Metabolism of Lignin Model Compounds of the Arylglycerol-β-Aryl ...

Vol. 53, No. 11

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1987, p. 2605-2609

0099-2240/87/112605-05$02.00/0 Copyright © 1987, American Society for Microbiology

Metabolism of Lignin Model Compounds of the Arylglycerol-p-Aryl Ether Type by Pseudomonas acidovorans D3 Laboratorio de

R. VICUNA,' B. GONZALEZ,' M. D. MOZUCH,2 AND T. KENT KIRK2* Bioquimica, Pontificia Universidad Catolica de Chile, Santiago, Chile,' and Forest Products Laboratory,

Forest Service, U.S.

Department of Agricultire, Madison,

Wisconsin 53705-23982

Received 12 June 1987/Accepted 6 August 1987

A natural bacterial isolate that we have classified as Pseudomonas acidovorans grows on the lignin model compounds 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)propane-1,3-diol (compound 1) and 1-(4-hydroxy3-methoxyphenyl)-2-(2-methoxyphenoxy)propane-1,3-diol (compound 1'), as well as on the corresponding 1-oxo compounds (2 and 2') as sole sources of carbon and energy. Metabolic intermediates present in cultures growing on compound 1 included compound 2, 2-methoxyphenol (guaiacol [compound 3]), ,-hydroxypropioveratrone (compound 4), acetoveratrone (compound 5), and veratric acid (compound 6). Also identified were compounds 1', 2', ,-hydroxypropiovanillone (compound 4'), and acetovanillone (compound 5'), indicating that 4-0 demethylation also occurs. The phenolic intermediates were the same as those found in cultures growing on compound 1'. Compounds 2 and 2' were in part also reduced to compounds 1 and 1', respectively. Compound 3 was shown to be derived from the 2-juethoxyphenoxy moiety. A suggested degradation scheme is as follows: compound 1-*2---*(3 + 4)-*5--+6 (and similarly for 1'). In this scheme, the key reaction is cleavage of the ether linkage between C-2 (Cp) of the phenylpropane moiety and the 2methoxyphenoxy moiety in compounds 2 and 2' (i.e., 1-aryl ether cleavage). On the basis of compounds identified, viz., 3 and 4 (4'), cleavage appears formally to be reductive. Because this is unlikely, the initial cleavage products probably were not detected. The implications of these results for the enzyme(s) responsible are discussed.

MATERIALS AND METHODS

The arylglycerol- -aryl ether (1B-0-4) linkage accounts for approximately 50% of the linkages between the phenylpropane units of lignin (1). Cleavage of this linkage is expected to be essential to lignin biodegradation. Ligninase (lignin peroxidase), an extracellular peroxidase isolated from basidiomycetous fungi, is the only enzyme described so far that is able to cleave the 1-0-4 linkage (12, 14, 16, 22). Cleavage indirectly follows ligninase oxidation of the aromatic nuclei to cation radicals and occurs in more than one way (12, 14, 16). Some bacterial strains proliferate in synthetic media containing lignin-related compounds of the 1-0-4 type as the only source of carbon and energy (7, 17, 18, 20, 23). The metabolism of the compounds clearly involves cleavage of the ,B-ether linkage at some point. Enzymes from procaryotes responsible for catalyzing this cleavage, however, have not been identified. Essential to the search for such an enzyme is the identification of the degradation pathway in ,B-0-4 model substrates, especially of the products that immediately precede and follow the cleavage. To this end, we describe the partial catabolic pathways of two lignin model compounds, 1 and 1' (Fig. 1), by Pseudomonas acidovorans D3, isolated from decaying wood. Previously, Crawford et al. (8) described the initial transformations of the same substrates by a strain of P. acidovorans able to grow on the model compounds. Recently, Samejima et al. (20) identified several degradation intermediates in the degradation of a ,B-0-4 model by Pseudomonas putida FK-2, which was able to grow on compound 1.

