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.     

4-sulfomuconolactone hydrolases from Hydrogenophaga intermedia S1 and Agrobacterium radiobacter S2

The 4-carboxymethylen-4-sulfo-but-2-en-olide (4-sulfomuconolactone) hydrolases from Hydrogenophaga intermedia strain S1 and Agrobacterium radiobacter strain S2 are part of a modified protocatechuate pathway responsible for the degradation of 4-sulfocatechol. In both strains, the hydrolase-encoding genes occur downstream of those encoding the enzymes that catalyze the lactonization of 3-sulfomuconate. The deduced amino acid sequences of the 4-sulfomuconolactone hydrolases demonstrated the highest degree of sequence identity to 2-pyrone-4,6-dicarboxylate hydrolases, which take part in the meta cleavage pathway of protocatechuate. The 4-sulfomuconolactone hydrolases did not convert 2-pyrone-4,6-dicarboxylate, and the 2-pyrone-4,6-dicarboxylate hydrolase from Sphingomonas paucimobilis SYK-6 did not convert 4-sulfomuconolactone. Nevertheless, the presence of highly conserved histidine residues in the 4-sulfomuconolactone and the 2-pyrone-4,6-dicarboxylate hydrolases and some further sequence similarities suggested that both enzymes belong to the metallo-dependent hydrolases (the "amidohydrolase superfamily"). The 4-sulfomuconolactone hydrolases were heterologously expressed as His-tagged enzyme variants. Gel filtration experiments suggested that the enzymes are present as monomers in solution, with molecular weights of approximately 33,000 to 35,000. 4-Sulfomuconolactone was converted by sulfomuconolactone hydrolases to stoichiometric amounts of maleylacetate and sulfite. The 4-sulfomuconolactone hydrolases from both strains showed pH optima at pH 7 to 7.5 and rather similar catalytic constant (k(cat)/K(M))values. The suggested 4-sulfocatechol pathway from 4-sulfocatechol to maleylacetate was confirmed by in situ nuclear magnetic resonance analysis using the recombinantly expressed enzymes.     

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.     

Isolation, characterization, and systematic significance of 2-pyrone-4,6-dicarboxylic acid in Rosaceae

2-Pyrone-4,6-dicarboxylic acid was isolated from Potentilla anserina. Until now this substance was only found in bacteria and not in higher plants. By sterile cultivation it was verified that this compound is genuine also in plants. In addition the systematic relevance of 2-pyrone-4,6-dicarboxylic acid within the Rosaceae was tested. The compound seems to be a chemotaxonomic marker for the Rosoideae sensu stricto proposed by Morgan et al. (Morgan, D.R., Soltis, D.E., Robertson, K.R., 1994. Systematic and evolutionary implications of rbcL sequence variation in Rosaceae. American Journal of Botany 81, 890-903).     

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.     

Gentisate 1,2-dioxygenase from pseudomonas. Purification, characterization, and comparison of the enzymes from Pseudomonas testosteroni and Pseudomonas acidovorans

The 3-hydroxybenzoate inducible gentisate 1,2-dioxygenases have been purified to homogeneity from P. acidovorans and P. testosteroni, the two divergent species of the acidovorans group of Pseudomonas. Both enzymes exhibit a 40-fold higher specific activity than previous preparations and have an (alpha Fe)4 quaternary structure (holoenzyme Mr = 164,000 and 158,000, respectively). The enzymes have different amino terminal sequences, amino acid contents, and isoelectric points. Each enzyme contains essential active site iron that is EPR silent but binds nitric oxide quantitatively to give an EPR active complex (S = 3/2), showing that the iron is Fe2+ with coordination sites for exogenous ligands. The EPR spectra of these complexes are altered uniquely for each enzyme when gentisate is bound. This suggests that substrate binds to or near the iron and shows that the substrate-iron interactions of each enzyme are subtly different. The kinetic parameters for turnover of gentisate by the enzymes are nearly identical (kcat/Km = 4.3 x 10(6) s-1 M-1). Both enzymes cleave a wide range of gentisate analogs substituted in the 3 or 4 ring position, although at reduced rates relative to gentisate. Of the two enzymes, P. testosteroni gentisate 1,2-dioxygenase exhibits substantially lower kcat/Km values for the turnover of these compounds. Evidence for both steric and electronic substituent effects is obtained. In accord with the results of Wheelis et al. (Wheelis, M. L., Palleroni, N. J., and Stanier, R. Y. (1967) Arch. Mikrobiol. 59, 302-314), 3-hydroxybenzoate is shown to be metabolized by P. acidovorans through the gentisate pathway, and gentisate 1,2-dioxygenase is the only ring cleavage dioxygenase induced. In contrast, 3-hydroxybenzoate is metabolized by P. testosteroni exclusively through the protocatechuate pathway utilizing protocatechuate 4,5-dioxygenase, although gentisate 1,2-dioxygenase is coinduced. Growth of P. testosteroni on 3-O-methylbenzoate or 5-O-methylsalicylate is shown to result in a approximately 10-fold increase in the amount of gentisate 1,2-dioxygenase relative to protocatechuate 4,5-dioxygenase. Together, these results suggest that induction of gentisate 1,2-dioxygenase by 3-hydroxybenzoate in P. testosteroni may be adventitious and that this enzyme may function in fundamentally different metabolic pathways in the two related Pseudomonas species.     

