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Journal of Bacteriology, January 2003, p. 41-50, Vol. 185, No. 1
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.1.41-50.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Characterization of the 4-Carboxy-4-Hydroxy-2-Oxoadipate Aldolase Gene and Operon Structure of the Protocatechuate 4,5-Cleavage Pathway Genes in Sphingomonas paucimobilis SYK-6

Hirofumi Hara,1 Eiji Masai,1* Keisuke Miyauchi,1 Yoshihiro Katayama,2 and Masao Fukuda1

Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata 940-2188,1 and Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan2

Received 29 July 2002/ Accepted 4 October 2002


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ABSTRACT
 
The protocatechuate (PCA) 4,5-cleavage pathway is the essential metabolic route for degradation of low-molecular-weight products derived from lignin by Sphingomonas paucimobilis SYK-6. In the 10.5-kb EcoRI fragment carrying the genes for PCA 4,5-dioxygenase (ligAB), 2-pyrone-4,6-dicarboxylate hydrolase (ligI), 4-oxalomesaconate hydratase (ligJ), and a part of 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase (ligC), we found the ligK gene, which encodes 4-carboxy-4-hydroxy-2-oxoadipate (CHA) aldolase. The ligK gene was located 1,183 bp upstream of ligI and transcribed in the same direction as ligI. We also found the ligR gene encoding a LysR-type transcriptional activator, which was located 174 bp upstream of ligK. The ligK gene consists of a 684-bp open reading frame encoding a polypeptide with a molecular mass of 24,131 Da. The deduced amino acid sequence of ligK showed 57 to 88% identity with those of the corresponding genes recently reported in Sphingomonas sp. strain LB126, Comamonas testosteroni BR6020, Arthrobacter keyseri 12B, and Pseudomonas ochraceae NGJ1. The ligK gene was expressed in Escherichia coli, and the gene product (LigK) was purified to near homogeneity. Electrospray-ionization mass spectrometry indicated that LigK catalyzes not only the conversion of CHA to pyruvate and oxaloacetate but also that of oxaloacetate to pyruvate and CO2. LigK is a hexamer, and its isoelectric point is 5.1. The Km for CHA and oxaloacetate are 11.2 and 136 µM, respectively. Inactivation of ligK in S. paucimobilis SYK-6 resulted in the growth deficiency of vanillate and syringate, indicating that ligK encodes the essential CHA aldolase for catabolism of these compounds. Reverse transcription-PCR analysis revealed that the PCA 4,5-cleavage pathway genes of S. paucimobilis SYK-6 consisted of four transcriptional units, including the ligK-orf1-ligI-lsdA cluster, the ligJAB cluster, and the monocistronic ligR and ligC genes.


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INTRODUCTION
 
Protocatechuate (PCA) is one of the most important intermediate metabolites in the bacterial degradation pathways for various aromatic compounds, including low-molecular-weight products derived from lignin. Sphingomonas paucimobilis SYK-6 is able to degrade a wide variety of dimeric lignin compounds, including ß-aryl ether (24, 25), biphenyl (33, 34), pinoresinol, phenylcoumarane, and diarylpropane. In S. paucimobilis SYK-6, dimeric lignin compounds with guaiacyl (4-hydroxy-3-methoxyphenyl) and syringyl (4-hydroxy-3,5-dimethoxyphenyl) moieties are thought to be converted to vanillate and syringate, respectively (26). Vanillate and syringate are converted into PCA and 3-O-methylgallate (3MGA), respectively. It is known that the aromatic ring opening of PCA is catalyzed by one of the three dioxygenase species: PCA 3,4-dioxygenase (3,4-PCD) (4, 9, 12, 51), 4,5-PCD (31, 44), and 2,3-PCD (49). The 3,4-PCD is the most extensively characterized enzyme, and the metabolic pathway for the 3,4-PCD product, ß-carboxy-cis,cis-muconate into succinyl coenzyme A and acetyl coenzyme A (the ß-ketoadipate pathway) has been well characterized (7, 13, 14, 32). On the other hand, the PCA 4,5- and PCA 2,3-cleavage pathways are poorly understood.

In the case of S. paucimobilis SYK-6, PCA is degraded via the PCA 4,5-cleavage pathway (Fig. 1). This pathway was enzymatically characterized by Kersten et al. (16) and Maruyama and coworkers (18-22). In this pathway, PCA is initially trans-formed to 4-carboxy-2-hydroxymuconate-6-semialdehyde(CHMS) by 4,5-PCD (LigAB). CHMS is nonenzymatically converted to an intramolecular hemiacetal form and then oxidized by CHMS dehydrogenase. The resulting intermediate, 2-pyrone-4,6-dicarboxylate (PDC), is hydrolyzed by PDC hydrolase to yield the keto form and enol form of 4-oxalomesaconate (OMA), which are in equilibrium. OMA is converted to 4-carboxy-4-hydroxy-2-oxoadipate (CHA) by OMA hydratase. Finally, CHA is cleaved by CHA aldolase to produce pyruvate and oxaloacetate. Recently, we have identified and characterized all of the gene products and genes except the CHA aldolase gene in the SYK-6 PCA 4,5-cleavage pathway (11, 27, 28, 31, 44). These genes are essential to PCA degradation, while the 3MGA degradation is suggested to go through both the PCA 4,5-cleavage pathway and an alternative ring-cleavage pathway. In this alternative pathway, 3MGA was finally converted to OMA and then entered the PCA 4,5-cleavage pathway. Thus, the PCA 4,5-cleavage pathway genes play a key role in PCA and 3MGA degradation (11).



