Degradation of 3-O-methylgallate in Sphingomonas paucimobilis SYK-6 by pathways involving protocatechuate 4,5-dioxygenase

Sphingomonas paucimobilis SYK-6 converts vanillate and syringate to protocatechuate and 3-O-methylgallate (3MGA), respectively. 3MGA is metabolized via multiple pathways involving 3MGA 3,4-dioxygenase, protocatechuate 4,5-dioxygenase (LigAB), and gallate dioxygenase whereas protocatechuate is degraded via the protocatechuate 4,5-cleavage pathway. Here the secondary role of LigAB in syringate metabolism is investigated. The reaction product of 3MGA catalyzed by His-tagged LigAB was identified as 4-carboxy-2-hydroxy-6-methoxy-6-oxohexa-2,4-dienoate (CHMOD) and 2-pyrone-4,6-dicarboxylate (PDC), indicating that 3MGA is transformed to CHMOD and PDC by both reactions catalyzed by DesZ and LigAB. Mutant analysis revealed that the 3MGA catabolic pathways involving LigAB are functional in SYK-6.     

A tetrahydrofolate-dependent O-demethylase, LigM, is crucial for catabolism of vanillate and syringate in Sphingomonas paucimobilis SYK-6

Vanillate and syringate are converted into protocatechuate (PCA) and 3-O-methylgallate (3MGA), respectively, by O-demethylases in Sphingomonas paucimobilis SYK-6. PCA is further degraded via the PCA 4,5-cleavage pathway, while 3MGA is degraded through multiple pathways in which PCA 4,5-dioxygenase (LigAB), 3MGA 3,4-dioxygenase (DesZ), and an unidentified 3MGA O-demethylase and gallate dioxygenase are participants. For this study, we isolated a 4.7-kb SmaI fragment that conferred on Escherichia coli the activity required for the conversion of vanillate to PCA. The nucleotide sequence of this fragment revealed an open reading frame of 1,413 bp (ligM), the deduced amino acid sequence of which showed 49% identity with that of the tetrahydrofolate (H4folate)-dependent syringate O-demethylase gene (desA). The metF and ligH genes, which are thought to be involved in H4folate-mediated C1 metabolism, were located just downstream of ligM. The crude LigM enzyme expressed in E. coli converted vanillate and 3MGA to PCA and gallate, respectively, with similar specific activities, and only in the presence of H4folate; however, syringate was not a substrate for LigM. The disruption of ligM led to significant growth retardation on both vanillate and syringate, indicating that ligM is involved in the catabolism of these substrates. The ability of the ligM mutant to transform vanillate was markedly decreased, and this mutant completely lost the 3MGA O-demethylase activity. A ligM desA double mutant completely lost the ability to transform vanillate, thus indicating that desA also contributes to vanillate degradation. All of these results indicate that ligM encodes vanillate/3MGA O-demethylase and plays an important role in the O demethylation of vanillate and 3MGA, respectively.     

Characterization of the 3-O-methylgallate dioxygenase gene and evidence of multiple 3-O-methylgallate catabolic pathways in Sphingomonas paucimobilis SYK-6

Sphingomonas paucimobilis SYK-6 is able to grow on various lignin-derived biaryls as the sole source of carbon and energy. These compounds are degraded to vanillate and syringate by the unique and specific enzymes in this strain. Vanillate and syringate are converted to protocatechuate (PCA) and 3-O-methylgallate (3MGA), respectively, by the tetrahydrofolate-dependent O-demethylases. Previous studies have suggested that these compounds are further degraded via the PCA 4,5-cleavage pathway. However, our subsequent analysis of the ligB insertion mutant, which encodes the beta subunit of PCA 4,5-dioxygenase, suggested that at least one alternative route is involved in 3MGA degradation. In the present study, we isolated the desZ gene, which confers 3MGA degradation activity on Escherichia coli. The deduced amino acid sequence of desZ showed ca. 20 to 43% identity with the type II extradiol dioxygenases. Gas chromatography-mass spectrometry analysis suggested that DesZ catalyzes the 3,4-cleavage of 3MGA. Disruption of both desZ and ligB in SYK-6 resulted in loss of the dioxygen-dependent 3MGA transformation activity, but the resulting mutant retained the ability to grow on syringate. We found that the cell extract of the desZ ligB double mutant was able to convert 3MGA to gallate when tetrahydrofolate was added to the reaction mixture, and the cell extract of this mutant degraded gallate to the same degree as the wild type did. All these results suggest that syringate is degraded through multiple 3MGA degradation pathways in which ligAB, desZ, 3MGA O-demethylase, and gallate dioxygenase are participants.     

