Publications

• Unravelling the gallic acid degradation pathway in bacteria: the gal cluster from Pseudomonas putida.

  Nogales J., Canales A., Jimenez-Barbero J., Serra B., Pingarron J.M., Garcia J.L., Diaz E.

  Gallic acid (3,4,5-trihydroxybenzoic acid, GA) is widely distributed in nature, being a major phenolic pollutant and a commonly used antioxidant and building-block for drug development. We have characterized the first complete cluster (gal genes) responsible for growth in GA in a derivative of the ... >> More

  Gallic acid (3,4,5-trihydroxybenzoic acid, GA) is widely distributed in nature, being a major phenolic pollutant and a commonly used antioxidant and building-block for drug development. We have characterized the first complete cluster (gal genes) responsible for growth in GA in a derivative of the model bacterium Pseudomonas putida KT2440. GalT mediates specific GA uptake and chemotaxis, and highlights the critical role of GA transport in bacterial adaptation to GA consumption. The proposed GA degradation via the central intermediate 4-oxalomesaconic acid (OMA) was revisited and all enzymes involved have been identified. Thus, GalD is the prototype of a new subfamily of isomerases that catalyses a biochemical step that remained unknown, i.e. the tautomerization of the OMAketo generated by the GalA dioxygenase to OMAenol. GalB is the founding member of a new family of zinc-containing hydratases that converts OMAenol into 4-carboxy-4-hydroxy-2-oxoadipic acid (CHA). galC encodes the aldolase catalysing CHA cleavage to pyruvic and oxaloacetic acids. The presence of homologous gal clusters outside the Pseudomonas genus sheds light on the evolution and ecology of the gal genes in GA degraders. The gal genes were used for expanding the metabolic abilities of heterologous hosts towards GA degradation, and for engineering a GA cellular biosensor. << Less

  Mol. Microbiol. 79:359-374(2011) [PubMed] [EuropePMC]

  This publication is cited by 6 other entries.

• Enzymes responsible for degradation of 4-oxalmesaconic acid in Pseudomonas ochraceae.

  Maruyama K.

  The enzyme responsible for the degradation of 4-oxalmesaconate was partially purified from Pseudomonas ochraceae grown with phthalate. Column chromatography on DEAE-cellulose caused separation into two distinct enzymes, I and II. 4-Oxalmesaconate was converted into pyruvate and oxalacetate in the ... >> More

  The enzyme responsible for the degradation of 4-oxalmesaconate was partially purified from Pseudomonas ochraceae grown with phthalate. Column chromatography on DEAE-cellulose caused separation into two distinct enzymes, I and II. 4-Oxalmesaconate was converted into pyruvate and oxalacetate in the presence of MgCl2 and enzymes I and II. Optimum pH of the reaction was observed at pH 8.2 in Tris-HCl buffer. MgCl2 could be replaced by MnCl2 or CoCl2. Both enzymes were stable to heat-treatment at 65 degrees C for 10 min. Analyses of time course, products and substrate specificity of the enzyme reaction accounted for the functions of two enzymes. Enzyme I (molecular weight 55,000, isoelectric point 5.1) hydrated 4-oxalmesaconate to give 4-oxalcitramate and may be classified as a hydrolyase. Enzyme II (160,000, 5.0) catalyzed the aldolitic cleavage of 4-oxalcitramalate to pyruvate and oxalacetate in the presence of MgCl2. Enzyme II also cleaved 4-hydroxy-4-methyl-2-oxoglutarate into pyruvate. Stoichiometry of the enzyme reaction suggested that enzyme II-catalyzed cleavage occurred on only one enantiomer of the substrates. Furthermore, the metabolic pathway for the dissimilation of protocatechuate in P. ochraceae is presented and discussed in comparison with the pathway postulated previously by other workers. << Less

  J Biochem 93:567-574(1983) [PubMed] [EuropePMC]

• 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.

  Hara H., Masai E., Katayama Y., Fukuda M.

  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) ... >> More

  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. << Less

  J. Bacteriol. 182:6950-6957(2000) [PubMed] [EuropePMC]

• Purification and properties of gamma-oxalomesaconate hydratase from Pseudomonas ochraceae grown with phthalate.

  Maruyama K.

  Pseudomonas ochraceae produced inducibly a hydro-lyase which catalyzes the reversible conversion of gamma-oxalomesaconate into (-)-gamma-oxalocitramalate. The enzyme has been purified to homogeneity from the bacteria grown with phthalate. The enzyme was a dimeric protein (pI=4.9) with a Mr of 68,0 ... >> More

  Pseudomonas ochraceae produced inducibly a hydro-lyase which catalyzes the reversible conversion of gamma-oxalomesaconate into (-)-gamma-oxalocitramalate. The enzyme has been purified to homogeneity from the bacteria grown with phthalate. The enzyme was a dimeric protein (pI=4.9) with a Mr of 68,000 and showed a high specificity for gamma-oxalomesaconate (Km=14 microM) and (-)-gamma-oxalocitramalate (Km=6.4 microM). Equilibrium constant for the hydration of gamma-oxalomesaconate at pH 8.0 and 24 degrees C was 2.5. Various thiols activated the enzyme. << Less

  Biochem Biophys Res Commun 128:271-277(1985) [PubMed] [EuropePMC]