Paucimobilis SYK-6 Research Articles

Convenient synthesis of chiral lignin model compounds via optical resolution: four stereoisomers of guaiacylglycerol-β-guaiacyl ether and both enantiomers of 3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)-propan-1-one (erone)

Guaiacylglycerol-β-guaiacyl ether (GGE) is one of the most important phenolic compound for studying the chemistry and biochemistry of lignin. GGE contains two asymmetric carbons at the alpha and beta positions of its side chain; therefore, theoretically it can exist as four different stereoisomers. It has been proposed that a Gram-negative bacterium, Sphingobium sp. SYK-6 (formerly referred as Sphingomonas paucimobilis SYK-6), degrades GGE enantiospecifically via cleavage of the ether bond. The cleavage was thought to proceed in two steps, each catalyzed by a different enantiospecific enzyme. In the first step, the alcohol residue at the alpha position of the side chain in GGE was thought to be oxidized enantiospecifically by four distinct Cα-dehydrogenases (LigD, LigN, LigL and LigO), to produce two enantiomers of 3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)-propan-1-one (erone). To study this enantiospecific degradation step by the four dehydrogenases, we synthesized all four stereoisomers of GGE and both enantiomers of erone and separated them into enantiopure samples. The stereoisomers and our synthetic methods to prepare them are useful both for microbial and chemical investigations of lignin degradation.

Microbial conversion of glucose to a novel chemical building block, 2-pyrone-4,6-dicarboxylic acid

2-Pyrone-4,6-dicarboxylic acid (PDC) is a catabolic intermediate in Sphingobium sp. SYK-6 (previously characterized as Sphingomonas paucimobilis SYK-6), which is a degrader of lignin-derived aromatic compounds. Recently, PDC has been also characterized as a novel starting material for several potentially useful synthetic polymers. In a previous study, we constructed a biosynthetic system in which PDC was generated efficiently from a chemically synthesized compound, protocatechuate. In order to develop an alternative system for production of PDC, we tried to generate it from glucose, which is a low-cost sugar that can be obtained from abundant cellulosic wastes and biomass crops. We designed a metabolic bypass to PDC from the shikimate pathway in recombinant Escherichia coli cells. PDC accumulated in the medium of recombinant E. coli cells that had been transformed with genes isolated from Emericella niger, E. coli, Pseudomonas putida, and Sphingobium sp. SYK-6. The yield of PDC depended on the combination of genes that we introduced into the cells and on the specific of host strain. Under optimal conditions, the yield and titer of PDC were, respectively, 17.3% and 0.35 mg/l when the concentration of glucose was 2 g/l and the culture volume was 50 ml. Our results open up the possibility of novel utilization of biomass as the source of a useful chemical building block.

Degradation of 3-O-methylgallate inSphingomonas paucimobilisSYK-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.

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.

Efficient production of 2-pyrone 4,6-dicarboxylic acid as a novel polymer-based material from protocatechuate by microbial function

Sphingomonas paucimobilis SYK-6, which can degrade various low molecular weight compounds derived from plant polyphenols such as lignin, lignan, and tannin, metabolizes these substances via 2-pyrone-4,6-dicarboxylic acid (PDC). We focused on this metabolic intermediate as a potential raw material for novel, bio-based polymers. We cloned the ligAB and ligC genes of SYK-6, which respectively encode protocatechuate 4,5-dioxygenase and 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase, into a broad host range plasmid vector, pKT230MC. The resulting plasmid, pDVABC, was introduced into the PpY1100 strain of Pseudomonas putida, and we found that PDC could be stably produced from protocatechuate and accumulated. In addition, we examined the efficiency of production of PDC from protocatechuate on a 5-L scale in a Luria-Bertani medium containing 100 mM glucose and determined that PDC was stably produced from protocatechuate to yield 10 g/L or more.

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.

