome P-450 in Streptomyces setonii JOHN B. SUTHERLAND Institiute of Wood Research and the BioSoiurce Institiute, Michigan Technological University, Houghton, Michigan 49931 Received 27 January 1986/Accepted 15 April 1986

The actinomycete Streptomyces setonii 75Vi2 demethylates vanillic acid and guaiacol to protocatechuic acid and catechol, respectively, and then metabolizes the products by the ,-ketoadipate pathway. UV spectroscopy showed that this strain could also metabolize veratrole (1,2-dimethoxybenzene). When grown in veratrole- containing media supplemented with 2,2'-dipyridyl to inhibit cleavage of the aromatic ring, S. setonii accumulated catechol, which was detected by both liquid chromatography and gas chromatography. Reduced cell extracts from veratrole-grown cultures, but not sodium succinate-grown cultures, produced a carbon monoxide difference spectrum with a peak at 450 nm that indicated the presence of soluble cytochrome P-450. Addition of veratrole or guaiacol to oxidized cell extracts from veratrole-grown cultures produced difference spectra that indicated that these compounds were substrates for cytochrome P-450. My results suggest that S. setonii produces a cytochrome P-450 that is involved in the demethylation of veratrole and guaiacol to catechol, which is then catabolized by the 1-ketoadipate pathway.

Veratrole (1,2-dimethoxybenzene) is a xenobiotic cell mg of veratrole (3.6 mM), 200 mg of NaCl, 200 mg of depressant that reduces flagellar movement and cytoplasmic MgSO4- 7H20, 50 mg of CaCl2 .2H20, and 50 mg of Tween streaming in green algae (30) and inhibits glucose oxidation 80 (polyoxyethylene sorbitan monooleate). The pH was in fat cells (26). Kawakami (16) listed veratrole among the adjusted to 7.4. Flasks were inoculated and incubated with aromatic compounds metabolized by Pselidomonas ovialis; reciprocal shaking for 48 h at 37°C. Aromatic compounds in Kaiser and Hanselmann (15) found a mixed culture of spent culture media were measured by UV spectroscbpy, anaerobic bacteria that metabolized it to catechol. Veratrole using a Varian Techtron 635 spectrophotometer. Veratrole is metabolized by several species of fungi in the genus was omitted in some experiments; in others, it was replaced Fusarium (13) and serves as a substrate for the ligninase of by 3.6 mM guaiacol or sodium succinate. the white-rot fungus Phanerochaete chrysosporiurm (17). To detect intermediate products of veratrole metabolism, The lignin-degrading actinomycete Streptomyces setonii the veratrole-containing medium was modified by adding 0.5 75Vi2 metabolizes several aromatic compounds, including mM 2,2'-dipyridyl to inhibit catechol 1,2-dioxygenase (9, benzoic acid, catechol, gentisic acid, guaiacol, m-hy- 32). Catabolic intermediates were detected by reversed- droxybenzoic acid, p-hydroxybenzoic acid, protocatechuic phase high-performance liquid chromatography with a Var- acid, and vanillic acid (21); phenol (1); benzaldehyde, ian model 5020 liquid chromatograph. The column was a cinnamic acid, p-coumaric acid, ferulic acid, p- Varian MicroPak MCH-10 column (30 cm by 4 mm) operated hydroxybenzaldehyde, and vanillin (32); and homogentisic at 26°C. The mobile phase contained 12.5 mM KH2PO4 acid, p-hydroxyphenylacetic acid, p-hydroxyphenylpyruvic (adjusted to pH 3.0 with perchloric acid) and acetonitrile acid, phenylacetic acid, L-phenylalanine, phenylpyruvic (75:25, vol/vol). The flow rate was 1.5 ml/min, and the UV acid, and L-tyrosine (20). Demethylation processes are in- detector was set at 270 nm. Gas chromatography was volved in the metabolism of guaiacol (21) and of vanillic acid performed with a Hewlett-Packard model 5840A gas chro- in cultures that have been grown with ferulic acid (32). matograph fitted with a type DB-5 fused-silica capillary In this paper I report the ability of S. setonii to metabolize column (30 m; wide bore; film thickness, 0.25 pLm) obtained veratrole by converting it to guaiacol. Additional experi- from J & W Scientific. The carrier gas was H2 at a pressure ments were performed to determine whether veratrole was of 34 kPa, and the make-up gas was N2 at a flow rate of 30 demethylated by an iron-sulfur protein of the putid- ml/min. The injector and flame ionization detector tempera- amonooxin type, as in the metabolism of p-anisic acid by tures were 175 and 275°C, respectively. Pseudomonas piutida (3); a cytochrome P-450, as in the Induction of cytochrome P-450 was demonstrated in cul- metabolism of isovanillic acid by Nocatrdia sp. (5) and tures grown in media containing 3.6 mM veratrole, guaiacol, guaiacol by Moraxella sp. (10); or an H202-dependent or sodium succinate. Mycelia were harvested by low-speed ligninase, as in the metabolism of lignin by Phanerochaete centrifugation, washed in 114 mM KCI, and disrupted by chrysosporium (12, 17). grinding with alumina (type 305; Sigma Chemical Co.) (18). The extract was suspended in 24 mM Na2HPO4-KH2PO4 MATERIALS AND METHODS buffer (pH 7.4) and centrifuged at 25,000 x g for 20 min. The Stock cultures of S. setonii 75Vi2 (= ATCC 39116) were supernatant was centrifuged again at 105,000 x g for 2 h (29) maintained on slants of glycerol-asparagine agar (22). in a Beckman model L8-55 ultracentrifuge equipped with a For growth studies, spores from agar slant cultures were type SW-55TI rotor and then used to measure difference inoculated into 250-ml Erlenmeyer flasks containing 100 ml spectra. Protein was determined by the method of Bradford of a liquid medium. This medium contained (per liter of (4). distilled water) 4.5 g of Na,HPO4 7H2O, 3.0 g of Difference spectra were obtained with a Hewlett-Packard (NH4)2SO4, 1.0 g of KH2PO4, 500 mg of yeast extract, 500 model 8451A diode array spectrophotometer. Carbon mon- 98 VOL. 52, 1986 VERATROLE DEMETHYLATION BY S. SETONII 99

co .0 ------0

-0.1n~ / 350 375 400 425 450 475 500 400 425 450 475 500 Wavelength (nm) Wavelength ( nm) FIG. 2. Difference spectra for cell extracts (1.1 mg of protein per FIG. 1. Carbon monoxide difference spectra of cell extracts. ml) from S. setonii grown with veratrole after the addition of 1.35 Cultures of S. setonii were grown with veratrole ( ) or succinate mM veratrole ( ) or 0.55 mM metyrapone (-----). ( ); the- protein contents were 1.0 and 1.1 mg/ml, respectively. produced a difference spectrum showing a type I interaction. oxide difference spectra (6, 7) were obtained after sodium The peak was at 390 nm, and the trough was at 418 nm (data dithionite reduction of cell extracts from 48-h cultures grown not shown). However, the cytochrome P-450 inhibitor with veratrole, guaiacol, or sodium succinate. Aerated cell metyrapone produced a difference spectrum showing a type extracts from 48-h veratrole-grown cultures were used to II interaction with a peak at 426 nm and a trough at 392 nm obtain the difference spectra resulting from the addition of (Fig. 2). 1.35 mM veratrole or guaiacol (10, 28). Difference spectra were also obtained with 0.55 mM metyrapone (2-methyl-1,2- DISCUSSION P-450 di-3-pyridyl-1-propanone), an inhibitor of cytochrome veratrole to catechol by S. setonii, in bacteria (19). Spectral changes were classified as type I or The conversion of and troughs presumably via guaiacol, makes further metabolism possible II by the wavelengths of the absorbance peaks via the 3-ketoadipate pathway (21). It seems likely that (14). guaiacol is demethylated to catechol as fast as it is produced. Figure 3 shows the steps involved in the transformation of RESULTS veratrole to the aliphatic product cis,cis-muconate. by Other methoxylated compounds that are 0-demethylated Catabolism of veratrole by S. setonii was demonstrated by various species of Streptomyces include aconitine (2), measuring the UV absorption spectra of inoculated and anisole (27), 10,11-dimethoxyaporphine (25), glaucine (11), noninoculated liquid media before and after incubation for 3 and veratrate (31). Vanillate may be either days. Noninoculated media