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.

• Biochemical and structural analysis of RraA proteins to decipher their relationships with 4-hydroxy-4-methyl-2-oxoglutarate/4-carboxy-4-hydroxy-2-oxoadipate aldolases.

  Mazurkewich S., Wang W., Seah S.Y.

  4-Hydroxy-4-methyl-2-oxoglutarate (HMG)/4-carboxy-4-hydroxy-2-oxoadipate (CHA) aldolases are class II (divalent metal ion dependent) pyruvate aldolases from the meta cleavage pathways of protocatechuate and gallate. The enzyme from Pseudomonas putida F1 is structurally similar to a group of protei ... >> More

  4-Hydroxy-4-methyl-2-oxoglutarate (HMG)/4-carboxy-4-hydroxy-2-oxoadipate (CHA) aldolases are class II (divalent metal ion dependent) pyruvate aldolases from the meta cleavage pathways of protocatechuate and gallate. The enzyme from Pseudomonas putida F1 is structurally similar to a group of proteins termed regulators of RNase E activity A (RraA) that bind to the regulatory domain of RNase E and inhibit the ribonuclease activity in certain bacteria. Analysis of homologous RraA-like proteins from varying species revealed that they share sequence conservation within the active site of HMG/CHA aldolase. In particular, the P. putida F1 HMG/CHA aldolase has a D-X20-R-D motif, whereas a G-X20-R-D-X2-E/D motif is observed in the structures of the RraA-like proteins from Thermus thermophilus HB8 (TtRraA) and Saccharomyces cerevisiae S288C (Yer010Cp) that may support metal binding. TtRraA and Yer010Cp were found to contain HMG aldolase and oxaloacetate decarboxylase activities. Similar to the P. putida F1 HMG/CHA aldolase, both TtRraA and Yer010Cp enzymes required divalent metal ions for activity and were competitively inhibited by oxalate, a pyruvate enolate analogue, suggesting a common mechanism among the enzymes. The RraA from Escherichia coli (EcRraA) lacked detectable C-C lyase activity. Upon restoration of the G-X20-R-D-X2-E/D motif, by site-specific mutagenesis, the EcRraA variant was able to catalyze oxaloacetate decarboxylation. Sequence analysis of RraA-like gene products found across all the domains of life revealed conservation of the metal binding motifs that can likely support a divalent metal ion-dependent enzyme reaction either in addition to or in place of the putative RraA function. << Less

  Biochemistry 53:542-553(2014) [PubMed] [EuropePMC]

• 4-Hydroxy-4-methyl-2-ketoglutarate aldolase from Pseudomonas putida.

  Dagley S.

  Methods Enzymol 90 Pt E:272-276(1982) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

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

  Maruyama K., Miwa M., Tsujii N., Nagai T., Tomita N., Harada T., Sobajima H., Sugisaki H.

  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 t ... >> More

  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. << Less

  Biosci. Biotechnol. Biochem. 65:2701-2709(2001) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Purification and properties of 4-hydroxy-4-methyl-2-oxoglutarate aldolase from Pseudomonas ochraceae grown on phthalate.

  Maruyama K.

  4-Hydroxy-4-methyl-2-oxoglutarate aldolase [4-hydroxy-4-methyl-2-oxoglutarate pyruvate-lyase: EC 4.1.3.17] has been purified to homogeneity (about 770-fold purification, yield 11.4%) from Pseudomonas ochraceae grown on phthalate. The enzyme has a molecular weight of 160,000 (gel filtration on Bio- ... >> More

  4-Hydroxy-4-methyl-2-oxoglutarate aldolase [4-hydroxy-4-methyl-2-oxoglutarate pyruvate-lyase: EC 4.1.3.17] has been purified to homogeneity (about 770-fold purification, yield 11.4%) from Pseudomonas ochraceae grown on phthalate. The enzyme has a molecular weight of 160,000 (gel filtration on Bio-Gel A-1.5m), a subunit molecular weight of 26,000 (SDS-PAGE) and an isoelectric point of 5.0 (isoelectric focusing). The enzyme requires divalent metal ions such as Mg2+, Mn2+, Co2+, Zn2+, and Cd2+ for activity. The enzyme actively cleaves 4-carboxy-4-hydroxy-2-oxoadipate, a physiological substrate of the enzyme, to give pyruvate and oxaloacetate, but shows much lower affinity for 4-hydroxy-4-methyl-2-oxoglutarate. 4-Hydroxy-2-oxoglutarate is cleaved at a low rate to pyruvate and glyoxylate. The l-isomers of the substrates are preferentially cleaved rather than the d-isomers as determined polarimetrically. The enzyme reactions are reversible: the equilibrium constants (pH 8.0, 25 C) for the HMG and HG cleavage reactions are about 0.07 and 0.03 M, respectively, whereas no equilibrium is observed with CHA due to oxaloacetate beta-decarboxylase activity associated with the enzyme. The enzyme activity is hardly affected by thiols and thiol reagents. The non-enzymatic cleavage reaction caused by various metal ions has also been studied to examine the mechanistic similarity to the enzymatic reaction. << Less

  J. Biochem. 108:327-333(1990) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Purification and properties of 4-hydroxy-4-methyl-2-oxoglutarate aldolase.

  Tack B.F., Chapman P.J., Dagley S.

  J Biol Chem 247:6444-6449(1972) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.