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J Bacteriol. 2005 August; 187(15): 5067–5074.
PMCID: PMC1196043

Characterization of the Gallate Dioxygenase Gene: Three Distinct Ring Cleavage Dioxygenases Are Involved in Syringate Degradation by Sphingomonas paucimobilis SYK-6

Abstract

Sphingomonas paucimobilis SYK-6 converts vanillate and syringate to protocatechuate (PCA) and 3-O-methylgallate (3MGA) in reactions with the tetrahydrofolate-dependent O-demethylases LigM and DesA, respectively. PCA is further degraded via the PCA 4,5-cleavage pathway, whereas 3MGA is metabolized via three distinct pathways in which PCA 4,5-dioxygenase (LigAB), 3MGA 3,4-dioxygenase (DesZ), and 3MGA O-demethylase (LigM) are involved. In the 3MGA O-demethylation pathway, LigM converts 3MGA to gallate, and the resulting gallate appears to be degraded by a dioxygenase other than LigAB or DesZ. Here, we isolated the gallate dioxygenase gene, desB, which encodes a 418-amino-acid protein with a molecular mass of 46,843 Da. The amino acid sequences of the N-terminal region (residues 1 to 285) and the C-terminal region (residues 286 to 418) of DesB exhibited ca. 40% and 27% identity with the sequences of the PCA 4,5-dioxygenase β and α subunits, respectively. DesB produced in Escherichia coli was purified and was estimated to be a homodimer (86 kDa). DesB specifically attacked gallate to generate 4-oxalomesaconate as the reaction product. The Km for gallate and the Vmax were determined to be 66.9 ± 9.3 μM and 42.7 ± 2.4 U/mg, respectively. On the basis of the analysis of various SYK-6 mutants lacking the genes involved in syringate degradation, we concluded that (i) all of the three-ring cleavage dioxygenases are involved in syringate catabolism, (ii) the pathway involving LigM and DesB plays an especially important role in the growth of SYK-6 on syringate, and (iii) DesB and LigAB are involved in gallate degradation.

Lignin is the most abundant aromatic substance in nature, and its mineralization is an important step in the terrestrial carbon cycle. The use of lignin as a bioresource is anticipated, although few practical uses for lignin have been established to date. In nature, it is believed that the degradation of native lignin is initiated by white rot fungi, which secrete extracellular degradation enzymes, such as lignin peroxidase, manganese peroxidase, and laccase (11, 32). Bacteria contribute to the process of mineralization of the abundant lignin-derived compounds in soil (33). Sphingomonas paucimobilis SYK-6 is one of the best-characterized degraders of lignin-derived compounds, and this strain is capable of utilizing various lignin-derived biaryls, including β-aryl ether (16), biphenyl (21), and diarylpropane, as sole sources of carbon and energy. Therefore, the lignin degradation enzymes in SYK-6 could be used as practical tools for the conversion of lignin-derived compounds into valuable intermediate metabolites, such as 2-pyrone-4,6-dicarboxylate (PDC), which has been found to be useful as a starting material for the synthesis of biodegradable polymers (26).

S. paucimobilis SYK-6 degrades lignin-derived compounds possessing guaiacyl (4-hydroxy-3-methoxyphenyl) and syringyl (4-hydroxy-3,5-dimethoxyphenyl) moieties to vanillate and syringate, respectively. Vanillate and syringate are converted to protocatechuate (PCA) and 3-O-methylgallate (3MGA) by the tetrahydrofolate (H4folate)-dependent O-demethylases LigM and DesA, respectively (1, 17). PCA is further degraded through the PCA 4,5-cleavage pathway (Fig. (Fig.1).1). In contrast, 3MGA is degraded via three different pathways, in which PCA 4,5-dioxygenase (LigAB), 3MGA 3,4-dioxygenase, and 3MGA O-demethylase participate. Disruption of the 4-oxalomesaconate (OMA) hydratase gene (ligJ) leads to defects in the growth of SYK-6 on syringate; therefore, it appears that 3MGA is ultimately metabolized through OMA. In these multiple pathways, we have characterized the desZ gene, which encodes 3MGA 3,4-dioxygenase (12), in addition to the PCA 4,5-cleavage pathway genes (10). A desZ ligB double mutant was able to grow on syringate, despite the finding that this mutant completely lost dioxygen-dependent 3MGA transformation activity (12). Recently, our investigations indicated that 3MGA is converted to gallate by vanillate/3MGA O-demethylase (LigM) (1). Disruption of both desZ and ligB in SYK-6 had no effect on the gallate degradation activity, suggesting that the gallate dioxygenase gene is present in SYK-6 (12).

