Catabolism of tryptophan, anthranilate, and 2,3-dihydroxybenzoate in Trichosporon cutaneum

Abstract

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.

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 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. The 4-hydroxybenzoate decarboxylase from C. hydroxybenzoicurn differs in its properties from the above aryl decarboxylases.…”

Degradation of organic nitrogen compounds by yeasts

“…The majority of catabolic pathways that have been discussed in sections 1-9 do not take place in a single cellular compartment but may involve enzymes located in the m i t~c h o n d r i a ,~~ the microbodies or peroxisomes (the terms will be used without di~tinction),~'-'~' the endoplasmic reticulum3' and the v a~u o l e .~~~, '~~ It is thus likely that in nearly every pathway discussed above, transfer of metabolic intermediates between different subcellular compartments will occur, and this will pose both permeability and energetic problems to the cell. Schemes involving such changes of compartment play an essential role in the breakdown, for example, of n-alkane~,~' arginine," ethanol' 8 8 and methylated a m i n e~.~' Figure 11 illustrates examples of this transfer between cellular compartments. The modifications that some compounds undergo may be associated with permeability problems.…”

The morphology, physiology, and fine structure of a toluene-oxidizing strain of Pseudomonas putida

“…The benzene nucleus is exceedingly abundant in the biosphere and, because of its resonance structure, is extremely stable. Most of the degradation of the benzene nucleus is done by microorganisms (14,15).…”