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  		FIG. 1. Catabolic pathway of vanillate and syringate by S. paucimobilis SYK-6 (A) and organization of the PCA 4,5-cleavage pathway genes (B). (A) LigA and LigB, the small and large subunits of 4,5-PCD (31, 44); LigH, an essential gene product for vanillate and syringate O demethylations (30); LigC, CHMS dehydrogenase (27); LigI, PDC hydrolase (28); LigJ, OMA hydratase (11); LigK, CHA aldolase/oxaloacetate (OA) decarboxylase (in this study). The degradation pathway for syringate indicated by a dashed line was suggested in our previous study (11, 27, 28). (B) orf1, orf2, ligI, ligJ, ligK, ligA, ligB, ligC, ligR, and lignostilbene {alpha},ß-dioxygenase homolog (lsdA) genes are demonstrated by the filled arrows. Vertical bars above the restriction map indicate the positions of the Kmr gene insertion of orf1 mutant (DF1), ligK mutant (DLK), ligR mutant (DLR), and orf2 mutant (DF2). Double-headed arrows indicate locations of amplified RT-PCR products shown in Fig. 5. Abbreviations for restriction enzymes: C, ClaI; E, EcoRI; Ec, Eco47III; P, PstI; Pu, PpuMI; Sh, SphI; Sl, SalI; Sm, SmaI; X, XhoI; Xb, XbaI.

Recently, cloning of the PCA 4,5-cleavage pathway genes has been reported in Sphingomonas sp. strain LB126 (48), Arthrobacter keyseri 12B (6), Comamonas testosteroni BR6020 (36), and Pseudomonas ochraceae NGJ1 (23). However, detailed information is not available in regard to the actual role and property of each of the corresponding gene products. In this study, we characterized the structure and functions of the CHA aldolase gene, which is involved in the final step of the PCA 4,5-cleavage pathway. We also examined the involvement of the two open reading frames (ORFs) found among the genes which encode the PCA 4,5-cleavage pathway enzymes, and the operon structure of this pathway genes was estimated.


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MATERIALS AND METHODS
 
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 (33) containing 10 mM vanillate or syringate or in Luria-Bertani (LB) medium (1).


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  		TABLE 1. Strains and plasmids used in this study

Preparation of substrate. PDC and OMA were prepared as described earlier (28). CHA was prepared by incubating 1 mmol of OMA with 500 U of purified OMA hydratase for 5 min (11). Electrospray-ionization mass spectrometry (ESI-MS) analysis revealed that the m/z 201 showing [M-H]- of OMA (where M is a molecular ion of OMA) was completely converted into m/z 219, indicating [M-H]- of CHA by LigJ. Then, the reaction product of OMA catalyzed by purified LigJ was used as a substrate.

DNA manipulations and nucleotide sequencing. DNA manipulations were carried out essentially as described in references 1 and 38. 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.).

A Sanger reaction (39) was carried out by using the Thermosequenase fluorescence-labeled primer cycle sequencing kit with 7-deaza-dGTP (Amersham Pharmacia Biotech, Little Chalfont, United Kingdom). Sequence analysis and homology alignment were carried out with the GeneWorks programs (IntelliGenetics, Inc., Mountain View, Calif.). The DDBJ database was used for searching homologous proteins.