FIG. 1.
Proposed catabolic pathways for vanillate and syringate in S. paucimobilis SYK-6. Enzymes: LigM, vanillate/3MGA O-demethylase; LigA and LigB, small and large subunits, respectively, of PCA 4,5-dioxygenase; LigC, 4-carboxy-2-hydroxymuconate-6-semialdehyde ...

In this study, we isolated the gallate dioxygenase gene from SYK-6 and characterized its function. Specific ring cleavage dioxygenase activity with gallate has been reported in a syringate degrader, Pseudomonas putida (28), but the enzyme and the gene have not been characterized yet. This is the first report on characterization of the gallate dioxygenase gene, and the role played by each of the three distinct dioxygenases in syringate degradation was examined in this study.

MATERIALS AND METHODS

Strains and plasmids.

The strains and plasmids used in this study are listed in Table Table1.1. S. paucimobilis SYK-6 was grown in W minimal salt medium (20) containing 10 mM syringate or in Luria-Bertani (LB) medium at 30°C. P. putida PpY1100 was grown in LB medium. The SYK-6 mutants were grown in LB medium. If necessary, 50 mg of kanamycin/liter and 300 mg of carbenicillin/liter were added to the cultures. Escherichia coli strains were grown in LB medium at 37°C. For cultures of cells carrying antibiotic resistance markers, the media were supplemented with 100 mg of ampicillin/liter or 25 mg of kanamycin/liter.

TABLE 1.
Strains and plasmids used in this study

Cloning of the desB gene.

A partially SalI-digested gene library of SYK-6 constructed with pVK100 as the vector was introduced into P. putida PpY1100 by triparental mating (6). The resulting transconjugants were grown in LB medium containing 50 mg of kanamycin/liter. When the turbidity of the culture at 600 nm reached 1.0, cells were harvested and washed with 50 mM Tris-HCl buffer (pH 7.5). Cells were resuspended in 1 ml of the same buffer. Each 1-ml reaction mixture contained 990 μl of the cell suspension and 10 μl of 100 mM gallate, and the mixtures were shaken at 30°C for 6 h. The cells were removed by centrifugation (15,000 × g for 5 min), and then the supernatant was filtered. The amounts of gallate in the filtrates were determined with a high-pressure liquid chromatography system (HP1100 series LC-MSD; Agilent Technologies Co., Palo Alto, Calif.) using a TSKgel ODS-80TM column (6 by 150 mm; Tosoh, Tokyo, Japan). The mobile phase was a mixture of water (79.2%), acetonitrile (19.8%), and acetic acid (1.0%), and the flow rate was 1.0 ml/min. Compounds were detected at 275 nm, and the retention time of gallate was 2.9 min.

A cosmid, pVK729, was obtained from a transconjugant that showed gallate degradation activity. The 10-kb EcoRV fragment of pVK729 was cloned into pBluescript II KS(+). The resulting plasmid, pEVK1G, was digested with KpnI and then self-ligated, which yielded pEVK3G. These plasmids were introduced into E. coli MV1184, and crude extracts of the transformants were prepared as described below. The gallate dioxygenase activity was assayed by measuring the substrate-dependent oxygen consumption rate.

DNA manipulations and nucleotide sequencing.

DNA manipulations were performed essentially as described by Ausubel et al. (2) and Sambrook et al. (23). A series of deletion derivatives of pEVK3G was constructed by using a Kilosequence kit (Takara Shuzo Co. Ltd., Kyoto, Japan). Nucleotide sequences were determined by the dideoxy termination method (24) with a CEQ 2000XL genetic analysis system (Beckman Coulter, Inc., Fullerton, Calif.). A Sanger reaction was carried out by using a CEQ dye terminator cycle sequencing quick start kit (Beckman Coulter, Inc.). Analysis of the nucleotide sequence was performed as described in a previous study (1).