Characterization of the gallate dioxygenase gene: three distinct ring cleavage dioxygenases are involved in syringate degradation by Sphingomonas paucimobilis SYK-6

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 beta and alpha 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 K(m) for gallate and the V(max) were determined to be 66.9 +/- 9.3 microM 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.     

Assimilation of aromatic compounds by Comamonas testosteroni: characterization and spreadability of protocatechuate 4,5-cleavage pathway in bacteria

Comamonas testosteroni strain CNB-1 was isolated from activated sludge and has been investigated for its ability to degrade 4-chloronitrobenzene. Results from this study showed that strain CNB-1 grew on phenol, gentisate, vanillate, 3-hydroxybenzoate (3HB), and 4-hydroxybenzoate (4HB) as carbon and energy sources. Proteomic data and enzyme activity assays suggested that vanillate, 3HB, and 4HB were degraded in strain CNB-1 via protocatechuate (PCA) 4,5-cleavage pathway. The genetics and biochemistry of the PCA 4,5-cleavage pathway were investigated. Results showed that the 4-oxalomesaconate (OMA) hydratase from C. testosteroni takes only enol-OMA as substrate. A previously functionally unknown gene pmdU encodes an OMA tautomerase and catalyzes conversion of OMA_keto into OMA_enol. The 4-carboxy-4-hydroxy-2-oxoadipate (CHA) aldolase is encoded by pmdF and catalyzes the last step of the PCA 4,5-cleavage pathway. We explored the 1,183 microbial genomes at GenBank for potential PCA 4,5-cleavage pathways, and 33 putative pmd clusters were found. Results suggest that PCA 4,5-cleavage pathways are mainly distributed in α- and β-Proteobacteria.     

A novel tetrahydrofolate-dependent O-demethylase gene is essential for growth of Sphingomonas paucimobilis SYK-6 with syringate

Sphingomonas paucimobilis SYK-6 degrades syringate to 3-O-methylgallate (3MGA), which is finally converted to pyruvate and oxaloacetate via multiple pathways in which protocatechuate 4,5-dioxygenase, 3MGA dioxygenase, and gallate dioxygenase are involved. Here we isolated the syringate O-demethylase gene (desA), which complemented the growth deficiency on syringate of a Tn5 mutant of the SYK-6 derivative strain. The desA gene is located 929 bp downstream of ferA, encoding feruloyl-coenzyme A synthetase, and consists of a 1,386-bp open reading frame encoding a polypeptide with a molecular mass of 50,721 Da. The deduced amino acid sequence of desA showed 26% identity in a 325-amino-acid overlap with that of gcvT of Escherichia coli, which encodes the tetrahydrofolate (H(4)folate)-dependent aminomethyltransferase involved in glycine cleavage. The cell extract of E. coli carrying desA converted syringate to 3MGA only when H(4)folate was added to the reaction mixture. DesA catalyzes the transfer of the methyl moiety of syringate to H(4)folate, forming 5-methyl-H(4)folate. Vanillate and 3MGA were also used as substrates for DesA; however, the relative activities toward them were 3 and 0.4% of that toward syringate, respectively. Disruption of desA in SYK-6 resulted in a growth defect on syringate but did not affect growth on vanillate, indicating that desA is essential to syringate degradation. In a previous study the ligH gene, which complements the growth deficiency on vanillate and syringate of a chemical-induced mutant of SYK-6, DC-49, was isolated (S. Nishikawa, T. Sonoki, T. Kasahara, T. Obi, S. Kubota, S. Kawai, N. Morohoshi, and Y. Katayama, Appl. Environ. Microbiol. 64:836-842, 1998). Disruption of ligH resulted in the same phenotype as DC-49; its cell extract, however, was found to be able to convert vanillate and syringate in the presence of H(4)folate. The possible role of ligH is discussed.     