Enzyme assay. According to the method of Maruyama (21), a coupled assay was used for CHA aldolase. The decrease in the absorbance at 340 nm derived from the oxidation of NADH ({varepsilon}340 = 6.6 x 103 M-1 cm-1; pH 8.0) in a reaction mixture containing 200 µM CHA, 140 µM NADH, coupled enzymes (30 U of lactate dehydrogenase and malate dehydrogenase), 1 mM MgCl2, and a suitable aliquot of LigK was measured in 0.1 M Tris-acetate buffer (pH 8.0). The enzyme reaction was carried out at 30°C in a cuvette. One unit of enzyme activity is defined as that causing the oxidation of 2 µmol of NADH/min in this assay. Specific activity was expressed as units per milligram of protein. Oxaloacetate decarboxylase activity was determined by measuring the decrease in absorbance at 340 nm derived from the oxidation of NADH. The 1-ml reaction mixture contained 200 µM oxaloacetate, 140 µM NADH, 30 U of lactate dehydrogenase, 1 mM MgCl2, and LigK enzyme in 0.1 M Tris-acetate buffer (pH 8.0). One unit of enzyme activity is defined as that causing the oxidation of 1 µmol of NADH/min in this assay. Under these conditions, the spontaneous oxaloacetate decarboxylase activity was detected (0.01 U). This spontaneous activity was subtracted from the raw data of oxaloacetate decarboxylase activity of LigK. Specific activity was expressed as units per milligram of protein. The Km and Vmax values were obtained from the Hanes-Woolf plots. The inhibition constant (Ki) for oxaloacetate was determined from the Dixon plot. These kinetic constants were 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. Escherichia coli BL21(DE3) harboring pETK was grown in 100 ml of LB medium containing 100 mg of ampicillin/liter. Expression of ligK was induced for 4 h at 37°C by the addition of isopropyl-ß-D-thiogalactopyranoside (final concentration, 1 mM) when the turbidity of the culture at 660 nm reached 0.5. Cells were harvested by centrifugation and resuspended in 20 mM Tris-HCl buffer (pH 8.0) (buffer A). The cells were broken by two passages through a French pressure cell. The cell lysate was centrifuged at 15,000 x g for 15 min. Streptomycin (final concentration, 1% [wt/vol]) was added to the supernatant, which was centrifuged again at 15,000 x g for 15 min to remove nucleic acids. The supernatant was recovered and then centrifuged again at 170,000 x g for 60 min at 4°C. The crude extract was obtained after concentration by ultrafiltration using a minicon B15 (Amicon, Beverly, Mass.).

(ii) POROS PI anion-exchange chromatography. The crude extract was applied to a POROS polyethyleneimine (PI) column (7.5 by 100 mm) (PerSeptive Biosystems) previously equilibrated with buffer A. The enzyme was eluted with 88 ml of linear gradient of 0 to 0.5 M NaCl. The CHA aldolase was eluted at approximately 0.20 M.

(iii) POROS HQ anion-exchange chromatography. The fractions containing CHA aldolase activity eluted from a PI column were pooled, desalted, and concentrated by ultrafiltration using a minicon B15. The resulting solution was applied to a POROS quaternized polyethyleneimine (HQ) 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 CHA aldolase activity that eluted at approximately 0.30 M were pooled.

(iv) POROS PE hydrophobic-interaction chromatography. The fractions containing CHA aldolase activity eluted from an HQ column were pooled, desalted, and concentrated. Ammonium sulfate was added to the enzyme solution to a final concentration of 2 M. After centrifugation at 15,000 x g for 10 min, the supernatant was recovered and applied to a POROS phenylether (PE) 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 CHA aldolase activity that eluted at approximately 1.3 M were pooled, desalted, and concentrated as described above. Glycerol was added to a final concentration of 10%, and the purified enzyme was stored at -80°C until use.

Analytical method. The protein concentration was determined by the method of Bradford (2). The purity of the enzyme preparation was examined by sodium dodecyl sulfate-15% polyacrylamide gel electrophoresis (SDS-15% PAGE) (17). The molecular mass of the native enzyme was estimated by Superdex200 HR10/30 (Pharmacia Biotech) gel filtration column chromatography using a BioCAD700E apparatus. Elution was performed with 50 mM potassium phosphate buffer (pH 7.0) containing 0.15 M NaCl at a flow rate of 0.8 ml/min. The molecular weight was estimated on the basis of calibration curve of reference proteins.

To determine the N-terminal amino acid sequence, the cell extract of E. coli BL21(DE3) harboring pETK was subjected to SDS-15% PAGE and electroblotted onto a polyvinylidene difluoride membrane (Bio-Rad, Hercules, Calif.). The area at 27 kDa was cut out and analyzed on a PPSQ-21 protein sequencer (SHIMADZU, Kyoto, Japan). The isoelectric point of LigK was determined by isoelectric focusing on an Ampholine PAG plate (pH 3.5 to 9.5; Pharmacia Biotech) using a model Multiphor II electrophoresis system (Pharmacia Biotech).

The substrate and the reaction products were detected and identified by gas chromatography (GC)-MS using model 5971A with an Ultra-2 capillary column (50 m by 0.2 mm; Agilent technologies, Palo Alto, Calif.) and ESI-MS using HP1100 series LC-MSD (Agilent technologies). The analytical conditions for GC-MS were the same as described previously (28). In ESI-MS analysis, mass spectra were obtained by negative-mode ESI, with a needle voltage of -3.5 kV and a source temperature at 350°C. The sample was injected directly into the mass spectrometer; the water/methanol ratio was 90:10 (vol/vol), and the flow rate was 0.2 ml/min.