Expression of desB in E. coli and preparation of cell extracts.

A 2.8-kb NdeI-XbaI fragment carrying desB of pEVK3G was inserted into pET21a(+) to generate pETB. E. coli BL21(DE3) cells harboring pETB were grown in 1 liter of LB medium containing 100 mg of ampicillin/liter at 30°C. Expression of desB was induced for 6 h by adding 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) when the absorbance of the culture at 600 nm reached 0.5. Cells were harvested and resuspended in FE2 buffer consisting of 50 mM Tris-HCl buffer (pH 7.0), 10% glycerol, 0.1 mM ferrous ammonium sulfate, and 2 mM l-cysteine hydrochloride. The cells were broken by two passages through a French pressure cell (Aminco, Urbana, Ill.) and centrifuged at 15,000 × g for 15 min. The resulting supernatant was then used as a crude enzyme.

Purification of DesB.

Enzyme purification was performed by using the method described previously (12) and a BioCAD700E apparatus (PerSeptive Biosystems, Framingham, Mass.). The enzyme solution was applied to a POROS polyethyleneimine (PI) column (16 by 100 mm; PerSeptive Biosystems) previously equilibrated with buffer A consisting of 50 mM Tris-HCl (pH 8.0), 0.1 mM ferrous ammonium sulfate, and 2 mM l-cysteine hydrochloride. The enzyme was eluted with 402 ml of a linear gradient of 0.1 to 0.4 M NaCl. The fractions containing gallate dioxygenase activity that eluted at approximately 0.28 M were pooled, desalted, and concentrated. The resulting solution was applied to a POROS quaternized PI column (4.6 by 100 mm; PerSeptive Biosystems) equilibrated with buffer A. The enzyme was eluted with 50 ml of a linear gradient of 0 to 0.4 M NaCl. The fractions containing gallate dioxygenase activity that eluted at approximately 0.19 M were pooled, desalted, and concentrated as described above.

Purification of LigAB.

E. coli harboring pELAB carrying ligAB was grown in 2.5 liters of LB medium containing 100 mg of ampicillin/liter at 30°C. Expression of ligAB was induced for 2 h by adding 1 mM IPTG. The crude extract was fractionated by two consecutive ammonium sulfate precipitation steps. The cell extract was added to 45% saturation, and then the pellet was removed by centrifugation at 15,000 × g for 15 min. The supernatant was then added to 60% saturation, and the pellet was recovered by centrifugation at 15,000 × g for 15 min and dissolved in 50 ml of FE2 buffer. The enzyme solution was desalted, concentrated, and applied to a POROS PI column previously equilibrated with buffer A. The enzyme was eluted with 241 ml of a linear gradient of 0 to 0.6 M NaCl. The fractions containing PCA 4,5-dioxygenase (4,5-PCD) activity that eluted at approximately 0.17 M were pooled, desalted, and concentrated. The resulting solution was applied to a POROS quaternized PI column equilibrated with buffer A. The enzyme was eluted with 66 ml of a linear gradient of 0 to 0.4 M NaCl. The fractions containing 4,5-PCD activity that eluted at approximately 0.14 M were pooled, desalted, and concentrated as described above.

Analytical methods.

The protein concentration was determined by the method of Bradford (4). The purity of the enzyme preparation was examined by sodium dodecyl sulfate-12% polyacrylamide gel electrophoresis. The molecular mass of the native enzyme was determined by Superdex200 10/300GL (Amersham Biosciences, Freiburg, Germany) gel filtration column chromatography using a BioCAD700E apparatus as described previously (12).

The N-terminal amino acid sequence of DesB was determined by using a PPSQ-21 protein sequencer (Shimadzu, Kyoto, Japan).

Enzyme assays.

