Phthalate and 4-hydroxyphthalate metabolism in Pseudomonas testosteroni: purification and properties of 4,5-dihydroxyphthalate decarboxylase

Abstract

Phthalate is degraded through 4,5-dihydroxyphthalate and protocatechuate in Pseudomonas testosteroni NH1000. The enzyme 4,5-dihydroxyphthalate decarboxylase, catalyzing the conversion of 4,5-dihydroxyphthalate to protocatechuate and carbon dioxide, was purified approximately 130-fold from phthalate-induced cells of a protocatechuate 4,5-dioxygenase-deficient mutant of P. testosteroni. The most purified preparation showed a single protein band on sodium dodecyl sulfate-acrylamide disc gel electrophoresis with a molecular weight of 38,000. The apparent molecular weight of the native enzyme determined by Sephadex G-200 column chromatography was 150,000. Among the substrate analogs tested, only 4-hydroxyphthalate served as a substrate, which was decarboxylated to form mhydroxybenzoate. The apparent Km values for 4,5-dihydroxyphthalate and 4hydroxyphthalate were estimated to be 10.5 ,uM and 1.25 mM, respectively, and the V., for the former was 10 times larger than that for the latter. Whereas the wild-type strain could utilize 4-hydroxyphthalate as a sole source of carbon, none of the following could grow with the compound: 4,5-dihydroxyphthalate decarboxylase-deficient, m-hydroxybenzoate-nondegradable, and protocatechuate 4,5dioxygenase-deficient mutants. Since one-step revertants of these mutants could utilize 4-hydroxyphthalate, the compound appears to be metabolized through mhydroxybenzoate and protocatechuate in P. testosteroni NH1000.

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).…”

“…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). The 2,3-dihydroxybenzoate de-carboxylases from Aspergillus niger (Kamath et al, 1987) and Trichosporon cutaneum (Anderson and Dagley, 1981) had molecular masses of 120 kDa with four subunits of 28 kDa, and 66.1 kDa with two subunits of 36.5 kDa, and optimum pH of 5.2 and 7.7, respectively.…”

Microbial degradation of phthalates: biochemistry and environmental implications

“…Here, decarboxylation of the carboxyl functionality in para ‐position to a phenolic group is largely facilitated, and consequently these decarboxylases do not require a special cofactor for catalysis (Fig. C), (Nakazawa and Hayashi, ; Pujar and Ribbons, ).…”

Bacterial Decarboxylation of o -Phthalic Acids

“…strain FO and the mixed culture ON-7, of a novel enzyme system that catalyzes the anaerobic conversion of phthalic acid to benzoic acid and which may conveniently be termed phthalic acid decarboxylase. The mixed culture ON-7 also possesses a 4,5-dihydroxyphthalic acid decarboxylase activity, similar to that in P. testosteroni and other pseudomonads and micrococci (7,15,21), that promotes the nonoxidative decarboxylation of 4,5-dihydroxyphthalic acid. The two decarboxylating enzyme systems are distinguishable by their substrate specificities and products ( Table 5).…”