Identification of the reaction product. 200 µM CHA was incubated with purified LigK (0.5 µg) in 0.1 M Tris-acetate buffer (pH 8.0) containing 1 mM MgCl2 for 1 min or 5 min, the reaction mixture was diluted to 1/10 with 10 mM Tris-acetate buffer (pH 8.0), and the portion of mixture (5 µl) was injected into the ESI-mass spectrometer.

In the case of GC-MS analysis, the reaction product was acidified and extracted with ethylacetate, and then the extract was trimethylsilylated. The resultant trimethylsilylated derivatives were analyzed.

The metabolites of vanillate and syringate by the ligK insertion mutant (DLK) were analyzed. DLK cells grown in 10 ml of LB medium were washed with 0.1 M Tris-acetate buffer (pH 8.0). The cells were resuspended in the same buffer and incubated with 10 mM vanillate and 10 mM syringate for 12 h at 30°C. After centrifugation, the supernatant was diluted 20-fold with 10 mM Tris-acetate buffer (pH 8.0) and analyzed by ESI-MS as described above. On the other hand, the metabolites were extracted by ethylacetate, trimethylsilylated, and analyzed by GC-MS.

Disruption of orf1, ligK, ligR, and orf2. The 4.0-kb XhoI-SmaI fragment carrying ligK and orf1 was cloned into pBluescript II SK(+) to generate pXS4, and it was digested with PpuMI for ligK disruption or with SalI for orf1 disruption. The 1.2-kb PstI fragment containing the kanamycin resistance gene from pUC4K (47) was inserted into the PpuMI or SalI site of the 4.0-kb XhoI-SmaI fragment to construct pXS4K and pXS4K2, respectively. pXS4K and pXS4K2 were digested with BamHI and KpnI, and their inserts were cloned into pK19mobsacB (40) to generate pLKD and pF1D, respectively. The 1.8-kb ClaI-SmaI fragment carrying ligR was cloned into pUC19 to generate pCS18, and it was digested with Eco47III. The kanamycin resistance gene was inserted into this Eco47III site. The resultant plasmid, pCS18K, was digested with KpnI and SacI, and the insert containing the inactivated ligR gene was cloned into pK19mobSacB to generate pLRD. The 1.7-kb PstI fragment carrying orf2 was cloned into pUC19 to generate pPS17, and it was digested with SmaI. The kanamycin resistance gene was inserted into the SmaI site. The resultant plasmid, pPS17K, was digested with BamHI and KpnI, and the insert containing the inactivated orf2 gene was cloned into pK19mobsacB to generate pF2D.

Each of plasmids, pLKD, pF1D, pLRD, and pF2D was introduced into SYK-6 cells by electroporation, and the candidates for mutants were isolated as described previously (28). To examine the disruption of each gene, Southern hybridization analysis was carried out. The total DNA of the candidates for ligK, ligR, and orf2 mutants were digested with PstI, and those for orf1 were digested with SmaI. The 1.2-kb PstI fragment carrying the kanamycin resistance gene, the 2.3-kb PstI fragment carrying ligR and ligK, the 4.0-kb XhoI-SmaI fragment carrying orf1, and the 1.7-kb PstI fragment carrying orf2 were labeled with the DIG system (Roche Diagnostics, Indianapolis, Ind.) and used as probes.

Reverse transcription (RT)-PCR. Cells of S. paucimobilis SYK-6 were grown in W minimal salt medium containing 10 mM vanillate until they reached the turbidity at 660 nm of 0.5. Total RNA was prepared from 10 ml of culture by using RNeasy Mini columns (Qiagen Inc, Chatsworth, Calif.). To remove any contaminating genomic DNA, the RNA samples were incubated with 1 U of RNase-free DNase (Takara Shuzo Co., Ltd.) in 40 mM Tris-HCl (pH 7.9) containing 1 U of RNase inhibitor (Takara Shuzo Co., Ltd.), 10 mM NaCl, 10 mM CaCl2, and 6 mM MgSO4 for 30 min at 37°C. RT-PCR was carried out with a BcaBEST RNA PCR kit (Takara Shuzo Co., Ltd.). A cDNA library was obtained by an RT reaction using a hexanucleotide random priming mix. The cDNA was used as a template for subsequent PCRs with specific primers, which amplify the boundaries of ligK-orf1-ligI-lsdA and ligR-orf2-ligJ-ligA-ligB-ligC. The forward and reverse primers used were as follows: lsdA-forward (nucleotide positions from 1,363 to 1,383 in the 10.5-kb EcoRI fragment) and ligI-reverse (positions 1,924 to 1,944); ligI-forward (positions 2,528 to 2,548) and orf1-reverse (positions 2,999 to 3,019); orf1-forward (positions 3,489 to 3,509) and ligK-reverse (positions 3,740 to 3,760); internal ligR-forward (positions 4,613 to 4,533) and internal ligR-reverse (positions 5,215 to 5,235); ligR-forward (positions 5,770 to 5,790) and orf2-reverse (positions 6,476 to 6,496); internal orf2-forward (positions 5,843 to 5,863) and orf2-reverse; orf2-forward (positions 6,536 to 6,556) and ligJ-reverse (positions 6,943 to 6,963); ligJ-forward (positions 7,662 to 7,682) and ligA-reverse (positions 8,162 to 8,182); ligA-forward (positions 8,162 to 8,182) and ligB-reverse (positions 8,706 to 8,726); ligB-forward (positions 9,119 to 9,139) and ligC-reverse (positions 9,598 to 9,608); internal ligC-forward (positions 9,609 to 9,629) and internal ligC-reverse (positions 10,098 to 10,118). Control samples in which reverse transcriptase was omitted in RT-PCR and in which genomic DNA was used as a template in PCRs were run in parallel with RT-PCRs.