Characterization of Sphingomonas paucimobilis SYK-6 genes involved in degradation of lignin-related compounds

Sphingomonas paucimobilis SYK-6 is able to grow on a wide variety of dimeric lignin compounds. These compounds are degraded via vanillate and syringate by a unique enzymatic system, composed of etherases, O demethylases, ring cleavage oxygenases and side chain cleaving enzymes. These unique and specific lignin modification enzymes are thought to be powerful tools for utilization of the most abundant aromatic biomass, lignin. Here, we focus on the genes and enzymes involved in β-aryl ether cleavage and biphenyl degradation. Two unique etherases are involved in the reductive cleavage of β-aryl ether. These two etherases have amino acid sequence similarity with the glutathione S-transferases, and use glutathione as a hydrogen donor. It was found that 5,5′-dehydrodivanillate, which is a typical lignin-related biphenyl structure, was transformed into 5-carboxyvanillate by the reaction sequence of O-demethylation, meta-ring cleavage, and hydrolysis, and the genes involved in the latter two reactions have been characterized. Vanillate and syringate are the most common intermediate metabolites in lignin catabolism. These compounds are initially O-demethylated and the resulting diol compounds, protocatechuate (PCA) and 3-O-methylgallate, respectively, are subjected to ring cleavage catalyzed by PCA 4,5-dioxygenase. The ring cleavage products generated are further degraded through the PCA 4,5-cleavage pathway. We have isolated and characterized genes for enzymes involved in this pathway. Disruption of a gene for 2-pyrone-4,6-dicarboxylate hydrolase (ligI) in this pathway suggested that an alternative route for 3-O-methylgallate degradation, in which ligI is not involved, would play a role in syringate catabolism. In this article, we describe the genetic and biochemical features of the S. paucimobilis SYK-6 genes involved in degradation of lignin-related compounds. A possible application of the SYK-6 lignin degradation system to produce a valuable chemical material is also described. Received 01 May 1999/ Accepted in revised form 29 July 1999     

Genetic and biochemical characterization of 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase and its role in the protocatechuate 4,5-cleavage pathway in Sphingomonas paucimobilis SYK-6

Protocatechuate (PCA) is the key intermediate metabolite in the lignin degradation pathway of Sphingomonas paucimobilis SYK-6 and is metabolized to pyruvate and oxaloacetate via the PCA 4,5-cleavage pathway. We characterized the 4-carboxy-2-hydroxymuconate-6-semialdehyde (CHMS) dehydrogenase gene (ligC). CHMS is the 4,5-cleavage product of PCA and is converted into 2-pyrone-4,6-dicarboxylate (PDC) by LigC. We found that ligC was located 295 bp downstream of ligB, which encodes the large subunit of the PCA 4,5-dioxygenase. The ligC gene consists of a 945-bp open reading frame encoding a polypeptide with a molecular mass of 34,590 Da. The deduced amino acid sequence of ligC showed 19 to 20% identity with 3-chlorobenzoate cis-dihydrodiol dehydrogenase of Alcaligenes sp. strain BR60 and phthalate cis-dihydrodiol dehydrogenases of Pseudomonas putida NMH102-2 and Burkholderia cepacia DBO1, which are unrelated to group I, II, and III microbial alcohol dehydrogenases (M. F. Reid and C. A. Fewson, Crit. Rev. Microbiol. 20:13-56, 1994). The ligC gene was expressed in Escherichia coli and LigC was purified to near homogeneity. Production of PDC from CHMS catalyzed by LigC was confirmed in the presence of NADP(+) by electrospray ionization-mass spectrometry and gas chromatography-mass spectrometry. LigC is a homodimer. The isoelectric point, optimum pH, and optimum temperature were estimated to be 5.3, 8.0, and 25 degrees C, respectively. The K(m) for NADP(+) was estimated to be 24.6 +/- 1.5 microM, which was approximately 10 times lower than that for NAD(+) (252 +/- 3.9 microM). The K(m)s for CHMS in the presence of NADP(+) and NAD(+) are 26.0 +/- 0.5 and 20.6 +/- 1.0 microM, respectively. Disruption of ligC in S. paucimobilis SYK-6 prevented growth with vanillate. Only PCA was accumulated during the incubation of vanillate with the whole cells of the ligC insertion mutant (DLC), indicating a lack of PCA 4,5-dioxygenase activity in DLC. However, the introduction of ligC into DLC restored its ability to grow on vanillate. PDC was suggested to be an inducer for ligAB gene expression.     