The dioxygenase activity of DesB was assayed by measuring the substrate-dependent oxygen consumption rate. Each 2-ml assay mixture contained 50 mM GTA buffer (pH 8.5) consisting of 50 mM 3,3-dimethylglutarate, 50 mM Tris, and 50 mM 2-amino-2-methyl-1,3-propanediol, purified DesB (25 μg of protein), and 500 μM substrate (gallate, PCA, 3MGA, gentisate, 2,3-dihydroxybiphenyl, 4-methylcatechol, 2,3-dihydroxybenzoate, methylgallate, or pyrogallol). The reaction mixture was incubated at 30°C, and the oxygen consumption rate was determined with an oxygen electrode (B-505; Iijima Electronics Manufacturing Co., Ltd., Aichi, Japan). One unit of enzyme activity was defined as the amount of activity that resulted in consumption of 1 μmol of O2 per 1 min at 30°C. Specific activity was expressed in units per milligram of protein. The optimal pH and optimal temperature for DesB were determined at pH and temperature ranges of 6.0 to 9.0 and 15 to 40°C, respectively, by using 50 mM GTA buffer.

Km and Vmax values were obtained from Hanes-Woolf plots and were expressed as means ± standard deviations based on at least three independent experiments. Kinetic parameters were determined at substrate ranges of 0.005 to 5 mM (gallate), 0.01 to 5 mM (3MGA), and 0.005 to 1 mM (PCA). The DesZ and LigAB reactions were carried out essentially as described above.

Identification of the reaction product.

Each 2-ml assay mixture contained 50 mM GTA buffer (pH 8.5), 100 μM gallate, and purified DesB (200 μg of protein). The reaction mixture was incubated at 30°C for 2 h. The reaction mixture was then acidified with 6 N hydrochloric acid to pH 1 and extracted with ethyl acetate. The extract was trimethylsilylated (TMS) with the TMSI-H reagent (GL Science Inc., Tokyo, Japan). The resulting TMS derivatives were analyzed by gas chromatography-mass spectrometry (GC-MS) using a model 5971A instrument with an Ultra-2 capillary column (50 m by 0.2 mm; Agilent Technologies). The analytical conditions were the same as those described previously (12).

Construction of mutants.

The 3.9-kb HindIII-XbaI fragment carrying desB of pEVK4G was inserted into pK19mobsacB to generate pKGBE. The 0.7-kb EcoRI-BamHI fragment in the desB gene of pKGBE was deleted to form pDGBE.

pDGBE was introduced into SYK-6 cells by electroporation, and candidates for a desB mutant (strain RB) were isolated as described previously (18). The ligB disruption plasmid, pAAB, was introduced into cells of the RB strain, and candidates for the desB ligB double mutant (strain RBB) were isolated. In the same way, the desZ disruption plasmid, pKDDZ, was introduced into RB and RBB cells, respectively, and candidates for the desB desZ double mutant (strain RBZ) and the desB desZ ligB triple mutant (strain RBBZ) were isolated (see Fig. 4A to C).

FIG. 4.
Disruption of desB, desZ, and ligB in SYK-6. (A to C) Schematic diagrams of the desB, desZ, and ligB disruptions, respectively. (A) The open box in the thick arrow indicates the region deleted in the desB mutant (RB), desB desZ mutant (RBZ), desB ligB ...

To obtain the ligM desB ligB triple mutant (strain RBBM) and the ligM ligI double mutant (strain DLIM), the ligM disruption plasmids, pDLM and pDALM, were introduced into RBB and the ligI mutant (strain DLI) (18), respectively, and candidates for mutants were isolated (see Fig. Fig.5A5A).

FIG. 5.
Disruption of ligM in the desB ligB mutant and ligI mutant. (A) Schematic diagrams of the ligM disruption in the desB ligB mutant (RBB) and ligI mutant (DLI). Vertical bars above the restriction map indicate the positions of kan and bla gene insertions ...

Disruption of each gene was examined by Southern hybridization analysis. To confirm disruptions of desB, desZ, ligB, and ligM, total DNA of candidates for mutants were digested with KpnI-ApaI, XhoI, PvuII, and SmaI, respectively. The 2.1-kb BglII-ApaI fragment carrying desB, the 1.2-kb SmaI-PvuII fragment carrying desZ, the 1.5-kb XbaI-SmaI fragment carrying ligB, the 2.8-kb Eco47III fragment carrying ligM, the 1.3-kb EcoRV fragment carrying kan, and the 1.0-kb BspHI fragment carrying bla were labeled with the digoxigenin system (Roche Molecular Biochemicals, Mannheim, Germany) and used as probes.

Analysis of mutants.