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. AB073227.


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RESULTS AND DISCUSSION
 
Nucleotide sequence analysis. We detected the CHA aldolase activity in E. coli JM109 harboring pHN139F, which contained the 10.5-kb EcoRI fragment carrying ligAB (31), ligI (28), ligJ (11), and a part of ligC (27) (Fig. 1B). In the deletion analysis, the DNA region that conferred CHA aldolase activity to E. coli was limited to the 1.0-kb SalI-SphI fragment. The nucleotide sequences of the 5.0-kb SmaI fragment and the overlapping 1.7-kb PstI fragment were determined, and an ORF of 684 bp was revealed in the 1.0-kb SalI-SphI fragment. This ORF encodes 228 amino acid residues with a molecular mass of 24,131 Da and was designated ligK. The deduced amino acid sequence of ligK shared the highest degree of identity (88%) with that of fldZ, which is located in the putative PCA 4,5-cleavage pathway genes of the fluorene degrader Sphingomonas sp. strain LB126 (48). The deduced amino acid sequence of ligK also showed 66, 66, and 57% identity with those of the CHA aldolase genes recently identified in C. testosteroni BR6020 (36), P. ochraceae NGJ1 (23), and A. keyseri 12B (6), respectively. Three ORFs were also found adjacent to ligK (Fig. 1B). orf1 was located downstream of ligK with a direction of transcription identical to that of ligK. On the other hand, ligR and orf2 were located upstream of ligK with a transcription orientation opposite that of ligK. The orf1 product showed 78 and 44% identity to FldA of LB126 and a putative transmembrane protein of YbhH in E. coli (GenBank accession no. D90715), whose functions are unknown. This similarity may suggest that orf1 encodes a transporter for substrates such as PCA, vanillate, and/or syringate. LigR has 28% identity with various LysR-type transcriptional regulators, including PcaQ of {alpha} proteobacterium Y3F (3), and 22% identity with SdsB, which positively regulates alkyl sulfatase (SdsA) of Pseudomonas sp. strain ATCC 19151 (5). LigR might be involved in the transcriptional control of ligK-orf1-ligI-lsdA, because LysR-type transcriptional regulators generally control the divergently transcribed operon (41). We are unable to conjecture about the function of orf2, although its deduced amino acid sequence showed 50% identity with FldX from Sphingomonas sp. strain LB126 (48) and 39% identity with a conserved hypothetical protein from Sinorhizobium meliloti (8), whose functions are unknown.

The PCA 4,5-cleavage pathway genes of S. paucimobilis SYK-6 consisted of the two divergently transcribed gene clusters of ligK-orf1-ligI-lsdA and ligR-orf2-ligJ-ligA-ligB-ligC (Fig. 1B). Recently, all or partial sequences of the PCA 4,5-cleavage pathway genes have been reported in Sphingomonas sp. strain LB126 (48), A. keyseri 12B (6), C. testosteroni BR6020 (36), and P. ochraceae NGJ1 (23). Two types of gene clusters can be clearly seen. Interestingly, the gene organization of the fluorene degrader, Sphingomonas sp. strain LB126, is essentially the same as that of SYK-6, although the identities of the corresponding genes between SYK-6 and LB126 vary from 50 to 88%. On the other hand, C. testosteroni BR6020 and A. keyseri 12B have a packed single gene cluster that seems to be an operon.

Purification of CHA aldolase. The 1.0-kb SalI-SphI fragment carrying ligK was cloned in pET21(+) to construct pETK, and ligK was expressed in E. coli BL21(DE3) under the control of the T7 promoter. Production of the 27-kDa protein was observed by SDS-PAGE (data not shown). The size of the product is close to the molecular mass calculated from the deduced amino acid sequence of ligK. LigK was purified from a cell extract of E. coli BL21(DE3) harboring pETK by a series of column chromatography procedures with PI, HQ, and PE. LigK was purified approximately 52-fold to near homogeneity (>99%) with a recovery of 20%. N-terminal amino acid sequencing revealed that the first 15 residues, with the exception of the first methionine, Arg-Gly-Ala-Ala-Met-Gly-Val-Val-Val-Gln-Asn-Ile-Glu-Arg-Ala, corresponded to the deduced amino acid sequence of ligK.