Generation of transgenic hybrid aspen that express a bacterial gene for feruloyl-CoA hydratase/lyase (FerB), which is involved in lignin degradation in Sphingomonas paucimobilis SYK-6

Transgenic hybrid aspens that expressed a bacterial gene for an enzyme involved in the degradation of hydroxycinnamates, feruloyl-CoA hydratase/lyase (FerB), were produced by Agrobacterium-mediated transformation. The gene was isolated from the bacterium Sphingomonas paucimobilis SYK-6, which is able to degrade a wide variety of monomeric and dimeric lignin-related compounds. The introduced gene was expressed and FerB (vanillin-forming) activity was detected in cell-free extracts obtained from most of the plants. There were no detectable differences between the transgenic plants and wild-type plants, except in one of the transformants 33 that had chlorotic leaves and a lower growth rate. The results of reverse transcription-polymerase chain reaction analysis of endogenous genes for phenylpropanoid biosynthesis suggested that expression of the FerB transgene did not have a significant effect on the expression of endogenous genes in transformants at an early stage of growth, namely in 2-month-old plantlets, even though levels of expression of genes for 4-coumarate:CoA ligase and ferulate 5-hydroxylase were higher in several of the FerB transformants than in the wild-type plants. Our data indicate that genes derived from bacteria can be expressed in woody plants and suggest a new method for manipulating phenylpropanoid biosynthesis and for producing transgenic woody plants with enhanced economic value.

Roles of the enantioselective glutathione S-transferases in cleavage of beta-aryl ether.

Cleavage of the beta-aryl ether linkage is the most important process in lignin degradation. Here we characterize the three tandemly located glutathione S-transferase (GST) genes, ligF, ligE, and ligG, from low-molecular-weight lignin-degrading Sphingomonas paucimobilis SYK-6, and we describe the actual roles of these genes in the beta-aryl ether cleavage. Based on the identification of the reaction product by electrospray ionization-mass spectrometry, a model compound of beta-aryl ether, alpha-(2-methoxyphenoxy)-beta-hydroxypropiovanillone (MPHPV), was transformed by LigF or LigE to guaiacol and alpha-glutathionyl-beta-hydroxypropiovanillone (GS-HPV). This result suggested that LigF and LigE catalyze the nucleophilic attack of glutathione on the carbon atom at the beta position of MPHPV. High-pressure liquid chromatography-circular dichroism analysis indicated that LigF and LigE each attacked a different enantiomer of the racemic MPHPV preparation. The ligG gene product specifically catalyzed the elimination of glutathione from GS-HPV generated by the action of LigF. This reaction then produces an achiral compound, beta-hydroxypropiovanillone, which is further degraded by this strain. Disruption of the ligF, ligE, and ligG genes in SYK-6 showed that ligF is essential to the degradation of one of the MPHPV enantiomers, and the alternative activities which metabolize the substrates of LigE and LigG are present in this strain.

Specific degradation ofβ-aryl ether linkage in synthetic lignin (dehydrogenative polymerizate) by bacterial enzymes ofSphingomonas paucimobilis SYK-6 produced in recombinantEscherichia coli

Sphingomonas paucimobilis SYK-6 produces unique and specific enzymes, such asβ-etherases,O-demethylases, and ring fission dioxygenases, for lignin degradation. Cleavage of arylglycerol-β-aryl ether linkage is the most important process in the lignin metabolic pathway ofS. paucimobilis SYK-6. We reported the genes (ligD, ligE, ligF) for enzymes that cleavedβ-aryl ether linkage of dimeric compounds in previous studies. In this study we synthesized the fluorescent high-molecular-weight lignin (UBE-DHP) by dehydrogenative polymerization. We investigated theβ-aryl ether cleavage ability of these enzymes produced in recombinantEscherichia coli. When UBE-DHP was incubated with LigF, 4-methylumbeliferone was released as a result ofβ-aryl ether cleavage of αO-methylumbelliferyl-β-hydroxypropiovanillone (compound III) incorporated in UBE-DHP. Here, we report thatβ-etherase ofS. paucimobilis SYK-6 can be expressed inE. coli and is able to cleave theβ-aryl ether linkage in synthetic high-molecular-weight lignin.

