Molecular Characterization of the Gallate Dioxygenase from Pseudomonas putida KT2440

Abstract

In this work we have characterized the galA gene product from Pseudomonas putida KT2440, a ring-cleavage dioxygenase that acts specifically on gallate to produce 4-oxalomesaconate. The protein is a trimer composed by three identical subunits of 47.6 kDa (419 amino acids) that uses Fe 2؉ as the main cofactor. The gallate dioxygenase showed maximum activity at pH 7.0, and the K m and V max values for gallate were 144 M and 53.2 mol/min/mg of protein, respectively. A phylogenetic study suggests that the gallate dioxygenase from P. putida KT2440 is the prototype of a new subgroup of type II extradiol dioxygenases that share a common ancestor with protocatechuate 4,5-dioxygenases and whose two-domain architecture might have evolved from the fusion of the large and small subunits of the latter. A three-dimensional model for the N-terminal domain (residues 1-281) and C-terminal domain (residues 294 -420) of the gallate dioxygenase from P. putida KT2440 was generated by comparison with the crystal structures of the large (LigB) and small (LigA) subunits of the protocatechuate 4,5-dioxygenase from Sphingomonas paucimobilis SYK-6. The expression of the galA gene was specifically induced when P. putida KT2440 cells grew in the presence of gallate. A P. putida KT2440 galA mutant strain was unable to use gallate as the sole carbon source and it did not show gallate dioxygenase activity, suggesting that the GalA protein is the only dioxygenase involved in gallate cleavage in this bacterium. This work points to the existence of a new pathway that is devoted to the catabolism of gallic acid and that remained unknown in the paradigmatic P. putida KT2440 strain.The variety and number of complete microbial genome sequences is increasing at an unprecedented rate. To date, more than 200 bacterial genomes have been successfully sequenced and nearly 1000 genomes are currently in progress, which provides an enormous amount of data for comparative genomic analysis and for identifying new enzymes, regulators, and pathways, including key aspects of genome evolution and bacterial adaptation (1). Among bacteria, Pseudomonas species constitute a large diverse group of ubiquitous ␥-proteobacteria that are well known for their broad metabolic versatility and genetic plasticity. Pseudomonas putida is one of the best studied species of the genus and many aspects of its biology have been unraveled (2, 3). P. putida strain KT2440 is a paradigm of a metabolically versatile bacterium used as a workhorse for genetic and physiological studies as well as for the development of biotechnological applications (3). This strain is particularly renowned for its ability to metabolize aromatic compounds (4). The recent sequencing of the P. putida KT2440 genome (5) permitted a genomic analysis of the global catabolic potential of this bacterium toward aromatic compounds (4, 6). This search revealed the presence of three central pathways, the ortho pathway for the catabolism of protocatechuate (pca genes) and catechol (cat genes), the phenylacetate pathway (pha ...

Structural and Kinetic Characterization of the 4-Carboxy-2-hydroxymuconate Hydratase from the Gallate and Protocatechuate 4,5-Cleavage Pathways of Pseudomonas putida KT2440

“…The molecular mass of the GalA, GalB, GalD, and LigJ CsE6 subunits, as determined by SDS-PAGE, was ϳ45, ϳ25, ϳ40, and ϳ40 kDa (data not shown), which is consistent with the predicted molecular mass values of 47.6, 27.5, 37.6, and 38.2 kDa, respectively, from previous reports (9,10,19,20).…”

Global Regulation of Food Supply by P seudomonas p utida DOT-T1E

“…The results also confirm the ability of P. putida to use organic acids (such as acetic, citric, glutaric, quinic, lactic, and succinic), certain L-amino acids (Ala, Arg, Asn, Glu, His, Ile, Lys, Pro, Tyr, and Val), various amino organic compounds, and aromatic compounds (3,4-dihydroxyphenylacetate [14], 4-hydroxybenzoate, homogentisate, sodium benzoate, 2-phenylethylamine, toluene, ethylbenzene, propylbenzene, gallate [37], 4-aminophenol, and salicylate [51]). Importantly, a number of the carbon source compounds on which wild-type P. putida DOT-T1E can grow have not been previously described, including decanoate, 3,4-dihydroxyphenylacetate, 4-hydroxyphenylpyruvate, phenylethylamine, phenylpyruvate, quinate, salicylate, and gallate (see Fig.…”

Uncovering the Protocatechuate 2,3-Cleavage Pathway Genes

“…Type I extradiol dioxygenases belong to the vicinal oxygen superfamily, including a number of 2,3-dihydroxybiphenyl 1,2-dioxygenases (3,27) and catechol 2,3-dioxygenases (40). Type II dioxygenases include several homoprotocatechuate 2,3-dioxygenases (49), the ␤ subunit of PCA 4,5-dioxygenase (41,47), gallate dioxygenase (30,42), and 2-aminophenol 1,6-dioxygenase (61). Type III dioxygenases belong to the cupin superfamily, which includes enzymes such as the gentisate dioxygenase, homogentisate dioxygenase, and 1-hydroxy-2-naphthoate dioxygenase (12).…”