INTRODUCTION

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Protocatechuate (PCA) is one of the most important intermediate metabolites in the bacterial pathways for various phenolic compounds, including lignin, which is the most abundant aromatic material in nature. Sphingomonas paucimobilis SYK-6 is able to degrade a wide variety of dimeric lignin compounds, including -aryl ether, biphenyl, and diarylpropane (20). The resulting lignin degradation enzymes are expected to be useful tools for the utilization of lignin as biomass. Dimeric lignin compounds with guaiacyl (4-hydroxy-3-methoxyphenyl) moieties are converted into PCA by the action of the various lignin degradation enzymes, including -etherase (LigF and LigE) (20, 21), ring cleavage dioxygenase for biphenyl (LigZ) (27), and demethylases for 5,5'-dehydrodivanillic acid (LigX) (unpublished data) and vanillic acid (LigH) (23), as well as side chain-cleaving enzymes. Thus, PCA is the key intermediate metabolite in the lignin degradation pathway in S. paucimobilis SYK-6, and the PCA metabolic pathway plays a key role in lignin degradation by this strain. It is generally known that the aromatic ring of PCA is opened in reactions catalyzed by three kinds of dioxygenases: PCA 3,4-dioxygenase (3,4-PCD) (5, 6, 40), PCA 4,5-dioxygenase (4,5-PCD) (24), and PCA 2,3-dioxygenase (2,3-PCD) (38). Among these dioxygenases, 3,4-PCD is the most commonly characterized enzyme, and its three-dimensional structure has been elucidated (25). The -ketoadipate pathway genes (pca genes), including that for 3,4-PCD, have been characterized in detail in Acinetobacter calcoaceticus, Pseudomonas putida, and Agrobacterium tumefaciens (8).

In the case of S. paucimobilis SYK-6, PCA is subjected to ring cleavage by 4,5-PCD and metabolized through the PCA 4,5-cleavage pathway proposed by Kersten et al. (13). In the PCA 4,5-cleavage pathway (Fig. 1), 4,5-PCD catalyzes 4,5-cleavage of PCA to form 4-carboxy-2-hydroxymuconate-6-semialdehyde (CHMS), which is nonenzymatically converted into an intramolecular hemiacetal form and then dehydrogenated by CHMS dehydrogenase (19). The resulting intermediate, 2-pyrone-4,6-dicarboxylic acid (PDC) (15), is hydrolyzed by PDC hydrolase to yield 4-oxalomesaconic acid (OMA) or its tautomer, 4-carboxy-2-hydroxymuconic acid (CHM) (13, 16). OMA is converted into 4-carboxy-4-hydroxy-2-oxoadipic acid (CHA) by OMA hydratase (17). Finally, CHA is cleaved by CHA aldolase to produce pyruvate and oxaloacetate (18, 36). Each enzyme catalyzing the last four steps in Pseudomonas ochraceae (15-19) has been purified and characterized. Additionally, the PDC hydrolase and CHA aldolase of Comamonas testosteroni (13, 36) have been purified. However, little is known about the genes encoding these four enzymes.

View larger version (22K): [in this window] [in a new window]   FIG. 1.   The proposed degradation pathway of vanillic acid, including the protocatechuate 4,5-cleavage pathway in S. paucimobilis SYK-6. LigA and B, small and large subunits of 4,5-PCD (24); LigH, O-demethylase for vanillic acid and syringic acid (23); LigI, PDC hydrolase (this study). The PCA 4,5-cleavage pathway is illustrated according to findings from previous studies (13, 15-18).

In this paper, we present the structure of the PDC hydrolase gene and the biochemical properties of the gene product. The actual role of PDC hydrolase in the degradation of model lignin compounds by S. paucimobilis SYK-6 is also discussed.

	MATERIALS AND METHODS

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Strains and plasmids. The strains and plasmids used in this study are listed in Table 1. S. paucimobilis SYK-6 was grown at 30°C in W minimal salt medium (27) containing 0.2% model lignin compounds, including vanillic acid and syringic acid, and in LB medium (Bacto Tryptone, 10 g/liter; yeast extract, 5 g/liter; NaCl, 5 g/liter).