The 4-oxalomesaconate hydratase gene, involved in the protocatechuate 4,5-cleavage pathway, is essential to vanillate and syringate degradation in Sphingomonas paucimobilis SYK-6

Sphingomonas paucimobilis SYK-6 is able to grow on various dimeric lignin compounds, which are converted to vanillate and syringate by the actions of unique lignin degradation enzymes in this strain. Vanillate and syringate are degraded by the O-demethylase and converted into protocatechuate (PCA) and 3-O-methylgallate (3MGA), respectively. PCA is further degraded via the PCA 4,5-cleavage pathway, while the results suggested that 3MGA is degraded through another pathway in which PCA 4,5-dioxygenase is not involved. In a 10.5-kb EcoRI fragment carrying the genes for PCA 4,5-dioxygenase (ligAB), 2-pyrone-4,6-dicarboxylate hydrolase (ligI), and a portion of 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase (ligC), we found the ligJ gene encoding 4-oxalomesaconate (OMA) hydratase, which catalyzes the conversion of OMA into 4-carboxy-4-hydroxy-2-oxoadipate. The ligJ gene is transcribed in the same direction as ligABC genes and consists of an 1,023-bp open reading frame encoding a polypeptide with a molecular mass of 38,008 Da, which is located 73-bp upstream from ligA. The ligJ gene product (LigJ), expressed in Escherichia coli, was purified to near homogeneity and was estimated to be a homodimer (69.5 kDa) by gel filtration chromatography. The isoelectric point was determined to be 4.9, and the optimal temperature is 30 degrees C. The K(m) for OMA and the V(max) were determined to be 138 microM and 440 U/mg, respectively. LigJ activity was inhibited by the addition of thiol reagents, suggesting that some cysteine residue is part of the catalytic site. The ligJ gene disruption in SYK-6 caused the growth defect on and the accumulation of common metabolites from both vanillate and syringate, indicating that the ligJ gene is essential to the degradation of these two compounds. These results indicated that syringate is converted into OMA via 3MGA, and it enters the PCA 4,5-cleavage pathway.     

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.     

Alternative routes of aromatic catabolism in Pseudomonas acidovorans and Pseudomonas putida: gallic acid as a substrate and inhibitor of dioxygenases.

When 3,4-dihydroxyphenylacetic acid (homoprotocatechuic acid) was added to Pseudomonase acidovorans growing at the expense of succinate, enzymes required for degrading homoprotocatechuate to pyruvate and succinate semialdehyde were strongly induced. These enzymes were effectively absent from cell extracts of the organism grown with 4-hydroxyphenylacetic acid, and this substrate was metabolized by the catabolic enzymes of the homogentisate pathway. Two separate ring-fission dioxygenases for 3,4,5-trihydroxybenzoic acid (gallic acid) were present in cell extracts of Pseudomonas putida when grown with syringic acid, and gallate was degraded by reactions associated with meta fission. One of the two gallate dioxygenases also attacked 3-O-methylgallic acid; the other, which did not, was induced when cells were exposed to gallate. This organism possessed ortho fission enzymes, including protocatechuate 3,4-dioxygenase (EC 1.13.11.3) and cis,cis-carboxymuconate-lactonizing enzyme (EC 5.5.1.2), after induction with 3,4-dihydroxybenzoic acid (protocatechuic acid). Gallate was a substrate for protocatechuate 3,4-dioxygenase, with a Vmax about 3% of that of protocatechuate and with an apparent Km slightly lower. Gallate was a powerful competitive inhibitor of protocatechuate oxidation.     

