Trichosporon cutaneum readily metabolized protocatechuate, homoprotocatechuate, and gentisate, but lacked ring fission dioxygenases for these compounds. Benzoic, salicylic, 2,3-dihydroxybenzoic, and gentisic acids were converted into beta-ketoadipic acid before entry into the Krebs cycle. Benzoic acid gave rise successively to 4-hydroxybenzoic acid, protocatechuic acid, and hydroxyquinol (1,3,4-trihydroxybenzene), which underwent ring fission to maleylacetic acid. Salicylate and 2,3-dihydroxybenzoate were both initially metabolized to give catechol. 2,3-Dihydroxybenzoate was the substrate for a specific nonoxidative decarboxylase induced by salicylate, although 2,3-dihydroxybenzoate was not a catabolite of salicylate. Gentisate was metabolized to maleylacetic acid and was also readily attacked by salicylate hydroxylase at each stage of a partial purification procedure. Phenylacetic acid was degraded through 3-hydroxyphenylacetic, homogentisic, and maleylacetoacetic acids to acetoacetic and fumaric acids. All the reactions of these catabolic sequences were catalyzed by cell extracts, supplemented with reduced pyridine nucleotide coenzymes where necessary, except for the hydroxylations of benzoic and phenylacetic acids which were demonstrated with cell suspensions and isotopically labeled substrates.
Trichosporon cutaneum degraded L-tryptophan by a reaction sequence that included L-kynurenine, anthranilate, 2,3-dihydroxybenzoate, catechol, and fl-ketoadipate as catabolites. All of the enzymes of the sequence were induced by both L-tryptophan and salicylate, and those for oxidizing kynurenine and its catabolites were induced by anthranilate but not by benzoate; induction was not coordinate. Molecular weights of 66,100 and 36,500 were determined, respectively, for purified 2,3-dihydroxybenzoate decarboxylase and its single subunit. Substrates for this enzyme were restricted to benzoic acids substituted with hydroxyl groups at C-2 and C-3; no added coenzyme was required for activity. Partially purified anthranilate hydroxylase (deaminating) catalyzed the incorporation of one atom of 180, derived from either '"02 or H2150, into 2,3-dihydroxybenzoic acid. In a previous study of the catabolism of aromatic acids in Trichosporon cutaneum (1), we showed that the enzyme 2,3-dihydroxybenzoate decarboxylase (EC 4.1.1.46) is strongly derepressed in cells grown on 2-hydroxybenzoate (salicylate), despite the fact that the substrate of this enzyme is not a catabolite of salicylate. An explanation for this observation has emerged from the present work. We have shown that salicylate derepresses the whole series of enzymes, including 2,3-dihydroxybenzoate decarboxylase, which the organism uses for degrading L-tryptophan. This catabolic pathway was found to be similar to that demonstrated by previous workers (5, 20, 21) for tryptophan catabolism in Aspergillus niger and Claviceps paspali. MATERIALS AND METHODS Organism and cell extracts. The organism used, T. cutaneum, was grown in shake cultures at 300C as described earlier (1, 16) with single aromatic acids (0.05%), including L-tryptophan, serving as major sources of carbon. For preparing cell extracts that contained about 10 mg of protein per ml, the organism was grown to the stationary phase in 16-liter batches under vigorous forced aeration. The inoculum used was 1 liter of a shake culture. For purification of 2,3dihydroxybenzoate decarboxylase, larger quantities of cells (150 g [wet weight]) were grown at the expense of salicylate (2-hydroxybenzoate) in a 100-liter fermentor which was provided with a mechanical stirrer and aeration and contained 90 liters of growth medium.
Trichosporon cutaneum degraded 4-hydroxyphenylacetic acid to acetoacetic and malic acids. 3,4-Dihydroxyphenylacetic acid, an intermediate in the reaction sequence, underwent hydroxylation before the benzene ring was cleaved.
Low erythrocyte activities of the selenium-containing enzyme glutathione peroxidase were found in Merino lambs in an area of the Strathbogie Ranges in central Victoria where selenium-responsive conditions have previously been reported. Body weight gain trials conducted over 10 properties in the above area demonstrated that the severity of selenium-responsive unthriftiness was significantly correlated with the erythrocyte glutathione peroxidase activity (r = – 0.95, P <: 0.001). Positive body weight responses to selenium treatment were only observed in lambs with erythrocyte glutathione peroxidase activities less than 30 U/g Hb prior to selenium treatment. These findings indicate that measurement of erythrocyte glutathione peroxidase activity provides a convenient index of the selenium status in sheep. ________________ *Part I, Aust. J. Agric. Res., 30: 695 (1979).
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