Identification of the reaction product. To identify the reaction product of CHA catalyzed by the purified LigK, the reaction mixture was analyzed by ESI-MS. The fragment at m/z 219 in Fig. 2B was estimated to be the deprotonated molecular ion ([M-H]-) of CHA (where M is a molecular ion). The other peaks in the spectrum of CHA were originated from components of the reaction buffer containing LigK enzyme (Fig. 2A). After 1 min of reaction, the intensity of the fragment at m/z 219 of CHA decreased to 42% of its initial intensity, and the generation of two fragments at m/z 87 and at m/z 131 corresponding to [M-H]- of pyruvate and [M-H]- of oxaloacetate, respectively, was observed (Fig. 2C). This result indicated that LigK catalyzes the conversion of CHA to pyruvate and oxaloacetate. After 5 min of reaction, the intensity of the fragment at m/z 87 increased to 144% of that of the corresponding fragment in the 1-min reaction mixture, whereas the intensity of the fragment at m/z 131 decreased to 38% of that of the corresponding fragment in the 1-min reaction mixture (Fig. 2D). These results strongly suggested that oxaloacetate was converted into pyruvate by LigK. The activity for ß-decarboxylation of oxaloacetate has been reported in CHA aldolase of P. ochraceae (21), 4-hydroxy-4-methyl-2-oxoglutarate aldolase of P. putida (45), and 2-keto-4-hydroxyglutarate aldolase of E. coli (29). To examine whether LigK has this activity, oxaloacetate was incubated with LigK in the presence of lactate dehydrogenase and NADH. A decrease in absorbance at 340 nm derived from NADH was observed. It is concluded that LigK is able to decarboxylate oxaloacetate to generate pyruvate.



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  		FIG. 2. Identification of the reaction product from CHA catalyzed by LigK. CHA (200 µM) was incubated with 0.5 µg of LigK in 0.1 M Tris-acetate buffer (pH 8.0) containing 1 mM MgCl2. (A) Negative-ion ESI-MS spectrum of the reaction mixture without CHA. (B to D) Negative-ion ESI-MS spectrum of the reaction mixture at 0, 1, and 5 min of incubation with LigK, respectively.

Enzyme properties. In accord with the previous study by Maruyama (21), the CHA aldolase activity was observed only when a divalent cation such as Mg2+ was present in the reaction mixture. We examined the effect of the various divalent cations for the enzyme activities of LigK. Addition of 1 mM Co2+, Zn2+, Ca2+, or Mn2+ resulted in 85, 65, 20, or 0% of the activity resulting from addition of 1 mM Mg2+, respectively. A similar metal dependency was observed in the decarboxylation of oxaloacetate by LigK. When 1 mM EDTA was added to the reaction mixture, both enzyme activities were completely lost in the presence of 1 mM metal ion. Aldolases are categorized as class I or class II based on their metal dependency. LigK was suggested to be one of the class II aldolases, which require the metal ion. Most class II aldolases show a significant rate enhancement in the presence of phosphate ion (37). Addition of 0.5 mM phosphate ion in the LigK reaction mixture caused 3.0- and 1.7-fold activation of CHA aldolase and oxaloacetate decarboxylase activity, respectively.

Gel filtration column chromatography using the Superdex200 indicated that the molecular mass of the native LigK was 160 kDa. This result suggested that LigK is a homohexamer. The isoelectric point of LigK was determined to be 5.1 by isoelectric focusing gel electrophoresis. The optimal temperature of LigK for aldolase activity on CHA, and the decarboxylase activity on oxaloacetate were both determined to be 25°C. The optimal pH for aldolase activity and decarboxylase activity were estimated to be 8.0 and 7.0, respectively. The Km for oxaloacetate (136 µM) is 12 times higher than that for CHA (11.2 µM). The Vmax for CHA aldol cleavage (265 U/mg) is 20 times higher than that for oxaloacetate decarboxylation (13.2 U/mg).

We also examined the influence of sulfhydryl reagents on LigK. One microgram of purified LigK was preincubated with 1 mM sulfhydryl reagents for 10 min. HgCl2 and N-ethylmaleimide inhibited 60 and 62% of the CHA aldolase and 92 and 88% of the oxaloacetate decarboxylase activities, respectively. These results suggested that some cysteine residues might be involved in the enzyme reaction. CHA aldolase activity was inhibited by oxaloacetate with a Ki value of 23 µM. As suggested by Maruyama (21), the amount of oxaloacetate in the cells might control the production of oxaloacetate from CHA.