containing veratrole had UV papaverine (24), lacked demethylated or decarboxylated by S. setonii, depending on absorption peaks at 222 and 272 nm; inoculated media the original growth substrate (21, 32). However, veratrate these peaks, producing spectra that were nearly identical to this extract as does not serve as a growth substrate for organism (21). the spectrum of a culture medium containing yeast It is not known whether one or more distinct monooxy- the only carbon source. genases are involved in demethylation reactions in the Both veratrole and catechol were identified by high- streptomycetes; some bacterial 0-demethylases are fairly performance liquid chromatography and gas chromatogra- specific for methoxyl groups at particular positions on aro- phy in spent culture media after the growth of S. setonii on matic rings (11, 23, 25). veratrole for 3 days in the presence of 2,2'-dipyridyl. The to be associated with retention times for Cytochrome P-450 has been reported high-performance liquid chromatography a specific hydroxylase activity in Streptomyces erythraeus veratrole and catechol were 12.84 and 3.14 min, respec- that were observed in the were 3.66 (8). The type I spectral changes tively; the gas chromatography retention times difference spectra of aerated cell extracts of S. setonii after and 4.06 min, respectively. Guaiacol, the presumed interme- the addition of veratrole or guaiacol suggest that both diate, was not positively identified in the media. veratrole and guaiacol are substrates for the demethylase Cell extracts from cultures grown for 48 h with veratrole, activity of cytochrome P-450 (14). The type II spectral after reduction with sodium dithionite, produced a carbon of is similar at change observed after the addition metyrapone monoxide difference spectrum with an absorbance peak to that observed after the addition of metyrapone to the 450 nm that indicated the presence of soluble cytochrome cytochrome P-450 of Moraxella sp. (10). P-450 (Fig. 1). Reduced cell extracts from cultures grown Thus, in S. setonii 75Vi2, veratrole and guaiacol induce with guaiacol produced a CO difference spectrum (data not the production of soluble cytochrome P-450, which appears shown) that also indicated the presence of cytochrome P-450, but cell extracts from cultures grown with sodium without an absorbance OCH3 OH OH COOH succinate produced a spectrum peak O -3 ~ 3CH3 H KjOOH at 450 nm (Fig. 1). glCa [CH3 3 O ¢^fCOOH The difference spectrum produced by the addition of veratrole to aerated cell extracts of cultures that had been h a I interaction with Veratrole G u a iacol Catechol cdiacs- grown on veratrole for 48 showed type acid a peak at 394 nm and a trough at 424 nm (Fig. 2). Guaiacol, Muconic when added to extracts of veratrole-grown cultures, also FIG. 3. Pathway of veratrole catabolism by S. setonii. 100 SUTHERLAND APPL. ENVIRON. MICROBIOL. to be involved in the demethylation of these methoxylated spectroscopy. Methods Enzymol. 52:258-279. aromatic compounds. 15. Kaiser, J.-P., and K. W. Hanselmann. 1982. Aromatic chemicals through anaerobic microbial conversion of lignin monomers. Experientia 38:167-176. ACKNOWLEDGMENTS 16. Kawakami, H. 1975. Bacterial degradation of lignin model I thank P. J. Walcheski and L. W. Severs for assistance with compounds. II. On the degradation of model monomers by chromatography and A. D. Williams and J. R. Grant for shaking culture. Mokuzai Gakkaishi 21:309-314. (In Japanese.) help with 17. Kersten, P. J., M. Tien, B. Kalyanaraman, and T. K. Kirk. 1985. spectrophotometry. I am also grateful to A. L. Pometto III of the The University of Idaho for helpful comments and to J. S. Bainbridge for ligninase of Phanerochaete chrysosporium generates cation assistance in preparing the manuscript. radicals from methoxybenzenes. J. Biol. Chem. 260:2609-2612. 18. McIlwain, H. 1948. Preparation of cell-free bacterial extracts with powdered alumina. J. Gen. 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