Genetic and biochemical investigations on bacterial catabolic pathways for lignin-derived aromatic compounds

Lignins are the most abundant aromatic compounds in nature, and their decomposition is essential to the terrestrial carbon cycle. White rot fungi secreting phenol oxidases are assumed to be involved in the initial degradation of native lignin, whereas bacteria play a main role in the mineralization of lignin-derived low-molecular-weight compounds in soil. There are a number of reports on the degradation pathways for lignin-derived aromatic compounds, but their catabolism has not been enzymatically or genetically characterized. Sphingomonas paucimobilis SYK-6 is one of the best-characterized lignin-degrading bacteria. It can grow on a wide variety of lignin-related biaryls and monoaryls, including beta-aryl ether, biphenyl, diarylpropane, and phenylpropane. These compounds are degraded via the protocatechuate (PCA) 4,5-cleavage pathway or multiple 3-O-methylgallate (3MGA) catabolic pathways. In this review, the enzyme systems for beta-aryl ether and biphenyl degradation, O demethylation linked with one carbon metabolism, the PCA 4,5-cleavage pathway, and the multiple 3MGA catabolic pathways in SYK-6 are outlined.     

Coexistence of two different O demethylation systems in lignin metabolism by Sphingomonas paucimobilis SYK-6: cloning and sequencing of the lignin biphenyl-specific O-demethylase (LigX) gene

Sphingomonas paucimobilis SYK-6 can grow on several dimeric model compounds of lignin as a carbon and energy source. It has O demethylation systems on three kinds of substrates: 5, 5'-dehydrodivanillic acid (DDVA), syringate, and vanillate. We previously reported the cloning of a gene involved in the tetrahydrofolate-dependent O demethylation of syringate and vanillate. In the study reported here, we cloned the gene responsible for DDVA O demethylation. Using nitrosoguanidine mutagenesis, a mutant strain, NT-1, which could not degrade DDVA but could degrade syringate and vanillate, was isolated and was used to clone the gene responsible for the O demethylation of DDVA by complementation. Sequencing analysis showed an open reading frame (designated ligX) of 1,266 bp in this fragment. The deduced amino acid sequence of LigX had similarity to class I type oxygenases. LigX was involved in O demethylation activity on DDVA but not on vanillate and syringate. DDVA O demethylation activity in S. paucimobilis SYK-6 cell extracts was inhibited by addition of the LigX polyclonal antiserum. Thus, LigX is an essential enzyme for DDVA O demethylation in SYK-6. S. paucimobilis SYK-6 has two O demethylation systems: one is an oxygenative demethylase system, and the other is a tetrahydrofolate-dependent methyltransferase system.     