CHA aldolase has been biochemically characterized only in P. ochraceae (21) and P. putida (45). The molecular mass, subunit structure, and pI of LigK are very similar to those of aldolases of P. ochraceae and P. putida. In the P. ochraceae enzyme, the kinetics parameters are measured using the substrates d-CHA and l-CHA. The Km and Vmax values of the P. ochraceae enzyme for l-CHA were similar to those for LigK. In our experiment, CHA was prepared from OMA by using the purified OMA hydratase from E. coli carrying the SYK-6 ligJ gene. The physiological substrate for LigK might be an l-isomer. The Km for oxaloacetate of the P. ochraceae enzyme was twofold higher than that of LigK, although the Vmax values of these strains are similar. LigK has a significantly higher affinity for oxaloacetate than did the P. ochraceae enzyme.

Disruption of ligK in S. paucimobilis SYK-6. The ligK gene was disrupted to clarify the actual role of ligK in the catabolism of vanillate and syringate by SYK-6. Gene inactivation was carried out using the ligK disruption plasmid, pLKD. The ligK insertional mutation was confirmed by Southern hybridization analysis using the 2.3-kb PstI fragment carrying ligK and the 1.2-kb PstI fragment carrying the kanamycin resistance gene as probes (data not shown). The ligK and kanamycin resistance gene probes revealed that the ligK gene was inactivated by homologous recombination through the double crossover. This mutant strain was designated DLK and used for the following experiments. The obtained mutant strain DLK completely lost the ability to grow on both vanillate and syringate. This result is compatible with the deduced catabolic pathways of vanillate and syringate by SYK-6 shown in Fig. 1A.

To determine the accumulated products from vanillate and syringate incubated with DLK, 10 mM concentrations of vanillate and syringate were independently incubated with the LB-grown whole cells of DLK in the W minimal medium, and the metabolites were identified by GC-MS and ESI-MS. As shown in the gas chromatogram (Fig. 3A and B), vanillate and syringate detected with retention times of 21.2 and 25.2 min, respectively, disappeared completely, and the accumulation of PDC, the enol form of OMA, product I with a retention time of 30.5 min, and product II with a retention time of 26.1 min was observed in both cultures. In a previous study, we identified product I as the compound generated from OMA by addition of two atoms of hydrogen by NADPH-dependent reductase in the ligJ insertion mutant of SYK-6 (11). The mass spectrum of product II accumulated in both cultures are identical but could not be assigned (data not shown). On the other hand, ESI-MS analysis indicated accumulation of the products whose deprotonated molecular ions appeared at m/z 203 and at m/z 221 in the metabolite from vanillate and syringate (Fig. 3C and D). In this analytical condition, neither PDC nor the enol form of OMA could be detected. We previously suggested that the ion at m/z 203 was a deprotonated molecular ion of product I. We therefore estimated that the ion at m/z 221 was generated from CHA accumulated by addition of two hydrogen atoms catalyzed by unidentified reductase(s) in DLK. To examine this hypothesis, CHA was incubated with the DLK crude extract prepared from cells grown in LB. ESI-MS of the reaction product after 10 min incubation showed that the peak at m/z 219 derived from CHA was converted to that at m/z 221 only in the presence of NADPH (data not shown). These results strongly suggested that product II was produced from accumulated CHA. Our preliminary experiment indicated that LigJ activity was not inhibited by the presence of CHA, and thus the reason why a large amount of OMA (product I) and PDC were also accumulated from vanillate and syringate in DLK is unknown.



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  		FIG. 3. Identification of accumulated products from vanillate and syringate by DLK. (A and B) Gas chromatograms of trimethylsilylated derivatives of the accumulated products from vanillate and syringate, respectively. In both cultures, PDC, the enol form of OMA, product I, and unidentified product II were observed. (C and D) Negative-ion ESI-MS spectrum of the same reaction mixture used in panels A and B, respectively.

Disruption of ligR, orf1, and orf2 in S. paucimobilis SYK-6. To investigate whether ligR, orf1, and orf2 are involved in the catabolism of vanillate and syringate, each of these genes in SYK-6 was disrupted. Gene inactivation was carried out using the ligR, orf1, and orf2 disruption plasmids, pLRD, pF1D, and pF2D, respectively. The growth rates of the ligR disruption mutant, DLR, on both vanillate and syringate were decreased compared with those of SYK-6 (Fig. 4). Based on this result and the fact that LigR has similarity with LysR-type transcriptional regulator, LigR may positively regulate the expression of the PCA 4,5-cleavage pathway genes, although it is not essential to growth of SYK-6 on vanillate and syringate. The orf1 insertion mutant, DF1, completely lost the ability to grow on both vanillate and syringate. This growth deficiency of DF1 on vanillate and syringate was complemented by introduction of pTS1210 carrying orf1 (pTSF1), indicating that orf1 is necessary for growth of SYK-6 on these compounds. Considering that orf1 has similarity with a putative transmembrane protein YbhH of E. coli, it is likely that orf1 encodes a transporter of vanillate and syringate. However, the actual role of orf1 is remained to clarify together with that of ligR. On the other hand, the disruption of orf2 did not affect the growth of SYK-6 on both vanillate and syringate.