Tetrahydrofolate-dependent vanillate and syringateO-demethylation links tightly to one-carbon metabolic pathway associated with amino acid synthesis and DNA methylation in the lignin metabolism ofSphingomonas paucimobilis SYK-6

Sphingomonas paucimobilis SYK-6 strain can degrade various lignin-related compounds. In the lignin metabolic pathway of this bacterium, vanillate and syringate are demethylated by the tetrahydrofolate (THF)-dependentO-demethylation system, which requires the enzymatic function of LigH. Upstream of theligH gene is the 5,10-methylene-THF reductase gene. Its gene product was essential for one-carbon metabolism involved in the amino acid synthesis and DNA methylation in all organisms. When themetF gene was inactivated in the genome of SYK-6, the resultant mutant, DLmetF, could not grow on vanillate and syringate as a sole carbon source. Furthermore, DLmetF showed significant accumulation of methyl-THF as a result of vanillate and syringateO-demethylation. We report here that THF-dependent vanillate and syringateO-demethylation links tightly to the one-carbon metabolic pathway that is associated with amino acid synthesis and DNA methylation, and the methyl group is the sole one-carbon source inS. paucimobilis SYK-6.

Cloning and characterization of the ferulic acid catabolic genes of Sphingomonas paucimobilis SYK-6.

Sphingomonas paucimobilis SYK-6 degrades ferulic acid to vanillin, and it is further metabolized through the protocatechuate 4,5-cleavage pathway. We obtained a Tn5 mutant of SYK-6, FA2, which was able to grow on vanillic acid but not on ferulic acid. A cosmid which complemented the growth deficiency of FA2 on ferulic acid was isolated. The 5.2-kb BamHI-EcoRI fragment in this cosmid conferred the transformation activity of ferulic acid to vanillin on Escherichia coli host cells. A sequencing analysis revealed the genes ferB and ferA in this fragment; these genes consist of 852- and 2,127-bp open reading frames, respectively. The deduced amino acid sequence of ferB showed 40 to 48% identity with that of the feruloyl-coenzyme A (CoA) hydratase/lyase genes of Pseudomonas and Amycolatopsis ferulic acid degraders. On the other hand, the deduced amino acid sequence of ferA showed no significant similarity to the feruloyl-CoA synthetase genes of other ferulic acid degraders. However, the deduced amino acid sequence of ferA did show 31% identity with pimeloyl-CoA synthetase of Pseudomonas mendocina 35, which has been classified as a new superfamily of acyl-CoA synthetase (ADP forming) with succinyl-CoA synthetase (L. B. Sánchez, M. Y. Galperin, and M. Müller, J. Biol. Chem. 275:5794-5803, 2000). On the basis of the enzyme activity of E. coli carrying each of these genes, ferA and ferB were shown to encode a feruloyl-CoA synthetase and feruloyl-CoA hydratase/lyase, respectively. p-coumaric acid, caffeic acid, and sinapinic acid were converted to their corresponding benzaldehyde derivatives by the cell extract containing FerA and FerB, thereby indicating their broad substrate specificities. We found a ferB homolog, ferB2, upstream of a 5-carboxyvanillic acid decarboxylase gene (ligW) involved in the degradation of 5,5'-dehydrodivanillic acid. The deduced amino acid sequence of ferB2 showed 49% identity with ferB, and its gene product showed feruloyl-CoA hydratase/lyase activity with a substrate specificity similar to that of FerB. Insertional inactivation of each fer gene in S. paucimobilis SYK-6 suggested that the ferA gene is essential and that ferB and ferB2 genes are involved in ferulic acid degradation.

Close association between the enzymes involved in the lignin metabolic pathway ofSphingomonas paucimobilis SYK-6: interaction ofO-demethylase (LigX) and ring fission dioxygenase (LigZ)

Close association between the enzymes involved in the lignin metabolic pathway ofSphingomonas paucimobilis SYK-6: interaction ofO-demethylase (LigX) and ring fission dioxygenase (LigZ)

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.

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 beta-aryl ether cleavage and biphenyl degradation. Two unique etherases are involved in the reductive cleavage of beta-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.

Crystal structure of an aromatic ring opening dioxygenase LigAB, a protocatechuate 4,5-dioxygenase, under aerobic conditions.

