Purification and characterization of a novel type of protocatechuate 3,4-dioxygenase with the ability to oxidize 4-sulfo
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© Springer-Verlag 1996
O R I G I N A L PA P E R
Angela Hammer · Andreas Stolz · Hans-Joachim Knackmuss
Purification and characterization of a novel type of protocatechuate 3,4-dioxygenase with the ability to oxidize 4-sulfocatechol Received: 5 February 1996 / Accepted: 18 April 1996
Abstract 4-Aminobenzenesulfonate is degraded via 4sulfocatechol by a mixed bacterial culture that consists of Hydrogenophaga palleronii strain S1 and Agrobacterium radiobacter strain S2. From the 4-sulfocatechol-degrading organism A. radiobacter strain S2, a dioxygenase that converted 4-sulfocatechol to 3-sulfomuconate was purified to homogeneity. The purified enzyme also converted protocatechuate with a similar catalytic activity to 3-carboxy-cis,cis-muconate. Furthermore, the purified enzyme oxidized 3,4-dihydroxyphenylacetate, 3,4-dihydroxycinnamate, catechol, and 3- and 4-methylcatechol. The enzyme had a mol. wt. of about 97,400 as determined by gel filtration and consisted of two different types of subunits with mol. wt. of about 23,000 and 28,500. The NH2-terminal amino acid sequences of the two subunits were determined. An isofunctional dioxygenase was partially purified from H. palleronii strain S1. A. radiobacter strain S2 also induced, after growth with 4-sulfocatechol, an „ordinary“ protocatechuate 3,4-dioxygenase that did not oxidize 4-sulfocatechol. This enzyme was also purified to homogeneity, and its catalytic and structural characteristics were compared to the „4-sulfocatechol-dioxygenase“ from the same strain. Key words 4-Aminobenzenesulfonate · 4-Sulfocatechol · Xenobiotics · Biodegradation · Agrobacterium radiobacter · Protocatechuate 3,4-dioxygenase · Intradiol cleavage Abbreviation P34O Protocatechuate 3,4-dioxygenase
Introduction A 4-aminobenzenesulfonate-degrading bacterial coculture currently studied in this laboratory consists of Hy-
drogenophaga palleronii strain S1 and Agrobacterium radiobacter strain S2. 4-Sulfocatechol was identified as a key intermediate in the degradation of 4-aminobenzenesulfonate. 4-Sulfocatechol was oxidized in both strains by an ortho-cleavage mechanism to 3-sulfomuconate, which is analogous to the intradiol ring cleavage of protocatechuate (Fig. 1). In partially purified enzyme fractions from both strains, enzyme activities that oxidized both 4-sulfocatechol and protocatechuate were found. The corresponding enzyme activity was tentatively termed protocatechuate 3,4-dioxygenase type II (Feigel and Knackmuss 1993). Protocatechuate (3,4-dihydroxybenzoate) is a central intermediate in the biodegradation of many aromatic compounds by bacteria. Protocatechuate 3,4-dioxygenase (protocatechuate: oxygen 3,4-oxidoreductase, EC 1.13.11.3) catalyzes the intradiol addition of molecular oxygen, cleaving the aromatic ring and forming 3-carboxy-cis,cismuconate. Protocatechuate 3,4-dioxygenases from several bacterial sources have been intensively studied (for a recent review, see Lipscomb and Orville 1992). The cleavage of the sulfonated aromatic ring of 4-sulfocatechol by protocatechuate 3,4-diox
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