*

Chemicals. The 1-0-4 model compounds 1 [1-(3,4-

dimethoxyphenyl)-2-(2-methoxyphenoxy)propane-1 ,3-diol], 2 [3,4-dimethoxy-a-(2-methoxyphenoxy)-,B-hydroxypropiophenone], 1' [1-(4-hydroxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)propane-1,3-diol], and 2' [4-hydroxy-3-me-

thoxy-a-(2-methoxyphenoxy)-1-hydroxypropiophenoneI prepared by the methods of Adler et al. (2) and Landucci et al. (15) (see also Fenn and Kirk [9]). Compounds 1, 1', 2, and 2' labeled in the aromatic nuclei were prepared as described elsewhere (9, 15); methylation with CH3IK2CO3, rather than ethylation with C2H51-K2CO3 (9), was used. Steps in the synthesis of compound 4 from 3-(3,4dimethoxyphenyl)-3-hydroxypropionic acid (obtained from G. Brunow, University of Helsinki, Helsinki, Finland) were as follows: (i) esterification with diazomethane in ether, (ii) reduction with lithium aluminum hydride in refluxing tetrahydrofuran, and (iii) oxidation with dichlorodicyanobenzoquinone in dioxane-methanol. Compound 4' was prepared in the same way from 3-(4-hydroxy-3-methoxyphenyl)-3-hydroxypropionic acid (also obtained from G. Brunow), except that the product of step 1 was benzylated to protect the phenolic hydroxyl group in step 2. The benzyl group was removed by catalytic hydrogenolysis after step 2 (15). Compounds 7 and 7' were prepared by catalytic reduction of compounds 2 and 2', respectively (H2 at 40 lb/in.2, 10% Pd on charcoal in 95% ethanol, 11 h). Compounds 8 and 8' were prepared by NaBH4 reduction of intermediates in the preparation of compounds 2 and 2' (15). Guaiacol (compound 3), acetoveratrone (compound 5), acetovanillone (compound 5'), veratric acid (compound 6), and vanillic acid (compound 6') were purchased from Aldrich Chemical Co.,

were

Corresponding author. 2605

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VICU&A

RESULTS

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Inc., Milwaukee, Wis. The structures of compounds 7, 7', 8, and 8' are given in Fig. 1; for other compounds, see Fig. 4. Bacterial strain. The biochemical properties of the bacterium used in these studies allowed us to classify it as a strain of P. acidovorans by following the procedures of Bergey's Manual (13) and Stanier et al. (21). Growth conditions. Liquid cultures of the bacterium were grown in a mineral salt medium described previously (10, 11) containing 3.3 mM lignin model compound as carbon source. Cultures were incubated at 35°C in a Gyrotory shaker (New Brunswick Scientific Co., Inc., Edison, N.J.) at 160 rpm. Identification of catabolic intermediates. Samples (1 ml) were taken from the cultures periodically after inoculation. They were filtered through Millex-GS filter units (0.22-Rm pore size; Millipore Corp., Bedford, Mass.) and acidified to pH 1 to 2 with 1 M HCl. Acidified samples were then extracted three times with 1.0 ml of ethyl acetate (total, 3 ml), and water was removed from the organic solvent with anhydrous Na2SO4. Solvent was removed under vacuum, and the residue was dissolved in 50 to 100 ,ul of 20% methanol in methylene chloride for thin-layer chromatography or in N,N-dimethylformamide for gas chromatographymass spectrometry (GC-MS) analysis. Analytical procedures. Thin-layer chromatography was on silica gel (Merck 60 F-254)-coated Al plates (EM Science, Gibbstown, N.J.) developed in hexanes-ethyl acetate (1:1). Compounds were visualized under short-wave UV light, or for 14C-labeled compounds, the plates were cut up and radioactivity was measured in scintillation fluid (4). Reverse-phase high-performance liquid chromatography (HPLC) was performed in a System 41 gradient analytical system (Gilson, Middleton, Wis.), a 3380 integrator (Hewlett-Packard Co., Palo Alto, Calif.), and a p.Bondapak C:18 column (Waters Associates, Inc., Milford, Mass.). Samples were withdrawn from the cultures and filtered as described above. Portions (20 p.l) were injected directly into the chromatograph. Compounds were eluted with a methanolwater gradient in which the concentration of methanol varied as follows: 0 to 5 min, 20%; 5 to 14 min, linear increase to 40%; 14 to 32 min, 40%; 32 to 33 min, decrease to 20%; and 33 to 45 min, 20%. GC-MS analyses were done with trimethylsilyl derivatives. Samples were dissolved in equal volumes of N,Ndimethylformamide and bis(trimethylsilyl)trifluoroacetamide (Sigma Chemical Co., St. Louis, Mo.) and heated at approximately 50°C for 1 min. Samples were analyzed without delay with a Finnigan MAT 4510 gas chromatograph-mass spectrometer (Finnigan, San Jose, Calif.). Gas chromatography was with a 60-m, 0.25-pum-film-thickness DB-5 (nonpolar silicone polymer) fused capillary column (J & W Scientific, Rancho Cordova, Calif.) operated at various temperatures. Electron impact mass spectra were obtained at 70 eV.