For growth tests with the mutants on syringate, all the mutants were pregrown in 10 ml of W medium containing 0.2% yeast extract for 36 h. The cells were harvested by centrifugation (5,000 × g for 15 min), washed with 5 ml of W medium, and suspended in 1 ml of the same medium. The cells were inoculated into W medium containing 10 mM syringate to a turbidity at 600 nm of 0.2. The growth of each mutant was periodically monitored by measuring the turbidity at 600 nm.

To determine the gallate dioxygenase activities of cell extracts of SYK-6 and mutants of this strain, the cells were grown in LB medium. Cells grown in LB medium until the turbidity of the culture at 600 nm was 1.5 were harvested by centrifugation (5,000 × g for 15 min), washed with W medium, and suspended in the same medium. To induce the gallate dioxygenase activities, the cells were inoculated into W medium containing 10 mM syringate to a turbidity at 600 nm of 0.5 and incubated for 20 h. The methods used for preparation of the cell extracts were essentially the same as the methods described above. Degradation of gallate by SYK-6 and mutants of this strain was assayed in 2-ml mixtures containing FE2 buffer, 1 mM gallate, and cell extract (5 mg of protein). Reaction mixtures were incubated at 30°C. Portions of each reaction mixture taken at sampling points were analyzed by GC-MS.

Nucleotide sequence accession number.

The nucleotide sequence reported in this paper has been deposited in the DDBJ, EMBL, and GenBank nucleotide sequence databases under accession no. AB190989.

RESULTS AND DISCUSSION

Cloning and nucleotide sequence of the gallate dioxygenase gene.

To isolate the SYK-6 gene responsible for the degradation of gallate, P. putida PpY1100, which is unable to degrade gallate, was employed as a host strain. A gene library of SYK-6 constructed with the cosmid vector pVK100 was introduced into P. putida PpY1100. Approximately 1,000 transconjugants were screened by high-pressure liquid chromatography analysis for the ability to degrade gallate. A cosmid from a transconjugant that exhibited gallate transformation activity was isolated and designated pVK729. A subcloning experiment with pVK729 revealed that pEVK3G containing the 3.9-kb KpnI-EcoRV fragment conferred gallate degradation activity on E. coli MV1184 (Fig. (Fig.22).

FIG. 2.
Deletion analysis of the 3.9-kb KpnI-EcoRV fragment. The gallate dioxygenase activity of E. coli MV1184 harboring each subclone is indicated on the right. The small arrows indicate the directions of transcription from the lac promoters. Abbreviations: ...

In the deletion analysis, the DNA region that conferred gallate-based oxygen consumption activity on E. coli was limited to the 3.1-kb XhoI-EcoRV and 2.6-kb KpnI-ApaI fragments (Fig. (Fig.2).2). Thus, the gallate dioxygenase gene appeared to be located in the 1.8-kb XhoI-ApaI DNA fragment. The nucleotide sequence of the 3.9-kb KpnI-EcoRV fragment was determined, and an open reading frame (orf1) and an incomplete open reading frame (orf2), which are encoded on opposite strands, were identified (Fig. (Fig.2).2). orf1 consists of 1,254 bp and encodes a polypeptide with a molecular mass of 46,843 Da. The amino acid sequence of the N-terminal region encoded by orf1 (residues 1 to 285) exhibited ca. 40% identity with the amino acid sequences encoded by 4,5-PCD β-subunit genes, including LigB (19), PmdB (22), FldU (35), and ProOb (15). On the other hand, the amino acid sequence of the C-terminal region encoded by orf1 (residues 286 to 418) exhibited 13 to 27% identity with the amino acid sequences encoded by 4,5-PCD α-subunit genes, including LigA (19), PmdA (22), FldV (35), and ProOa (15). These results suggested that orf1 encodes gallate dioxygenase, and this gene was designated desB. Based on sequence similarity, the primary structure of desB consists of contiguous DNA segments corresponding to the 4,5-PCD α- and β-subunit genes, which are joined to form a single gene in the order β-α.