Preparation of substrates. PDC was prepared from PCA by using cells of P. putida PpY1100 harboring pVAD4, which conferred transformation activity from PCA to PDC, but no PDC conversion activity. PCA appeared to be converted into PDC by 4,5-PCD and CHMS dehydrogenase encoded in pVAD4. PpY1100(pVAD4) was grown in 2 liters of W medium containing 0.2% succinate and 25 mg of kanamycin/liter for 20 h at 28°C. Cells were harvested by centrifugation, resuspended in 800 ml of W medium containing 10.4 mmol of PCA, and incubated for 20 h at 28°C. A culture was acidified to pH 1 and centrifuged to remove cells. Metabolites were extracted twice with 400 ml of ethyl acetate and dried in vacuo. The residue was dissolved in water, acidified to pH 1, and kept at 4°C for 3 days. The resultant white crystals were recovered, washed with 2 N HCl, and air dried. Gas chromatography and mass spectrometry (GC-MS) analysis of the trimethylsilylated (TMS) derivative of the product was carried out. The gas chromatogram showed a major peak at the retention time of 30.0 min. The mass spectrum of this peak had a molecular ion (M) at m/z 328, which corresponded to the expected molecular mass of the PDC TMS derivative. The product showed an absorption maximum at 312 nm in 50 mM Tris-HCl buffer (pH 8.5). In 1 N NaOH, the absorbance at 312 nm decreased and that at 353 nm increased. These characteristics correspond to the features of PDC reported by Maruyama (15, 16). Thus, it was concluded that the product obtained was PDC. Finally, 8.9 mmol of PDC was obtained from 10.4 mmol of PCA.

DNA manipulations and nucleotide sequencing. DNA manipulations were carried out essentially as described elsewhere (1, 29). A Kilosequence kit (Takara Shuzo Co., Ltd., Kyoto, Japan) was used to construct a series of deletion derivatives, whose nucleotide sequences were determined by the dideoxy termination method with an ALFexpress DNA sequencer (Pharmacia Biotech, Milwaukee, Wis.).

Enzyme assays. According to the method of Maruyama (16), PDC hydrolysis and synthesis were spectrophotometrically determined by measuring the decrease and increase in the absorbance at 312 nm (₃₁₂ = 6,600 M¹ cm¹; pH 8.5), respectively, with a DU-7500 spectrophotometer (Beckman, Fullerton, Calif.). The reaction was carried out at 30°C in a cuvette. The 1-ml reaction mixture for PDC hydrolysis contained 100 µM PDC and the enzyme in 50 mM Tris-HCl buffer (pH 8.5). That for PDC synthesis contained 100 µM CHM and the enzyme in 50 mM sodium phosphate buffer (pH 7.0). One unit of the enzyme was defined as the amount that degraded 1 µmol of substrate per min at 30°C. Specific activity was expressed as units per milligram of protein. The optimum pHs for PDC hydrolysis and synthesis were examined in the pH range of 4.0 to 10.0 by using buffers consisting of 50 mM sodium acetate (pH 4.0 to 5.0), GTA (16.7 mM [each] 3,3-dimethylglutaric acid, Tris, and 2-amino-2-methyl-1,3-propanediol) (pH 4.0 to 8.5), sodium phosphate (pH 6.0 to 8.0), Tris-HCl (pH 8.0 to 9.0), or sodium borate (pH 9.0 to 10.0). K_m and V_max values were obtained from the Hanes-Woolf plots and expressed as means from at least three independent experiments.

Enzyme purification. Enzyme purification was performed according to the method described below by using a BioCAD700E apparatus (PerSeptive Biosystems, Framingham, Mass.).