The isolation of four bacterial strains by using enrichment cultures containing compound 2' as carbon source was reported previously; the strains were designated D1, D2, D3, and D4 (10). Further experiments have now shown that only one of them, D3, is also able to consume quantitatively and grow on the nonphenolic model compounds 1 and 2. This bacterium (Fig. 2), which we have characterized as P. acidovorans, is also known to metabolize vanillic, pcoumaric, syringic, and trans-cinnamic acids as well as guaiacol and the ether-soluble (low-molecular-weight) fraction of kraft lignin (10). To identify catabolic intermediates, samples from cultures of P. acidovorans D3 growing on compound 2' were taken during the early logarithmic phase of growth and analyzed by HPLC. A representative HPLC pattern from these experiments (Fig. 3A), shows peaks with retention times that coincided with those of the starting material (peak 6), an authentic sample of a mixture of the erythro and threo forms of compound 1' (peak 5), and guaiacol (compound 3; peak 4). Identities were confirmed by GC-MS analysis of the trimethylsilyl derivatives. By GC-MS analysis we identified peak 3 as acetovanillone (compound 5'). The trimethylsilyl derivative of the compound giving peak 1 yielded major mass peaks at mlz 340, 325, 235, 223, and 73. A survey of the literature revealed an essentially identical mass spectrum which corresponded to that of ,-hydroxypropiovanillone (compound 4'), a catabolite in the degradation pathway of compound 1' by Pseudomonas sp. strain TMY1009 (20). We synthesized compound 4' and found it to have the same retention time as peak 1 on HPLC and a mass spectrum

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VOL. 53, 1987

ARYLGLYCEROL-p-ARYL ETHER METABOLISM BY P. ACIDOVORANS

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identical to that of the unknown peak 1. Peak 2 was not identified. Samples from cultures started with compound 1' instead of 2' exhibited a large peak corresponding to compound 2' on HPLC analysis together with peaks corresponding to compounds 3, 4', and 5' (data not shown). The identities of these intermediates were confirmed by GC-MS analysis of the trimethylsilyl derivatives. Vanillic acid (compound 6') was not detected. When cultures started with either substrate 1 or 2 (1' or 2') reached stationary phase, no aromatic products were detected by Thin-layer chromatography or HPLC in extracts of the medium. This indicates that all identified degradation products were further degraded. Studies on the metabolism of the nonphenolic lignin model compounds 1 and 2 followed the same experimental approach as those with compounds 1' and 2'. The HPLC pattern for culture fluid of P. acidovorans D3 grown on compound 1 (Fig. 3B) shows unreacted starting material (double peak 5). As was observed with cultures started with compound 1', peaks with retention times of the oxidized form of compound 2 (peak 6) and guaiacol (compound 3; peak 2), inter alia, were observed in cultures started with compound 1. By analogy with the products from compound 1', the intermediates 1-hydroxypropioveratrone (compound 4) and acetoveratrone (compound 5) were also expected to be present in the sample of culture started with compound 1. HPLC of authentic standards and GC-MS studies showed that this was indeed the case. In Fig. 3B, compounds 4 and 5 are seen as a shoulder on peak 1 and as peak 4, respectively. The mass fragmentation pattern of the trimethylsilyl