The crystallographic study revealed that the active site of LigB contains the Fe ion coordinated by His12, His61, and Glu242, and His195 is thought to act as an active site base to facilitate deprotonation of the hydroxyl group of the substrate (31). These residues are conserved among almost all of the type II extradiol dioxygenases (8). Alignment of the amino acid sequences between the N-terminal region of DesB (residues 1 to 285) and LigB indicated that residues His12, His59, Glu239, and His192 (DesB numbering), corresponding to His12, His61, Glu242, and His195, respectively, of LigB, were conserved in DesB. This result suggested that DesB belongs to the type II extradiol dioxygenase group, and the N-terminal region of DesB appears to form the active center. On the other hand, LigA, the α subunit of LigAB, forms a lid that closes the open end of the binding pocket for PCA (31). The C-terminal region of DesB (residues 286 to 418) is thought to be responsible for the function of LigA. It has been reported that the 4,5-PCD gene (pcmA) of Arthrobacter keyseri 12B is also a single gene corresponding to the 4,5-PCD α- and β-subunit genes, which are joined in the order α-β (7). Considering the high sequence similarity between DesB and each subunit of 4,5-PCD, desB and the 4,5-PCD genes appear to have originated from a common ancestor.

The deduced amino acid sequence encoded by orf2 (79 amino acids) exhibited 53% identity with the amino acid sequence of the 5,10-methylene-H4-folate dehydrogenase/methenyl-H4folate cyclohydrolase (folD) involved in one-carbon (C1) metabolism in E. coli K-12 (5) and Methylobacterium chloromethanicum CM4 (29). In the O demethylation of vanillate, syringate, and 3MGA by SYK-6, the methyl group of these compounds was transferred to H4folate by reactions catalyzed by LigM or DesA (1, 17), and the resulting 5-methyl-H4folate is thought to be metabolized through a C1 metabolic pathway consisting of 5,10-methylene-H4-folate reductase (MetF) and a putative 10-formyl-H4folate synthetase (LigH), the genes for which are located just downstream of ligM (1). To determine the participation of orf2 in C1 metabolism in SYK-6, isolation of the entire gene is currently under way.

Purification of DesB.

The desB gene was cloned in pET21a(+) to construct a plasmid, pETB. Production of the 45-kDa protein in E. coli BL21(DE3) cells harboring pETB was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular mass of this product was similar to the predicted molecular mass of the product of desB (Mr, 46,843). The sequence of the first 10 residues was determined to be Ala-Lys-Ile-Ile-Gly-Gly-Phe-Ala-Val-Ser, which corresponded to the deduced amino acid sequence of desB. The first methionine of DesB appeared to be processed.

To characterize the enzymatic properties of the gene product of desB, DesB was purified to near homogeneity by a combination of column chromatography procedures with PI and quaternized PI. DesB was purified approximately 1.4-fold, with a level of recovery of 8.2%.

Properties of DesB.

Gel filtration chromatography using Superdex 200 indicated that the molecular mass of native DesB was 86.0 kDa. This result suggested that DesB is a homodimer. The optimal temperature and optimal pH for the oxygen consumption activity of DesB with gallate were determined to be 30°C and 8.5, respectively.

Purified DesB (12.5 μg of protein/ml) was incubated with 500 μM gallate, PCA, 3MGA, gentisate, 2,3-dihydroxybiphenyl, 4-methylcatechol, 2,3-dihydroxybenzoate, methylgallate, or pyrogallol at 30°C in order to determine the substrate specificity. Oxygen consumption in the presence of gallate (41.8 U/mg) and pyrogallol (0.11 U/mg) was detected; however, no activity was observed when the other compounds were used as substrates.

The kinetic parameters of DesB, DesZ, and LigAB are summarized in Table Table2.2. The Km for gallate and the Vmax of DesB were determined to be 66.9 ± 9.3 μM and 42.7 ± 2.4 U/mg, respectively. The Vmax/Km of DesZ and LigAB for gallate were only 1.2% and 6.1% of the values of DesB for gallate, respectively, suggesting that DesB is mainly involved in gallate degradation among these ring cleavage dioxygenases.