(i) Preparation of cell extract. Cells were grown in 2 liters of LB medium containing 100 mg of ampicillin/liter. Expression of the ligI gene was induced for 3.5 h by adding isopropyl--D-thiogalactopyranoside (final concentration, 1 mM) when the optical density at 660 nm (OD₆₆₀) of the culture reached 0.5. Cells were harvested by centrifugation and sonicated in 50 mM Tris-HCl buffer (pH 8.0) (buffer A). The cell lysate was centrifuged at 15,000 × g for 15 min. Streptomycin (final concentration, 1%) was added to the supernatant, and it was recentrifuged at 15,000 × g for 15 min to remove nucleic acids. The supernatant was then centrifuged at 150,000 × g for 60 min at 4°C, and the crude extract was obtained after concentration by ultrafiltration using a YM-10 membrane (Amicon, Beverly, Mass.).

(ii) POROS PI anion-exchange chromatography. The crude extract was applied to a POROS PI (polyethyleneimine) column (4.6 by 100 mm) (PerSeptive Biosystems) previously equilibrated with buffer A. The enzyme was eluted with 25 ml of a linear gradient of 0 to 0.5 M NaCl. The PDC hydrolase was eluted at approximately 0.26 M.

(iii) POROS HQ anion-exchange chromatography. The fractions containing PDC hydrolase activity eluted from a PI column were pooled, desalted, and concentrated by ultrafiltration using a YM-10 filter. The resulting solution was applied to a POROS HQ (quaternized polyethyleneimine) column (4.6 by 100 mm) (PerSeptive Biosystems) previously equilibrated with buffer A. The enzyme was eluted with 33 ml of a linear gradient of 0 to 0.5 M NaCl. The fractions containing PDC hydrolase activity that eluted at approximately 0.2 M were pooled.

(iv) POROS HP2 hydrophobic interaction chromatography. The fractions containing PDC hydrolase activity were pooled, desalted, and concentrated by ultrafiltration using a YM-10 filter. Ammonium sulfate was added to the enzyme solution to a final concentration of 2 M. After centrifugation (at 3,000 × g for 10 min), the supernatant was collected and applied to a POROS HP2 (phenyl) column (4.6 by 100 mm) (PerSeptive Biosystems) equilibrated with buffer B (buffer A containing 2 M ammonium sulfate). The enzyme was eluted with 25 ml of a linear gradient of 2.0 to 0 M ammonium sulfate. The fractions containing PDC hydrolase activity that eluted at approximately 1.3 M were pooled. Glycerol was added to a final concentration of 10%, and the purified enzyme was stored at 80°C until use.

Analytical methods. The protein concentration was determined by the method of Bradford (3). The purity of the enzyme preparation was examined by sodium dodecyl sulfate-12% polyacrylamide gel electrophoresis (SDS-12% PAGE) (14). The molecular mass of the native enzyme was determined by Superdex200 HR10/30 (Pharmacia Biotech) gel filtration column chromatography using LC module 1 (Waters Corp., Milford, Mass.). Elution was performed with 50 mM potassium phosphate buffer (pH 7.0) containing 0.15 M NaCl with a flow rate of 0.25 ml/min. The molecular weight was estimated on the basis of the calibration curve of reference proteins.

Identification of the reaction product. PDC was incubated with purified LigI (0.5 µg) in 50 mM Tris-HCl buffer (pH 8.5) for 1 h. After the decrease of the absorbance at 312 nm derived from PDC, the reaction mixture was acidified, extracted with ethyl acetate, and then trimethylsilylated. GC-MS analysis was carried out as described above.

Insertional inactivation of the ligI gene. The 2.3-kb PstI-SmaI fragment carrying ligI was cloned into pUC19 to generate pUC1923. The 1.3-kb PstI fragment containing the kanamycin resistance gene from pUC4K was inserted into StuI in the middle of the ligI gene in pUC1923. The resultant plasmid, pUC1923K, was digested with EcoRI and PstI, and the insert containing the inactivated ligI gene was cloned into pK19mobsacB (31) to generate pLID1.

Nucleotide sequence accession number. The nucleotide sequence reported in this paper was deposited in the DDBJ, EMBL, and GenBank nucleotide sequence databases under accession no. AB015964.