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derivative of the unknown suspected to be compound 4 included peaks at 282, 267, 192, 165, 118, and 75 mlz, identical to those obtained for synthetic compound 4. GCMS analysis also revealed the presence of veratric acid (compound 6). In addition to these veratryl compounds, GC-MS analysis revealed small amounts of compounds 1', 2', 4', and 5' as well as vanillic acid (compound 6'), indicating that 4-0 demethylation occurs but is not required for the initial degradation. When cultures were started with compound 2 as carbon source instead of compound 1, small amounts of both the erythro and threo forms of compound 1 were detected by HPLC of samples taken during the early logarithmic phase of growth. This is analogous to the above-mentioned reduction of compound 2' to 1'. The origin of guaiacol (compound 3) was examined. Conceivably, it could arise from aromatic ring A or B (Fig. 4) or both. We grew P. acidovorans D3 with compound 1 uniformly labeled with 14C only in ring A or only in ring B. Cultures were extracted during the logarithmic phase of growth, and metabolic intermediates were resolved by Thinlayer chromatography. Guaiacol on the plates was found to be radioactive only in the case of the ring B-labeled substrate. Compound 4' supported growth of the bacterium, as did vanillic acid (compound 6'). Guaiacol (compound 3) was toxic and was metabolized only when added at concentrations below 1.0 mM. Surprisingly, compounds 5 and 5' were not degraded when supplied as sole carbon sources at concentrations from 0.6 to 30 mM. However, when added at 0.6 mM with a 3.3 mM concentration of compound 1, compounds 5 and 5' were partially consumed, while compound 1 was completely consumed. With compound 5 or 5' at 5 mM and compound 1' at 3.3 mM, no growth occurred, showing that compounds 5 and 5' are toxic. Because the intermediate products pointed to cleavage of the 1-0-4 linkage (see Discussion), we examined other dimeric models to gain insight into the nature of the cleavage reaction. The bacterium grew well with models of types 1 and 1' containing a C,aH2 group instead of a CaHOH group (compounds 7 and 7'); guaiacol (compound 3) was detected as an intermediate from both compounds 7 and 7'. Interestingly, the bacterium was unable to metabolize 13-0-4 compounds that lacked the y-methylol group (compounds 8 and 8'). DISCUSSION The information gained indicates the degradation pathway for compound 1' by P. acidovorans D3 shown in Fig. 4. The presence of a carbonyl group at Ca in compounds 4' and 5' points to cleavage of compound 2' instead of 1'. Vanillic acid (compound 6'), though not observed as a degradation product of compound 1' under the conditions used, is included because it was detected in the degradation of compound 1, as was the analogous compound veratric acid (compound 6). When supplied at the outset, vanillic acid (compound 6') was rapidly degraded by the bacterium; evidently, it is degraded as rapidly as it is formed from compound 1'. Why it was detected from compound 1 and not from 1' is not clear. The proposed degradation pathway for the veratryl model (compound 1) is analogous to that for the guaiacyl model

(compound 1') (Fig. 4). The initial transformations of compounds 1 and 1' agree with those observed by Crawford et al. (8) for another strain of P. acidovorans. Cell extracts contained an NAD+-linked dehydrogenase system that catalyzed oxidation of com-

VICUNA ET AL.

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APPL. ENVIRON. MICROgIOL.