TABLE 2.
Kinetic parameters of DesB, DesZ, and LigAB

In order to investigate the metal ion dependence of DesB, purified DesB was incubated with 500 μM EDTA at 0°C for 20 h, and the remaining activity was determined. No oxygen consumption was detected with gallate as the substrate in the reaction mixture, suggesting that a divalent cation is required by DesB. Fe2+, Fe3+, Co2+, Cu2+, Mg2+, Mn2+, and Zn2+ ions were added to the EDTA-treated enzyme preparation to a final concentration of 1 mM, and the resulting solutions were kept on ice for 5 min. The oxygen consumption activity was completely recovered by addition of Fe2+ (119%) and was partially recovered by addition of Mn2+ (14%) and Cu2+ (7%). These results suggested that DesB requires Fe2+ for activity.

Identification of the reaction product.

To identify the product formed from gallate, gallate (100 μM) was incubated with purified DesB (100 μg of protein/ml), and the reaction mixture was analyzed by GC-MS (Fig. (Fig.3).3). When the reaction mixture was analyzed immediately after the start of the reaction, the TMS derivative of gallate was detected with a retention time of 29.9 min (Fig. (Fig.3A).3A). DesB transformed 80% of the gallate during 2 h of incubation, and significant accumulation of compound I with a retention time of 30.1 min was observed (Fig. (Fig.3B).3B). The mass spectrum of compound I corresponded to that of the TMS derivative of the previously identified enol form of OMA (Fig. (Fig.3C)3C) (9, 18). However, the keto form of OMA was not detected with this detection method, because α-keto acid is generally unstable and the keto form of OMA might have been degraded during extraction (18). A small PDC peak with a retention time of 28.7 min was also observed (Fig. (Fig.3B).3B). It has been reported that some PDC was produced from OMA when OMA was incubated with hydrochloric acid (14). Because the DesB reaction mixture was acidified by addition of hydrochloric acid in the extraction process, it is very likely that a small amount of PDC was generated from OMA during this process. Therefore, we concluded that DesB is a gallate dioxygenase which catalyzes the ring cleavage of gallate to form OMA.

FIG. 3.
Conversion of gallate to OMA by DesB. Purified DesB (100 μg of protein/ml) was incubated with 100 μM gallate. (A and B) Gas chromatograms of TMS derivatives of the reaction products at the start of incubation and after 2 h of incubation, ...

Roles of desB, desZ, and ligB in syringate catabolism.

The desB gene was disrupted in order to examine its role in syringate degradation. desB in SYK-6 was inactivated by the gene replacement technique with the desB disruption plasmid pDGBE, which was constructed by deleting the 700-bp EcoRI-BamHI fragment of the internal region of desB in pK19mobsacB (Fig. (Fig.4A).4A). Because desZ and ligAB were found to be involved in syringate degradation, desZ, ligB, and both desZ and ligB in the desB mutant were inactivated with desZ and ligB disruption plasmids pKDDZ and pAAB, respectively, which were constructed in a previous study (Fig. 4B and C) (12). The desZ and ligB genes in pKDDZ and pAAB were inactivated by inserting the kan and bla genes into the structural genes, respectively. The resulting mutants were confirmed by Southern hybridization analysis with the desB, desZ, ligB, kan, and bla genes as probes (see Fig. S1 in the supplemental material).

In a previous study, we demonstrated that the rate of growth of the desZ ligB mutant (strain DBZ) on syringate was slightly less (k = 0.07/h) than that of SYK-6 (k = 0.09/h) (Fig. (Fig.4D).4D). In the case of the desB mutant (RB), the rate of growth on syringate was markedly decreased (k = 0.02/h), and the turbidity of the culture was ca. 54% that of the wild-type strain (Fig. (Fig.4E).4E). These results suggested that desB is primarily involved in the degradation of syringate. During the growth of RB cells with syringate, the culture darkened, suggesting that there was accumulation of an intermediate metabolite. However, no product was detected by GC-MS analysis. It is thought that because gallate is so unstable, the culture rapidly darkened when gallate was incubated in W medium without cells. The results of the GC-MS analysis revealed neither gallate nor derivatives of this compound in this culture (data not shown). Therefore, gallate appears to accumulate in an RB culture when it is incubated with syringate, and under these conditions gallate might be automatically oxidized and polymerized.