	RESULTS

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Nucleotide sequence of the PDC hydrolase gene. The ligAB genes are located near the 3' end of the SYK-6 10.5-kb EcoRI fragment (24). We examined PDC hydrolase activity on this 10.5-kb fragment, expecting to see evidence of the existence of the PDC hydrolase gene in it. PDC hydrolase activity was initially observed in P. putida PpY1100 containing pVA01, which has a 10.5-kb fragment inserted in a broad-host-range vector, pKT230. When SalI fragments in the middle of the 10.5-kb insert were deleted from pVA01, no PDC hydrolase activity was detected or in the resultant plasmid, pVAD4. PDC hydrolase activity was found in Escherichia coli JM109 harboring pHN139R, which contained the 10.5-kb EcoRI fragment (Fig. 2). Among the deletion derivatives, pSS50R, pSS32R, and pDS15 conferred PDC hydrolase activity. These depended on the transcription from the lac promoter. The results obtained with the deletion derivatives showed the following features of the PDC hydrolase gene: (i) its direction of transcription is the same as that indicated for pSS32R in Fig. 2 and opposite that of the ligAB genes; (ii) it resides in the SphI fragment of pDS15; and (iii) the 3'-terminal deletion beyond the SalI site, which was accomplished in pSS14R, eliminated its function. The first feature suggests that the PDC hydrolase gene belongs to a transcriptional unit different from that of ligAB.

View larger version (23K): [in this window] [in a new window]   FIG. 2.   Deletion analysis of the 10.5-kb EcoRI fragment and lig gene organization. The PDC hydrolase activities of the cells containing each subclone are presented on the right. The small arrows indicate the direction of transcription from the lac promoter. Large filled arrows, ligI, ligA, and ligB genes. A large partly filled arrow, the part of the ORF which showed a similarity with the LSD gene (lsdA) (9, 10). E. coli JM109 was used as a host strain except for pVA01 and pVAD4, for which P. putida PpY1100 was used. E, EcoRI; P, PstI; Sl, SalI; Sm, SmaI; Sp, SphI; St, StuI; X, XhoI; Xb, XbaI.

Purification of PDC hydrolase. E. coli JM109 harboring pDS15 was grown, and the ligI gene in pDS15 was expressed. The ligI gene product (LigI) was purified by a series of column chromatography with PI, HQ, and HP2. Table 2 and Fig. 3 summarize the results of a typical purification. LigI was purified approximately 38-fold, with a recovery of 10%. The purified enzyme was shown to be near homogeneity by SDS-PAGE (Fig. 3). The molecular mass of a subunit of LigI was estimated to be 38 kDa, which is close to the value deduced from the amino acid sequence of the ligI gene product (M_r, 32,737). The LigI protein in the HP2 fraction was subjected to N-terminal sequencing. The first six residues, M-T-N-D-E-R, corresponded to the deduced amino acid sequence of the ligI gene product.

View larger version (100K): [in this window] [in a new window]   FIG. 3.   SDS-PAGE analysis of protein fractions. Proteins were separated on an SDS-12% polyacrylamide gel and stained with Coomassie brilliant blue. Lanes: 1, molecular weight markers; 2, crude extract of E. coli JM109 harboring pBluescript II KS(+) (5 µg of protein); 3, crude extract of E. coli JM109 harboring pDS15 (5 µg of protein); 4, PI fraction (5 µg of protein); 5, HQ fraction (2 µg of protein); 6, HP2 fraction (2 µg of protein). Molecular masses are given on the left.

Identification of the reaction product. Figure 4 shows the gas chromatogram and mass spectra of TMS derivatives of the reaction product of LigI from PDC. Two product peaks with retention times of 31.5 (I) and 34.4 (II) min were observed. The mass spectra of products I and II were identical. We extracted and analyzed the PDC alkaline hydrolysate according to the procedure reported by Maruyama (16). Two kinds of products yielding the same retention times and mass spectra as products I and II in Fig. 4 were observed (data not shown). The two major fragments at m/z 475 and 373 seemed to correspond to (M-CH₃) and (M-COOTMS), respectively (where M is a molecular ion of the TMS derivative of CHM and COO represents a carboxyl group). The M-CH₃ ion is generally found in mass spectra of TMS derivatives of organic acids. Based on these results, we concluded that products I and II are the stereoisomers of CHM.

