Phthalate metabolism in Pseudomonas fluorescens PHK: purification and properties of 4,5-dihydroxyphthalate decarboxylase

Abstract

Pseudomonas fluorescens PHK uses 4,5-dihydroxyphthalate as the sole carbon source for o-phthalate catabolism. This intermediate is the substrate for a decarboxylase of the pathway yielding protocatechuate. The decarboxylase was purified to homogeneity by an affinity chromatography procedure in which the reaction product, protocatechuate, was used as a ligand. We describe some properties of the enzyme, including its apparent molecular weight of 420,000 as determined by gel filtration and of 66,000 after sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis, consistent with a hexameric functional protein. The apparent Km for the substrate 4,5-dihydroxyphthalate was 10.4 ,uM. The characteristics of this enzyme are compared with those described for the isofunctional enzyme from P. testosteroni.

Purification and Characterization of an Oxygen-Sensitive Reversible 4-Hydroxybenzoate Decarboxylase from Clostridium hydroxybenzoicum

“…To our knowledge, there have been no previous reports of purification of 4-hydroxybenzoate decarboxylase or a corresponding phenol carboxylase. There are reports of purifications of related enzymes, such as the 4,5-dihydroxyphthalate decarboxylase from pseudomonads (Nakazawa and Hayashi, 1978;Pujar and Gibson, 1985) and the 2,3-dihydroxybenzoate decarboxylase from the fungus AspergiZZus niger (Kamath et al, 1987) and yeast (Anderson and Dagley, 1981). The 4,5-dihydroxyphthalate decarboxylase from Pseudomonas puorescens had a molecular mass of 420 kDa with six subunits of 66 kDa and an optimum pH of 6.8 in phosphate buffer (Pujar and Gibson, 1985).…”

“…There are reports of purifications of related enzymes, such as the 4,5-dihydroxyphthalate decarboxylase from pseudomonads (Nakazawa and Hayashi, 1978;Pujar and Gibson, 1985) and the 2,3-dihydroxybenzoate decarboxylase from the fungus AspergiZZus niger (Kamath et al, 1987) and yeast (Anderson and Dagley, 1981). The 4,5-dihydroxyphthalate decarboxylase from Pseudomonas puorescens had a molecular mass of 420 kDa with six subunits of 66 kDa and an optimum pH of 6.8 in phosphate buffer (Pujar and Gibson, 1985). The enzyme from Pseudomonas testosteroni had a molecular mass of 150 kDa with four subunits of 38 kDa and an optimum pH of 7.5 in either phosphate or Trislacetate buffer (Nakazawa and Hayashi, 1978).…”

Purification and some properties of (1,2)-1,2-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylate dehydrogenase from T-2

“…The oxidation of the diem-dial to 4$dihydroxyphthalate (reaction II) has not been studied in detail. whereas the enzyme catalyzing decarboxylation to protocatechuatc (reaction III) has been purified from two organisms [27,28]. The terephthalate dioxygenase system (reaction A) has been partially characterized [2] and the diene-diol dehydrogenase (reaction B) was detected ip our preliminary experiments (cited in [6]): the reactions have also been detected in Pseudomonas putidu (cited in [6]).…”

Degradation of terephthalic acid by a Bacillus species

“…These chemicals have been found as environmental contaminants [2]. The degradation of phthalate isomers and phthalate esters has been studied extensively in various microorganisms [2][3][4][5]. In a soil Pseudomonas sp.…”