Cleavage of pyrogallol by non-heme iron-containing dioxygenases

Both intradiol and proximal extradiol dioxygenases are thought to produce the same product, alpha-hydroxymuconic acid, when pyrogallol (3-hydroxycatechol) is used as a substrate. However, when these enzymes were reacted with pyrogallol, they gave different products. A proximal extradiol dioxygenase, metapyrocatechase (catechol:oxygen 2,3-d-oxidoreductase (decyclizing), EC 1.13.11.2), gave a product having an absorption maximum at 290 nm, which was gradually converted to a more stable compound having an absorption maximum at 239 nm. On the other hand, an intradiol dioxygenase, protocatechuate 3,4-dioxygenase (protocatechuate:oxygen 3,4-oxidoreductase (decyclizing), EC 1.13.11.3), gave a product having an absorption maximum at 300 nm. Based on the spectral data and direct comparison with authentic samples, the primary products obtained by the action of the former and the latter enzymes were identified as alpha-hydroxymuconic acid and 2-pyrone-6-carboxylic acid, respectively. While another intradiol dioxygenase, pyrocatechase (catechol:oxygen 1,2-oxidoreductase (decyclizing), EC 1.13.11.1), gave a mixture of nearly equimolar amounts of these two compounds. Isotope labeling experiments indicated that 1 atom of oxygen was incorporated in 2-pyrone-6-carboxylic acid from the atmosphere. Based on these findings, the reaction mechanism for the formation of 2-pyrone-6-carboxylic acid is discussed. This may be the first experimental evidence indicating the presence of a seven-membered lactone intermediate during the oxygenative cleavage of catechols, proposed by Hamilton (Hamilton, G.A. (1974) in Molecular Mechanisms of Oxygen Activation (Hayaishi, O., ed) pp. 405-451, Academic Press, New York).     

Cloning, sequencing, and expression of the gene encoding 4-hydroxy-4-methyl-2-oxoglutarate aldolase from Pseudomonas ochraceae NGJ1

A DNA fragment that carried the gene (proA) encoding 4-hydroxy-4-methyl-2-oxoglutarate aldolase was cloned from the chromosomal DNA of Pseudomonas ochraceae NGJ1, and the coding region was assigned to the nucleotide sequence based on the N-terminal amino acid sequence of the enzyme purified from the organism. The proA gene was 684 bp long, corresponding to a protein of 227 amino acid residues with a calculated molecular mass of 24,067 Da. The genes encoding a putative transporter and a 4-oxalomesaconate hydratase were upstream, and a 3'-truncated gene encoding 2-pyrone-4,6-dicarboxylate lactonase was downstream from the proA gene in the same orientation on the DNA fragment. The proA gene product was overproduced in Escherichia coli and briefly purified to homogeneity from the crude extract by a two-step purification. The molecular and catalytic properties of the gene product were similar to those of the P. ochraceae enzyme.     

Cloning,Sequencing, and Expression of the Gene Encoding 4-Hydroxy-4-methyl-2-oxoglutarate Aldolase from Pseudomonas ochraceae NGJ1

A DNA fragment that carried the gene (proA) encoding 4-hydroxy-4-methyl-2-oxoglutarate aldolase was cloned from the chromosomal DNA of Pseudomonas ochraceae NGJ1, and the coding region was assigned to the nucleotide sequence based on the N-terminal amino acid sequence of the enzyme purified from the organism. The proA gene was 684 bp long, corresponding to a protein of 227 amino acid residues with a calculated molecular mass of 24,067 Da. The genes encoding a putative transporter and a 4-oxalomesaconate hydratase were upstream, and a 3'-truncated gene encoding 2-pyrone-4,6-dicarboxylate lactonase was downstream from the proA gene in the same orientation on the DNA fragment. The proA gene product was overproduced in Escherichia coli and briefly purified to homogeneity from the crude extract by a two-step purification. The molecular and catalytic properties of the gene product were similar to those of the P. ochraceae enzyme.     

Phthalate metabolism in Pseudomonas testosteroni: accumulation of 4,5-dihydroxyphthalate by a mutant strain.

A mutant strain of Pseudomonas testosteroni blocked in phthalate catabolism converted phthalate into 4,5-dihydroxyphthalate. The latter compound was isolated, and its physical properties were determined. A stoichiometric conversion of the compound to protocatechuate was demonstrated spectrophotometrically with crude extracts of a protocatechuate 4,5-dioxygenase-deficient mutant. Therefore, phthalate is metabolized through 4,5-dihydroxyphthalate and protocatechuate, which is further degraded by protocatechuate 4,5-dioxygenase in P. testosteroni. By using several mutants blocked in phthalate catabolism, 4,5-dihydroxyphthalate decarboxylase was shown to be induced by phthalate. A simple spectrophotometric assay for the enzyme is also reported.     