Genetic and Biochemical Characterization of a 2-Pyrone-4,6-Dicarboxylic Acid Hydrolase Involved in the Protocatechuate 4,5-Cleavage Pathway of Sphingomonas paucimobilis SYK-6

Sphingomonas paucimobilis SYK-6 is able to grow on a wide variety of dimeric lignin compounds with guaiacyl moieties, which are converted into protocatechuate by the actions of lignin degradation enzymes in this strain. Protocatechuate is a key metabolite in the SYK-6 degradation of lignin compounds with guaiacyl moieties, and it is thought that it degrades to pyruvate and oxaloacetate via the protocatechuate 4,5-cleavage pathway. In a 10.5-kb EcoRI fragment carrying the protocatechuate 4,5-dioxygenase gene (ligAB) (Y. Noda, S. Nishikawa, K. Shiozuka, H. Kadokura, H. Nakajima, K. Yoda, Y. Katayama, N. Morohoshi, T. Haraguchi, and M. Yamasaki. J. Bacteriol. 172:2704–2709, 1990), we found the ligI gene encoding 2-pyrone-4,6-dicarboxylic acid (PDC) hydrolase. PDC hydrolase is a member of this pathway and catalyzes the interconversion between PDC and 4-carboxy-2-hydroxymuconic acid (CHM). The ligI gene is thought to be transcribed divergently from ligAB and consists of an 879-bp open reading frame encoding a polypeptide with a molecular mass of 32,737 Da. The ligI gene product (LigI), expressed in Escherichia coli, was purified to near-homogeneity and was estimated to be a monomer (31.6 kDa) by gel filtration chromatography. The isoelectric point was determined to be 4.9. The optimum pH for hydrolysis of PDC is 8.5, the optimum pH for synthesis of PDC is 6.0 to 7.5, and the K_m values for PDC and CHM are 74 and 49 μM, respectively. LigI activity was inhibited by the addition of thiol reagents, suggesting that the cysteine residue is a catalytic site. LigI is more resistant to metal ion inhibition than the PDC hydrolases of Pseudomonas ochraceae (K. Maruyama, J. Biochem. 93:557–565, 1983) and Comamonas testosteroni (P. J. Kersten, S. Dagley, J. W. Whittaker, D. M. Arciero, and J. D. Lipscomb, J. Bacteriol. 152:1154–1162, 1982). The insertional inactivation of the ligI gene in S. paucimobilis SYK-6 led to the complete loss of PDC hydrolase activity and to a growth defect on vanillic acid; it did not affect growth on syringic acid. These results indicate that the ligI gene is essential for the growth of SYK-6 on vanillic acid but is not responsible for the growth of SYK-6 on syringic acid.     

A second 5-carboxyvanillate decarboxylase gene, ligW2, is important for lignin-related biphenyl catabolism in Sphingomonas paucimobilis SYK-6

A lignin-related biphenyl compound, 5,5'-dehydrodivanillate (DDVA), is degraded to 5-carboxyvanillate (5CVA) by the enzyme reactions catalyzed by DDVA O-demethylase (LigX), meta-cleavage oxygenase (LigZ), and meta-cleavage compound hydrolase (LigY) in Sphingomonas paucimobilis SYK-6. 5CVA is then transformed to vanillate by a nonoxidative 5CVA decarboxylase and is further degraded through the protocatechuate 4,5-cleavage pathway. A 5CVA decarboxylase gene, ligW, was isolated from SYK-6 (X. Peng, E. Masai, H. Kitayama, K. Harada, Y, Katayama, and M. Fukuda, Appl. Environ. Microbiol. 68:4407-4415, 2002). However, disruption of ligW slightly affected the 5CVA decarboxylase activity and the growth rate on DDVA of the mutant, suggesting the presence of an alternative 5CVA decarboxylase gene. Here we isolated a second 5CVA decarboxylase gene, ligW2, which consists of a 1,050-bp open reading frame encoding a polypeptide with a molecular mass of 39,379 Da. The deduced amino acid sequence encoded by ligW2 exhibits 37% identity with the sequence encoded by ligW. Based on a gas chromatography-mass spectrometry analysis of the reaction product from 5CVA catalyzed by LigW2 in the presence of deuterium oxide, LigW2 was indicated to be a nonoxidative decarboxylase of 5CVA, like LigW. After disruption of ligW2, both the growth rate on DDVA and the 5CVA decarboxylase activity of the mutant were decreased to approximately 30% of the wild-type levels. The ligW ligW2 double mutant lost both the ability to grow on DDVA and the 5CVA decarboxylase activity. These results indicate that both ligW and ligW2 contribute to 5CVA degradation, although ligW2 plays the more important role in the growth of SYK-6 cells on DDVA.     

