Purification and Properties of Catechol 1,2‐Oxygenase from Trichosporon cutaneum

Abstract

A highly purified preparation of catechol 1,2-oxygenase was obtained from a strain of Trichosporon cutaneum, isolated from soil as a predominant phenol utilizing yeast. The purified active enzyme was homogeneous upon polyacrylamide gel electrophoresis. The enzyme was gradually inactivated upon storage. The inactivation was followed by the appearance of a second, electrophoretically distinct band. The active enzyme is red, the inactive form is colourless. Its molecular weight is 109000 as determined by gel filtration; pH optimum 8.2. The Michaelis constants are 5.9 pM for catechol and 81 pM for oxygen. There is no cofactor requirement for activity. The enzyme is sensitive to iron chelating agents, reducing agents and quinones. The enzyme has a broad substrate specificity: besides catechol it is able to cleave hydroxyl-and methyl-substituted catechols. Several catechol derivatives inhibit the enzyme activity towards catechol.Catechol 1,2-oxygenase was originally purified from a Pseudomonas, and it was proved to be a dioxygenase, which introduces molecular oxygen directly into catechol, with the formation of cispismuconic acid [I-41. Recently the enzyme was isolated from Brevibacterium fuscurn [5]. The molecular weight of the catechol I ,2-oxygenases purified hitherto, is in the range of 70000-100000. The active enzymes are reported to have distinct red colour, and to be non-heme iron proteins.I n a previous work we reported the results of studies on the decomposition of phenols by Trichosporon cutaneum, a yeast isolated by us from soil [6,7]. Results of respirometric studies on intact cells and cell-free preparations indicated that cells adapted to either phenol or resorcinol contained ring cleaving enzyme(s) able to act on catechol and different catechol derivatives. This phenomenon might have been attributed to the presence of Werent ring cleaving enzymes, induced by phenol or resorcinol, as well as to a single enzyme with broad substratespecificity. We wanted to answer this question by purification and characterization of catechol 1,Zoxygenase from T . cutaneum.

Phenol hydroxylase from yeast

“…Enzyme purijication 100 c I Phenol hydroxylase was isolated from T. cutaneum, grown in 800-1 cultures in the medium described before [13]. The cell paste was stored at -70°C.…”

Activation enthalpies and pH dependence of phenol hydroxylase from Trichosporon cutaneum, in vitro and in situ

“…It also occurs with a second enzyme of the phenol degradative pathway, viz. catechol 1,2-oxygenase, for which Km at pH 8.3 is 3.3/zM in situ, but 5.9/zM in vitro [14]. The difference between phenol hydroxylase in situ and the enzyme in vitro extends to energetics of the reaction, the activation enthalpies for phenol hydroxylase in situ being about 75-8007o of those in vitro (table 1).…”

Characteristics of a New Catechol 1,2-Oxygenase fromTrichosporon cutaneumWY 2-2

“…The purified enzyme exhibited higher activities towards 4-methylcatechol (186%) and hydroxyquinol (159%) than towards catechol (100%), 3-methylcatechol (6%) and pyrogallol (9%), and it was obviously different in the substrate specificity from those isolated from Brevibacterium 5 ) and Trichosporon cutaneum. 6 ) All of the catechol 1,2-oxygenases so far purified and characterized contained nonheme iron as a sole cofactor. 19 ) The enzyme activity was inhibited 53% with KCN (1 mM), which might suggest that the enzyme, differently from other ring-cleaving oxygenases, is a heme iron protein.…”

“…6 ) In a previous paper, 7) we reported that catechol 1,2-oxygenase from Trichosporon cutaneum WY 2-2, which isolated by us from an activated sludge, was apparently different in its substrate specificity from the enzyme from T. cutaneum isolated by Neujahr et al 6 ) and the other such enzyme in bacteria.l~5)…”