Novel ring cleavage products in the biotransformation of biphenyl by the yeast Trichosporon mucoides

The yeast Trichosporon mucoides, grown on either glucose or phenol, was able to transform biphenyl into a variety of mono-, di-, and trihydroxylated derivatives hydroxylated on one or both aromatic rings. While some of these products accumulated in the supernatant as dead end products, the ortho-substituted dihydroxylated biphenyls were substrates for further oxidation and ring fission. These ring fission products were identified by high-performance liquid chromatography, gas chromatography-mass spectrometry, and nuclear magnetic resonance analyses as phenyl derivatives of hydroxymuconic acids and the corresponding pyrones. Seven novel products out of eight resulted from the oxidation and ring fission of 3,4-dihydroxybiphenyl. Using this compound as a substrate, 2-hydroxy-4-phenylmuconic acid, (5-oxo-3-phenyl-2,5-dihydrofuran-2-yl)acetic acid, and 3-phenyl-2-pyrone-6-carboxylic acid were identified. Ring cleavage of 3,4,4'-trihydroxybiphenyl resulted in the formation of [5-oxo-3-(4'-hydroxyphenyl)-2,5-dihydrofuran-2-yl]acetic acid, 4-(4'-hydroxyphenyl)-2-pyrone-6-carboxylic acid, and 3-(4'-hydroxyphenyl)-2-pyrone-6-carboxylic acid. 2,3,4-trihydroxybiphenyl was oxidized to 2-hydroxy-5-phenylmuconic acid, and 4-phenyl-2-pyrone-6-carboxylic acid was the transformation product of 3,4,5-trihydroxybiphenyl. All these ring fission products were considerably less toxic than the hydroxylated derivatives.     

Phthalate and 4-hydroxyphthalate metabolism in Pseudomonas testosteroni: purification and properties of 4,5-dihydroxyphthalate decarboxylase.

Phthalate is degraded through 4,5-dihydroxyphthalate and protocatechuate in Pseudomonas testosteroni NH1000. The ezyme 4,5-dihydroxyphthalate decarboxylase, catalyzing the conversion of 4,5-dihydroxyphthalate to protocatechuate and carbon dioxide, was purified approximately 130-fold from phthalate-induced cells of a protocatechuate 4,5-dioxygenase-deficient mutant of P. testosteroni. The most purified preparation showed a single protein band on sodium dodecyl sulfate-acrylamide disc gel electrophoresis with a molecular weight of 38,000. The apparent molecular weight of the native enzyme determined by Sephadex G-200 column chromatography was 150,000. Among the substrate analogs tested, only 4-hydroxyphthalate served as a substrate, which was decarboxylated to form m-hydroxybenzoate. The apparent Km values for 4,5-dihydroxyphthalate and 4-hydroxyphthalate were estimated to be 10.5 micrometer and 1.25 mM, respectively, and the Vmax for the former was 10 times larger than that for the latter. Whereas the wild-type strain could utilize 4-hydroxyphthalate as a sole source of carbon, none of the following could grow with the compound: 4,5-dihydroxyphthalate decarboxylase-deficient, m-hydroxybenzoate-nondegradable, and protocatechuate 4,5-dioxygenase-deficient mutants. Since one-step revertants of these mutants could utilize 4-hydroxyphthalate, the compound appears to be metabolized through m-hydroxybenzoate and protocatechuate in P. testosteroni NH1000.     

Phthalate and 4-hydroxyphthalate metabolism in Pseudomonas testosteroni: purification and properties of 4,5-dihydroxyphthalate decarboxylase.

Phthalate is degraded through 4,5-dihydroxyphthalate and protocatechuate in Pseudomonas testosteroni NH1000. The ezyme 4,5-dihydroxyphthalate decarboxylase, catalyzing the conversion of 4,5-dihydroxyphthalate to protocatechuate and carbon dioxide, was purified approximately 130-fold from phthalate-induced cells of a protocatechuate 4,5-dioxygenase-deficient mutant of P. testosteroni. The most purified preparation showed a single protein band on sodium dodecyl sulfate-acrylamide disc gel electrophoresis with a molecular weight of 38,000. The apparent molecular weight of the native enzyme determined by Sephadex G-200 column chromatography was 150,000. Among the substrate analogs tested, only 4-hydroxyphthalate served as a substrate, which was decarboxylated to form m-hydroxybenzoate. The apparent Km values for 4,5-dihydroxyphthalate and 4-hydroxyphthalate were estimated to be 10.5 micrometer and 1.25 mM, respectively, and the Vmax for the former was 10 times larger than that for the latter. Whereas the wild-type strain could utilize 4-hydroxyphthalate as a sole source of carbon, none of the following could grow with the compound: 4,5-dihydroxyphthalate decarboxylase-deficient, m-hydroxybenzoate-nondegradable, and protocatechuate 4,5-dioxygenase-deficient mutants. Since one-step revertants of these mutants could utilize 4-hydroxyphthalate, the compound appears to be metabolized through m-hydroxybenzoate and protocatechuate in P. testosteroni NH1000.