View larger version (17K): [in this window] [in a new window]   FIG. 4.   Identification of the reaction product from PDC catalyzed by LigI. (A) Gas chromatogram of the TMS derivative of the reaction product from PDC catalyzed by purified LigI. PDC indicates the TMS derivative of PDC. Compounds I and II were the TMS derivatives of the reaction products originating from a substrate, PDC. (B) Mass spectrum of the TMS derivative of PDC. (C) Mass spectrum of compound I. The mass spectra of compounds I and II are identical. Compounds I and II were identified as isomeric forms of CHM.

Enzyme properties. The molecular mass of the native enzyme was estimated to be 31.6 kDa on a Superdex 200 gel filtration column. This suggests that LigI is a monomer. The isoelectric point of LigI was determined by isoelectric focusing to be 4.9.

Effects of thiol reagents and metal ions. The influence of thiol reagents 5,5'-dithiobis(2-nitrobenzoic acid) (Ellman's reagent) and N-ethylmaleimide on the activity for PDC hydrolysis and PDC synthesis was examined (Table 3). These thiol reagents strongly inhibited both PDC hydrolysis and PDC synthesis activity, suggesting that the cysteine residue is involved in the enzyme reaction. The effects of metal ions on LigI activity are summarized in Table 3. Zn²⁺ strongly inhibited both PDC hydrolysis and PDC synthesis. Cu²⁺ inhibited only PDC synthesis. In the case of the P. ochraceae enzyme, Mn²⁺ and Co²⁺ inhibited PDC hydrolysis, and inhibition by Zn²⁺ and Cu²⁺ was similar to that for the SYK-6 enzyme (16). The SYK-6 PDC hydrolase was therefore shown to be more resistant to metal ions than the P. ochraceae enzyme.

ligI disruption in S. paucimobilis SYK-6. Inactivation of the ligI gene by the insertion of the kanamycin resistance gene was performed by using the ligI disruption plasmid pLID1 as described in Materials and Methods. The ligI insertion mutant was confirmed by Southern hybridization analysis (Fig. 5). The mutant strain DLI, grown on LB medium or syringic acid, showed no PDC transformation activity and no PDC hydrolysis. This strain completely lost the ability to grow on vanillic acid, even though it grew as well on syringic acid as the wild-type strain. The metabolite of vanillic acid generated by the whole cells of DLI grown on LB medium was examined by GC-MS analysis. After 22 h of incubation, vanillic acid was transformed completely, and only the accumulation of PDC was observed.

View larger version (22K): [in this window] [in a new window]   FIG. 5.   Insertional inactivation of the ligI gene in S. paucimobilis SYK-6. (A) Schematic representation of the insertional inactivation of ligI by the kanamycin resistance gene from pUC4K. Thick arrows, orientation of transcription of the ligI and kanamycin resistance genes. E, EcoRI; P, PstI; Sl, SalI; Sm, SmaI; Sp, SphI; St, StuI; X, XhoI. (B) Southern hybridization analysis of the ligI insertion mutant (DLI). Lanes 1 and 3, total DNA of SYK-6 digested with PstI; lanes 2 and 4, total DNA of DLI digested with PstI. The 2.3-kb PstI-SmaI fragment carrying ligI (lanes 1 and 2) and the 1.3-kb PstI fragment of the kanamycin resistance gene (lanes 3 and 4) were used as probes.

	DISCUSSION

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This is the first report on the genetic analysis of PDC hydrolase, which is one of the protocatechuate 4,5-cleavage pathway enzymes. The PDC hydrolase gene of S. paucimobilis SYK-6, designated ligI, encodes a protein of 32,737 Da (293 amino acids). There was no similarity between the LigI amino acid sequence and those of the proteins in the databases, including the dienelactone hydrolase (4, 28, 37) and the -ketoadipate enol-lactone hydrolase (7, 33), which seemed to be functionally related to LigI.