Characterization of the 5-carboxyvanillate decarboxylase gene and its role in lignin-related biphenyl catabolism in Sphingomonas paucimobilis SYK-6

Sphingomonas paucimobilis SYK-6 degrades a lignin-related biphenyl compound, 5,5'-dehydrodivanillate (DDVA), to 5-carboxyvanillate (5CVA) by the enzyme reactions catalyzed by the DDVA O-demethylase (LigX), the ring cleavage oxygenase (LigZ), and the meta-cleavage compound hydrolase (LigY). In this study we examined the degradation step of 5CVA. 5CVA was transformed to vanillate, O-demethylated, and further degraded via the protocatechuate 4,5-cleavage pathway by this strain. A cosmid clone which conferred the 5CVA degradation activity to a host strain was isolated. In the 7.0-kb EcoRI fragment of the cosmid we found a 1,002-bp open reading frame responsible for the conversion of 5CVA to vanillate, and we designated it ligW. The gene product of ligW (LigW) catalyzed the decarboxylation of 5CVA to produce vanillate along with the specific incorporation of deuterium from deuterium oxide, indicating that LigW is a nonoxidative decarboxylase of 5CVA. LigW did not require any metal ions or cofactors for its activity. The decarboxylase activity was specific to 5CVA. Inhibition experiments with 5CVA analogs suggested that two carboxyl groups oriented meta to each other in 5CVA are important to the substrate recognition by LigW. Gene walking analysis indicated that the ligW gene was located on the 18-kb DNA region with other DDVA catabolic genes, including ligZ, ligY, and ligX.     

Characterization of the 5-Carboxyvanillate Decarboxylase Gene and Its Role in Lignin-Related Biphenyl Catabolism in Sphingomonas paucimobilis SYK-6

Sphingomonas paucimobilis SYK-6 degrades a lignin-related biphenyl compound, 5,5′-dehydrodivanillate (DDVA), to 5-carboxyvanillate (5CVA) by the enzyme reactions catalyzed by the DDVA O-demethylase (LigX), the ring cleavage oxygenase (LigZ), and the meta-cleavage compound hydrolase (LigY). In this study we examined the degradation step of 5CVA. 5CVA was transformed to vanillate, O-demethylated, and further degraded via the protocatechuate 4,5-cleavage pathway by this strain. A cosmid clone which conferred the 5CVA degradation activity to a host strain was isolated. In the 7.0-kb EcoRI fragment of the cosmid we found a 1,002-bp open reading frame responsible for the conversion of 5CVA to vanillate, and we designated it ligW. The gene product of ligW (LigW) catalyzed the decarboxylation of 5CVA to produce vanillate along with the specific incorporation of deuterium from deuterium oxide, indicating that LigW is a nonoxidative decarboxylase of 5CVA. LigW did not require any metal ions or cofactors for its activity. The decarboxylase activity was specific to 5CVA. Inhibition experiments with 5CVA analogs suggested that two carboxyl groups oriented meta to each other in 5CVA are important to the substrate recognition by LigW. Gene walking analysis indicated that the ligW gene was located on the 18-kb DNA region with other DDVA catabolic genes, including ligZ, ligY, and ligX.     

A Second 5-Carboxyvanillate Decarboxylase Gene, ligW2, Is Important for Lignin-Related Biphenyl Catabolism in Sphingomonas paucimobilis SYK-6