Sphingomonas paucimobilis SYK-6 utilizes an extradiol-type catecholic dioxygenase, the LigAB enzyme (a protocatechuate 4,5-dioxygenase), to oxidize protocatechuate (or 3,4-dihydroxybenzoic acid, PCA). The enzyme belongs to the family of class III extradiol-type catecholic dioxygenases catalyzing the ring-opening reaction of protocatechuate and related compounds. The primary structure of LigAB suggests that the enzyme has no evolutionary relationship with the family of class II extradiol-type catecholic dioxygenases. Both the class II and class III enzymes utilize a non-heme ferrous center for adding dioxygen to the substrate. By elucidating the structure of LigAB, we aimed to provide a structural basis for discussing the function of class III enzymes. The crystal structure of substrate-free LigAB was solved at 2.2 A resolution. The molecule is an alpha2beta2 tetramer. The active site contains a non-heme iron coordinated by His12, His61, Glu242, and a water molecule located in a deep cleft of the beta subunit, which is covered by the alpha subunit. Because of the apparent oxidation of the Fe ion into the nonphysiological Fe(III) state, we could also solve the structure of LigAB complexed with a substrate, PCA. The iron coordination sphere in this complex is a distorted tetragonal bipyramid with one ligand missing, which is presumed to be the O2-binding site. The structure of LigAB is completely different from those of the class II extradiol-type dioxygenases exemplified by the BphC enzyme, a 2,3-dihydroxybiphenyl 1,2-dioxygenase from a Pseudomonas species. Thus, as already implicated by the primary structures, no evolutionary relationship exists between the class II and III enzymes. However, the two classes of enzymes share many geometrical characteristics with respect to the nature of the iron coordination sphere and the position of a putative catalytic base, strongly suggesting a common catalytic mechanism.

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 Km values for PDC and CHM are 74 and 49 microM, 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.

Cloning and sequencing of the gene for a Pseudomonas paucimobilis enzyme that cleaves beta-aryl ether

We isolated Pseudomonas paucimobilis SYK-6, which was able to degrade various dimeric lignin compounds (Y. Katayama, S. Nishikawa, M. Nakamura, K. Yano, M. Yamasaki, N. Morohoshi, and T. Haraguchi, Mokuzai Gakkaishi 33:77-79, 1987). This metabolic process is a distinct characteristic of this bacterium, which is equipped with an enzymatic modification system for various dimeric lignin compounds involved in the tricarboxylic acid cycle. Cleavage of the beta-aryl ether linkage is essential in this process, because this linkage is the most abundant (approximately 50%) in lignin. Here, we report the isolation and characterization of the beta-etherase gene, which contains an open reading frame of 843 bp and which we call ligE. This gene was expressed in Escherichia coli, and the enzyme had the same kinetic properties as the P. paucimobilis SYK-6 enzyme.

Detection and localization of a new enzyme catalyzing the β-aryl ether cleavage in the soil bacterium ( Pseudomonas paucimobilis SYK-6)

Cleavage of the arylglycerol-β-aryl ether linkage is the most important process in the biological degradation of lignin. We determined the activity of the enzyme cleaving the β-aryl ether linkage in membranes of Pseudomonas paucimobilis SYK-6. This enzyme was tightly associated with the cellular membrane and catalyzed the unique and reductive cleavage of compound II but not cleavage of compound I. This enzymatic activity was stimulated by addition of NADH. On the basis of this evidence, we present a model of the specific cellular assimilation of β-aryl ether by P. paucimobilis SYK-6.

The metabolism of biphenyl structures in lignin by the soil bacterium ( Pseudomonas paucimobilis SYK-6)

In the soil bacterium ( Pseudomonas paucimobilis SYK-6), the metabolism of DDVA (biphenyl structure of lignin) and syringic acid (characteristic aromatic ring in hardwood lignin) proceeds via a common intermediate, 3-methylgallic acid. Protocatechuate is also an intermediate in the metabolism of vanillate and p-hydroxybenzoic acid. 3-Methylgallic acid and protocatechuate are the final aromatic intermediates in lignin microbial degradation and these compounds are substrates of protocatechuate-4,5-dioxygenase, which is a key enzyme in obtaining metabolic energy from various structures of lignin in this bacterium.