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.

• Purification and properties of 2-pyrone-4,6-dicarboxylate hydrolase.

  Maruyama K.

  A hydrolase which catalyzes specifically the interconversion between 2-pyrone-4,6-dicarboxylate and 4-oxalmesaconate was purified about 410-fold with a 16% yield from cell-free extracts of Pseudomonas ochraceae grown with phthalate. Upon disc gel electrophoresis, the enzyme preparation gave a sing ... >> More

  A hydrolase which catalyzes specifically the interconversion between 2-pyrone-4,6-dicarboxylate and 4-oxalmesaconate was purified about 410-fold with a 16% yield from cell-free extracts of Pseudomonas ochraceae grown with phthalate. Upon disc gel electrophoresis, the enzyme preparation gave a single band which was coincident with the enzyme activity. The molecular weight of the enzyme was estimated to be 31,000 by gel filtration on Sephadex G-75 and 33,000 by sodium dodecyl sulfate gel electrophoresis. The isoelectric point of the enzyme was determined to be at pH 5.49 by isoelectric focusing. The enzyme is specific for 2-pyrone-4,6-dicarboxylate, and various other lactones did not serve as substrates. The stoichiometry of 2-pyrone-4,6-dicarboxylate hydrolysis, 4-oxalmesaconate formation and proton production was approximately 1:1:1. The optimum pHs are 8.5 and 6.0 for hydrolysis and synthesis of 2-pyrone-4,6-dicarboxylate, respectively. Km values are 87 and 26 microM for 2-pyrone-4,6-dicarboxylate and 4-oxalmesaconate, respectively. At pH 8.5, the ratio of 4-oxalmesaconate to 2-pyrone-4,6-dicarboxylate at equilibrium is about 2.2. Thiol reagents such as HgCl2 and p-chloromercuribenzoate strongly inhibit the enzyme activity. << Less

  J Biochem 93:557-565(1983) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• 2-pyrone-4,6-dicarboxylic acid, a catabolite of gallic acids in Pseudomonas species.

  Kersten P.J., Dagley S., Whittaker J.W., Arciero D.M., Lipscomb J.D.

  2-Pyrone-4,6-dicarboxylate hydrolase was purified from 4-hydroxybenzoate-grown Pseudomonas testosteroni. Gel filtration and electrophoretic measurements indicated that the preparation was homogeneous and gave a molecular weight of 37,200 for the single subunit of the enzyme. Hydrolytic activity wa ... >> More

  2-Pyrone-4,6-dicarboxylate hydrolase was purified from 4-hydroxybenzoate-grown Pseudomonas testosteroni. Gel filtration and electrophoretic measurements indicated that the preparation was homogeneous and gave a molecular weight of 37,200 for the single subunit of the enzyme. Hydrolytic activity was dependent upon a functioning sulfhydryl group(s) and was freely reversible; the equilibrium position was dependent upon pH, with equimolar amounts of pyrone and open-chain form present at pH 7.9. Since the hydrolase was strongly induced when the nonfluorescent organisms P. testosteroni and P. acidovorans grew with 4-hydroxybenzoate, it is suggested that 2-pyrone-4,6-dicarboxylate is a normal intermediate in the meta fission degradative pathway of protocatechuate. Laboratory strains of fluorescent pseudomonads did not metabolize 2-pyrone-4,6-dicarboxylate, but a strain of P. putida was isolated from soil that utilized this compound for growth; the hydrolase was then induced, but it was absent from extracts of 4-hydroxybenzoate-grown cells that readily catabolized protocatechuate by ortho fission reactions. 2-Pyrone-4,6-dicarboxylic acid was the major product formed when gallic acid was oxidized by purified protocatechuate 3,4-dioxygenase. Protocatechuate 4,5-dioxygenase gave only the open-chain ring fission product when gallic acid was oxidized, but the enzyme attacked 3-O-methylgallic acid, giving 2-pyrone-4,6-dicarboxylic acid as the major product. Cell suspensions of 4-hydroxybenzoate-grown P. testosteroni readily oxidized 3-O-methylgallate with accumulation of methanol. << Less

  J. Bacteriol. 152:1154-1162(1982) [PubMed] [EuropePMC]