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pound 1' to 2' (8). More recently, Pelmont et al. (18) isolated an NAD+-linked dehydrogenase from cells of a Pseudomonas sp. grown in compound 1; the enzyme converted 50% of compound 1 to 2, evidently being specific for either the R or S stereoisomer of Ca. The pathway indicated by our results differs from those described for mixed rumen bacteria (5) that degraded model compound 1 and for two gram-positive aerobic bacteria (Corynebacterium equi and Brevibacterium maris) that degraded 1-(3,4-dimethoxyphenyl)-2-phenoxypropane-1,3-diol (19). In the cases of rumen and aerobic bacteria, cleavage between Ca and Cp occurred; with the aerobes, and perhaps with the anaerobes as well, this cleavage was preceded by oxidation of C', to a carboxylic acid (5). As reported previously for other Pseudomonas strains (8, 17), our strain quantitatively consumed all stereoisomers of the ,-0-4 substrates. Compounds 1 and 1', with two asymmetric carbon atoms (C,, and Cp) each, can each have four stereoisomers. Thus, with respect to the dehydrogenase system catalyzing the initial Ca oxidation, we can envision the following alternatives: (i) a single enzyme lacking stereospecificity for C,a and Cp is functioning; (ii) two enzymes having stereospecificity for the two C, stereoisomers, but lacking specificity for C , are present; or (iii) four enzymes, each specific for one of the four stereoisomers, are operative. Because of the high degree of stereoselectivity found in dehydrogenases (3, 24), the first possibility seems

unlikely. The suspected requirement for more than one enzyme (the other two alternatives) would explain, at least in Further part, the difficulty in finding bacteria able to degrade comMetabolism w-~~~~~~~~~~~~~~~~~~~ VM pletely models of the ,B-0-4 type (7, 10). The reduction of compound 2' to 1' (and of compound 2 to OMe 1) observed here was also reported by Pelmont et al. (18) for OH the purified dehydrogenase mentioned above. In the presence of NADH, their enzyme catalyzed the total reduction of a racemic mixture of compound 2'. This indicates that their enzyme was insensitive to the stereochemistry at Cp, suggesting that the second alternative mentioned above is correct for their pseudomonad. (It is expected that the reduction of compounds 2 and 2' HC ~CH3 ACHZ] would be stereospecific and that the products compounds 1 and 1' would have optical activity. Also, the resolved 1~~~~~~~~~~~~l erythro and threo isomers are expected to have different optical activities; resolution would separate the two Cp OOMe TOM. enantiomers. The question of optical activity was not exanmOMe OH ined here or by Pelmont et al. (18).) In the second step of the degradation (Fig. 4), compound 2 (2') is cleaved to yield guaiacol (compound 3) and compound 4 (4'). If it is assumed that compound 4 (4') and guaiacol (compound 3), are in fact the initial products of this N, bacterial cleavage, then this cleavage is reductive, involving essentially a nucleophilic attack by a hydride ion at Cp. Such COOH ether cleavage reactions have not, to our knowledge, been COOH described in any enzymatic system and, in fact, seem rather unlikely. It seems more likely that the initial cleavage is oxygenative and that the initial C6-C3 product is reduced to OMe OMO OH OMe \ compound 4 (4'). Direct oxygenative cleavage was indicated by the work of Crawford et al. (8), who found that cultures lEI of P. acidovorans grown on compound 1, in the presence of NADH plus 2,2'-dipyridyl, released compound 3 from 2'. Further 2,2'-Dipyridyl is a chelator of iron and an inhibitor of Metabolism mixed-function oxygenases. That compound 2 (2') rather than compound 1 (1') is the substrate for cleavage was also FIG. 4. Proposed scheme for the degradation of model comindicated by the work of Crawford et al. (8), who found that pounds 1 and 1' by P. acidovorans D3. Compounds in brackets are crude cell extracts cleaved compound 2' much faster than suspected but were not identified. Dashed arrows represent unproven transformations (see text). they did compound 1'. OH

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