The ligI gene is located approximately 5.4 kb upstream of ligA. Interestingly, ligI is transcribed divergently from ligAB. This fact indicated that the PCA 4,5-cleavage pathway was composed of at least two distinct operons. We could not find other genes responsible for the PCA 4,5-cleavage pathway enzymes in the region sequenced. Downstream from ligI, an incomplete ORF which had the same direction of transcription as ligI was found. The predicted amino acid sequence of the product of this ORF showed significant similarity to those of the lignostilbene-,-dioxygenase (LSD) genes of Pseudomonas paucimobilis TMY1009 (9, 10). LSD has been reported to be a dioxygenase catalyzing the cleavage of the interphenyl double bond of lignostilbenes. The occurrence of the LSD gene homolog beside the PCA 4,5-cleavage pathway enzyme genes is interesting. Further nucleotide sequencing and functional analysis of the 10.5-kb EcoRI fragment may address the functions of a putative LSD and other enzymes of the PCA 4,5-cleavage pathway.

The reaction product from PDC, catalyzed by LigI, was estimated to be two stereoisomers of CHM. OMA, which is a tautomer of CHM was not detected. The production of OMA from PDC was suggested by Maruyama (16). OMA is an -keto acid, and its TMS derivative is easily distinguishable in MS from that of CHM, because a keto group of OMA is not trimethylsilylated. OMA might have been degraded during the process of extraction, since -keto acid is generally unstable.

The characteristics of the PDC hydrolases of S. paucimobilis SYK-6, P. ochraceae, and C. testosteroni are summarized in Table 4. All enzymes are monomeric proteins. The molecular mass and pH optima of the SYK-6 enzyme are very similar to those of the P. ochraceae enzyme. A higher affinity with CHM (or OMA) than with PDC and a higher V_max for PDC hydrolysis than for PDC synthesis were common features of the PDC hydrolases of SYK-6 and P. ochraceae. Thiol reagents, such as Ellman's reagent and N-ethylmaleimide, strongly inhibited activity, suggesting that the cysteine residue is the catalytic site of these three enzymes. A similar inhibition was also observed in the P. ochraceae and C. testosteroni enzymes. In the / hydrolase fold enzymes, the catalytic nucleophile is located in a highly conserved peptide, Gly-X-(Ser/Cys)-X-Gly. The three-dimensional structure of the dienelactone hydrolase from Pseudomonas sp. strain B13 was solved, and its catalytic nucleophile, cysteine residue 123, was shown to constitute part of a catalytic triad of residues (26). Recently, Schrag and Cygler reported that a consensus sequence of these enzymes might better be described as Sm (small amino acid)-X-Nuc (nucleophile)-X-Sm, since the glycine residues at Nuc 2 and Nuc +2 are sometimes substituted for other small amino acids, including alanine and serine (32). Among the three cysteine residues of LigI, Cys76 may be the catalytic site, since the sequence Ala74-Ser75-Cys76-His77-Gly78 corresponds to the consensus of Sm-X-Nuc-X-Sm.

The ligI gene insertion mutant, DLI, showed no PDC hydrolase activity when the DLI cells were grown on LB medium and on syringic acid. DLI was not able to grow on vanillic acid, and the cells of DLI grown on LB medium accumulated PDC from vanillic acid. These results indicated that the ligI gene is unique in conferring PDC hydrolysis and is essential for growth of SYK-6 on vanillic acid. According to the proposed metabolic pathway of syringic acid in SYK-6, syringic acid is converted to PDC via 3-O-methylgallic acid (11, 23). The production of 3-O-methylgallic acid from syringic acid was evident from the results obtained with the ligH gene. The mutation in the ligH gene, the product of which is involved in the conversion of syringic acid to 3-O-methylgallic acid, resulted in a growth defect on syringic acid (23). Kersten et al. reported the production of PDC from 3-O-methylgallic acid by 4,5-PCD (13). However, the ligI insertion mutant DLI grew on syringic acid and showed no PDC transformation activity and no PDC hydrolysis. These results obviously suggest that syringic acid is not metabolized via PDC and that neither 4,5-PCD nor LigI is involved in the metabolism of syringic acid.

The results obtained in this study strongly support the proposed PCA 4,5-cleavage pathway presented in Fig. 1. Further research is needed to elucidate the correct degradation pathway of syringic acid in SYK-6.