A lignin-related biphenyl compound, 5,5′-dehydrodivanillate (DDVA), is degraded to 5-carboxyvanillate (5CVA) by the enzyme reactions catalyzed by DDVA O-demethylase (LigX), meta-cleavage oxygenase (LigZ), and meta-cleavage compound hydrolase (LigY) in Sphingomonas paucimobilis SYK-6. 5CVA is then transformed to vanillate by a nonoxidative 5CVA decarboxylase and is further degraded through the protocatechuate 4,5-cleavage pathway. A 5CVA decarboxylase gene, ligW, was isolated from SYK-6 (X. Peng, E. Masai, H. Kitayama, K. Harada, Y, Katayama, and M. Fukuda, Appl. Environ. Microbiol. 68:4407-4415, 2002). However, disruption of ligW slightly affected the 5CVA decarboxylase activity and the growth rate on DDVA of the mutant, suggesting the presence of an alternative 5CVA decarboxylase gene. Here we isolated a second 5CVA decarboxylase gene, ligW2, which consists of a 1,050-bp open reading frame encoding a polypeptide with a molecular mass of 39,379 Da. The deduced amino acid sequence encoded by ligW2 exhibits 37% identity with the sequence encoded by ligW. Based on a gas chromatography-mass spectrometry analysis of the reaction product from 5CVA catalyzed by LigW2 in the presence of deuterium oxide, LigW2 was indicated to be a nonoxidative decarboxylase of 5CVA, like LigW. After disruption of ligW2, both the growth rate on DDVA and the 5CVA decarboxylase activity of the mutant were decreased to approximately 30% of the wild-type levels. The ligW ligW2 double mutant lost both the ability to grow on DDVA and the 5CVA decarboxylase activity. These results indicate that both ligW and ligW2 contribute to 5CVA degradation, although ligW2 plays the more important role in the growth of SYK-6 cells on DDVA.     

Cloning and Sequencing of the Sphingomonas (Pseudomonas) paucimobilis Gene Essential for the O Demethylation of Vanillate and Syringate

Sphingomonas (Pseudomonas) paucimobilis SYK-6 is able to grow on 5,5′-dehydrodivanillic acid (DDVA), syringate, vanillate, and other dimeric model compounds of lignin as a sole carbon source. Nitrosoguanidine mutagenesis of S. paucimobilis SYK-6 was performed, and two mutants with altered DDVA degradation pathways were isolated. The mutant strain NT-1 could not degrade DDVA, but could degrade syringate, vanillate, and 2,2′,3′-trihydroxy-3-methoxy-5,5′-dicarboxybiphenyl (OH-DDVA). Strain DC-49 could slowly assimilate DDVA, but could degrade neither vanillate nor syringate, although it could degrade protocatechuate and 3-O-methylgallate. A complementing DNA fragment of strain DC-49 was isolated from the cosmid library of strain SYK-6. The minimum DNA fragment complementing DC-49 was determined to be the 1.8-kbp insert of pKEX2.0. Sequencing analysis showed an open reading frame of 1,671 bp in this fragment, and a similarity search indicated that the deduced amino acid sequence of this open reading frame had significant similarity (60%) to the formyltetrahydrofolate synthetase of Clostridium thermoaceticum.     

Enzymatic release of halogens or methanol from some substituted protocatechuic acids.

Four strains of gram-negative bacteria capable of growing at the expense of 5-chlorovanillate were isolated from soil, and the metabolism of one strain was studied in particular detail. In the presence of alpha, alpha'-bipyridyl, a suspension of 5-chlorovanillate-grown cells accumulated 5-chloroprotocatechuate from 5-chlorovanillate; in the absence of inhibitor these compounds, and various other 5-substituted protocatechuates and vanillates, were oxidized to completion. Cell suspensions of this strain grown on 5-chlorovanillate or vanillate released chloride quantitatively from 5-chlorovanillate and released methanol from syringate. Extracts of cells grown with 4-hydroxybenzoate, vanillate, or syringate possessed high levels of both protocatechuate 4,5-dioxygenase and 2-pyrone-4,6-dicarboxylate hydrolase; extracts from acetate-grown cells did not. Protocatechuate 4,5-dioxygenase, purified from strains that could grow with 5-chlorovanillate, oxidized 5-halogeno-protocatechuates and 3-O-methylgallate with the formation of 2-pyrone-4,6-dicarboxylate. A crude extract converted 5-chloroprotocatechuate into pyruvate plus oxaloacetate. On the basis of these observations, a meta-fission reaction sequence is proposed for the bacterial degradation of vanillate and protocatechuate substituted at C-5 of the benzene ring with halogen or methoxyl.