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  		FIG. 4. Growth of SYK-6 and ligR insertion mutant (DLR) on vanillate and syringate. Growth of SYK-6 (open circles) and DLR (filled circles) on 10 mM vanillate (A) and 10 mM syringate (B). The results are the means of the representative of three independent experiments.

RT-PCR analysis of PCA 4,5-cleavage pathway genes. To determine the operon structure of the genes included in the 10.5-kb EcoRI fragment, RT-PCR experiments were performed with total RNA isolated from SYK-6 grown on vanillate and primers complementary to neighboring ORFs. The amplification products of lsdA-ligI (581 bp), ligI-orf1 (491 bp), orf1-ligK (271 bp), ligJ-ligA (520 bp), and ligA-ligB (564 bp) were obtained. However, RT-PCR products using primer which span the ligR-orf2, orf2-ligJ, and ligB-ligC regions were not obtained (Fig. 5), while PCR using their primers with SYK-6 total DNA as a template gave the expected PCR products (data not shown). In order to confirm the presence of the ligR, orf2, and ligC transcripts in the RNA samples, RT-PCR was carried out using primers to amplify inside of each ORF. RT-PCR products of ligR (622 bp) and ligC (509 bp) with the expected sizes were obtained (Fig. 5). On the other hand, the RT-PCR product of orf2 did not appear, indicating the DNA region of orf2 was not transcribed in SYK-6 cells grown on vanillate.



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  		FIG. 5. Agarose gel electrophoresis of RT-PCR products amplified from SYK-6 cells grown on vanillate. The sizes of molecular weight markers in lane M are indicated on the left side of the gels. Odd-numbered lanes are controls without reverse transcriptase. The primers for spanning each ORF intergenic region or ORF internal region are shown in the Materials and Methods. Lanes: 1 and 2, lsdA-ligI intergenic region (expected size, 581 bp); 3 and 4, ligI-orf1 intergenic region (expected size, 491 bp); 5 and 6, orf1-ligK intergenic region (expected size, 271 bp); 7 and 8, ligR-orf2 intergenic region (expected size, 726 bp); 9 and 10, orf2-ligJ intergenic region (expected size, 427 bp); 11 and 12, ligJ-ligA intergenic region (expected size, 520 bp); 13 and 14, ligA-ligB intergenic region (expected size, 564 bp); 15 and 16, ligB-ligC intergenic region (expected size, 489 bp); 17 and 18, ligC internal region (expected size, 509 bp); 19 and 20, ligR internal region (expected size, 622 bp); and 21 and 22, orf2 internal region (expected size, 653 bp).

In conclusion, the PCA 4,5-cleavage pathway genes of S. paucimobilis SYK-6 consist of four transcriptional units, including the ligK-orf1-ligI-lsdA cluster, the ligJAB cluster, and the monocistronic ligR and ligC genes (Fig. 1B). In the case of the PCA 3,4-cleavage pathway genes, the diversity in gene organization and transcriptional regulation has been found (7, 10, 13, 14, 32, 35, 46). In Acinetobacter sp. strain ADP1, all of the enzyme genes involved in this pathway constitute a single operon, pcaIJFBDKCHG, and their expression is regulated by the IclR-type transcriptional activator PcaU in concert with inducer PCA (10, 35, 46). On the other hand, the enzyme genes of P. putida PRS2000 (13) and Agrobacterium tumefaciens A348 (32) consist of several transcriptional units. In A348, the pcaDCHGB and pcaIJ clusters are independently regulated by the LysR-type transcriptional activator PcaQ, which responds to both ß-carboxy-cis,cis-muconate and {gamma}-carboxymuconolactone, and the IclR-type transcriptional activator PcaR, which responds to ß-ketoadipate, respectively (32). To gain better understanding of the PCA 4,5-cleavage pathway genes, it is essential to address their transcriptional regulation. Determination of the promoter regions and the actual role of ligR are currently under way in our laboratory.


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ACKNOWLEDGMENTS
 
We thank T. Nakazawa for providing pTS1210.

This work was supported in part by Grant-in-Aid for Encouragement of Young Scientists 11760057 from the Ministry of Education, Science, Sports, and Culture of Japan to E.M. H.H. was financially supported by research fellowship 2068 from the Japan Society for the Promotion of Science for Young Scientists.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Bioengineering, Nagaoka University of Technology, Kamitomioka, Nagaoka, Niigata 940-2188, Japan. Phone: 81-258-47-9428. Fax: 81-258-47-9450. E-mail: emasai{at}vos.nagaokaut.ac.jp. Back


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Journal of Bacteriology, January 2003, p. 41-50, Vol. 185, No. 1
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.1.41-50.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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