The desB desZ ligB triple mutant (strain RBBZ) and the desB ligB double mutant (strain RBB) completely lost the ability to grow on syringate (Fig. (Fig.4E).4E). In addition, the growth rate of the desB desZ double mutant (strain RBZ) on syringate was almost the same as that of RB. These results suggested that the role played by desZ in syringate degradation is quite minor. However, we could not exclude the possibility that the accumulation of gallate in RBB inhibited the growth of this strain on syringate. Our preliminary experiment showed that the growth of SYK-6 on syringate was almost completely inhibited by addition of 1 mM gallate (data not shown). In order to determine the actual contribution of desZ to syringate catabolism, ligM, which encodes vanillate/3MGA O-demethylase in RBB, was inactivated with the ligM disruption plasmid pDLM in order to avoid accumulation of gallate during incubation of the mutant with syringate (Fig. (Fig.5A;5A; see also Fig. S1 in the supplemental material). The resulting mutant strain, RBBM, was able to grow on syringate (Fig. (Fig.5B),5B), indicating that desZ does possess the ability to support the growth of SYK-6 on syringate.

DesZ catalyzes the 3,4-cleavage of 3MGA to form 4-carboxy-2-hydroxy-6-methoxy-6-oxohexa-2,4-dienoate (CHMOD), which is accompanied by generation of PDC. To confirm the presence of the syringate catabolic pathway via CHMOD, a ligM and ligI (PDC hydrolase gene) mutant (strain DLIM) was constructed by introduction of the ligM disruption plasmid pDLAM into the ligI insertion mutant strain DLI (Fig. (Fig.5A)5A) (18). This investigation revealed that DLIM was able to grow on syringate (Fig. (Fig.5B),5B), thus indicating that there was a pathway converting 3MGA to OMA via CHMOD catalyzed by DesZ and a hydrolase.

In order to estimate the level of participation of desB, desZ, and ligAB in gallate degradation, the gallate degradation activities of cell extracts of SYK-6 and disruption mutants of this strain incubated with syringate were determined by GC-MS. While the gallate degradation activities of DB, DZ, and DBZ were not affected, the activity of the cell extract of RB was found to decrease, and RBB and RBBZ were completely unable to degrade gallate (Fig. (Fig.6).6). These results indicated that desB and ligAB, but not desZ, are involved in the degradation of gallate. The kinetic properties of the three dioxygenases, the gallate degradation profiles of the mutants, and the observation that the desB mutants accumulated gallate strongly suggested that desB plays a major role in the degradation of gallate by SYK-6.

FIG. 6.
Degradation of gallate by insertion mutants. One millimolar gallate was incubated with cell extracts (2.5 mg of protein/ml) of SYK-6 (open diamonds), DZ (open squares), DB (open triangles), DBZ (open circles), RB (solid diamonds), RBZ (solid squares), ...

In conclusion, we characterized the gallate dioxygenase gene, desB, and determined the roles of the three ring cleavage dioxygenases in syringate degradation. The 3MGA catabolic pathways can be summarized as follows: (i) conversion of 3MGA to OMA via gallate in the reaction catalyzed by LigM and DesB or LigAB; (ii) conversion of 3MGA to OMA via CHMOD in the reaction catalyzed by DesZ and a hydrolase; and (iii) conversion of 3MGA to PDC in the reaction catalyzed by LigAB (and DesZ). The first pathway catalyzed by DesB is apparently the major pathway. LigAB also seemed to contribute to this pathway based on the finding that the RB cells exhibited gallate transformation activity (Fig. (Fig.6).6). However, considering the high Km value of LigAB for gallate, its contribution to the gallate degradation during growth of SYK-6 on syringate appeared to be minor. In addition, the Vmax/Km values of LigAB for gallate and 3MGA were only 6.1% and 5.8% of the Vmax/Km values of DesB for gallate and of DesZ for 3MGA, respectively. Based on the fact that LigAB is also involved in 3MGA transformation together with DesZ, the level of expression of ligAB might be higher than the levels of expression of desB and desZ when the cells are grown in the presence of syringate. Therefore, it is necessary to study the regulation of three dioxygenase genes in order to gain a better understanding of syringate catabolism by this strain.

Supplementary Material

[Supplemental material]

Acknowledgments

This work was supported in part by Grant-in-Aid for Encouragement of Young Scientists 13760062 from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

Footnotes

Supplemental material for this article may be found at http://jb.asm.org/.

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