Maleylacetate reductase from Trichosporon cutaneum.

The enzyme catalysing the reduction of maleylacetate to 3-oxoadipate was purified 150-fold from Trichosporon cutaneum, induced for aromatic metabolisms by growth with resorcinol as a major carbon source. The enzyme separated upon electrofocusing into three species with PI values 4.6, 5.1 and 5.6. They had similar catalytic properties and the same molecular weight.     

Uptake of phenol by Trichosporon cutaneum.

The soil yeast Trichosporon cutaneum, which is distinguished by having a strictly oxidative metabolism, can be induced to utilize phenol as a sole carbon source. The present paper shows that such phenol-induced cells contain a specific, energy-dependent uptake system for phenol. Phenol uptake is not directly linked to its o-hydroxylation inside the cell, the first step of phenol metabolism. The Km for uptake is 235 +/- 30 microM, that for hydroxylation only 4.5 +/- 0.5 microM. Further, the phenol analog 2,6-dimethylphenol, which can not be hydroxylated, competes with phenol for the uptake system. The pH dependence of uptake indicates that phenolate is an essential form during the uptake process. The energy requirement for phenol uptake is indicated by effects of various inhibitors of energy generation, including proton-conducting uncouplers. Direct monitoring of proton movements in a pH-stat during phenol uptake indicates a phenol-proton symport. One proton is cotransported with every phenol molecule. Phenol competes with the uptake of sucrose and glycerol by cells grown on these substrates. Under such conditions the uptake of phenol seems to proceed through a different system, with lower affinity for phenol than in phenol-grown cells.     

Catabolism of L-tyrosine in Trichosporon cutaneum.

Protocatechuic acid was a catabolite in the degradation of L-tyrosine by Trichosporon cutaneum. Intact cells oxidized to completion various compounds proposed as intermediates in this conversion, but they did not readily oxidize catabolites of the homogentisate and homoprotocatechuate metabolic pathways, which are known to function in other organisms. Cell extracts converted tyrosine first to 4-hydroxycinnamic acid and then to 4-hydroxybenzaldehyde and 4-hydroxybenzoic acid. The proposed hydration product of 4-hydroxycinnamic acid, namely, beta-(4-hydroxyphenyl)-hydracrylic acid, was synthesized chemically, and its enzymatic degradation to 4-hydroxybenzaldehyde was shown to be dependent upon additions of adenosine triphosphate and coenzyme A. The hydroxylase that attacked 4-hydroxybenzoate showed a specific requirement for reduced nicotinamide adenine dinucleotide phosphate. Protocatechuate, the product of this reaction, was oxidized by cell extracts supplemented with reduced nicotinamide adenine dinucleotide or, less effectively, with reduced nicotinamide adenine dinucleotide phosphate, but these extracts contained no ring fission dioxygenase for protocatechuate. Evidence is presented that the principal hydroxylation product of protocatechuate was hydroxyquinol, the benzene nucleus of which was cleaved oxidatively to give maleylacetic acid.     

Catabolism of aromatic acids in Trichosporon cutaneum.

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.     

Degradation of 4-hydroxyphenylacetic acid by Trichosporon cutaneum.

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.     

Metabolism of phenol and resorcinol in Trichosporon cutaneum.

Trichosporon cutaneum was grown with phenol or resorcinol as the carbon source. The formation of beta-ketoadipate from phenol, catechol, and resorcinol was shown by a manometric method using antipyrine and also by its isolation and crystallization. Metabolism of phenol begins with o-hydroxylation. This is followed by ortho-ring fission, lactonization to muconolactone, and delactonization to beta-ketoadipate. No meta-ring fission could be demonstrated. Metabolism of resorcinol begins with o-hydroxylation to 1,2,4-benzenetriol, which undergoes ortho-ring fission yielding maleylacetate. Isolating this product leads to its decarboxylation and isomerization to trans-acetylacrylic acid. Maleylacetate is reduced by crude extracts to beta-ketoadipate with either reduced nicotinamide adenine dinucleotide or reduced nicotinamide adenine dinucleotide phosphate as a cosubstrate. The enzyme catalyzing this reaction was separated from catechol 1,2-oxygenase, phenol hydroxylase, and muconate lactonizing enzyme on a diethyl-aminoethyl-Sephadex A50 column. As a result it was purified some 50-fold, as was the muconate-lactonizing enzyme. Methyl-, fluoro-, and chlorophenols are converted to a varying extent by crude extracts and by purified enzymes. None of these derivatives is converted to maleylacetate, beta-ketoadipate, or their derivatives. Cells grown on resorcinol contain enzymes that participate in the degradation of phenol and vice versa.     

Enzymology of the beta-ketoadipate pathway in Trichosporon cutaneum.

Cell extracts were prepared from Trichosporon cutaneum grown with phenol or p-cresol, and activities were assayed for enzymes catalyzing conversion of these two carbon sources into 3-ketoadipate (beta-ketoadipate) and 3-keto-4-methyladipate, respectively. When activities of each enzyme were expressed as a ratio, the rate for methyl-substituted substrate being divided by that for the unsubstituted substrate, it was apparent that p-cresol-grown cells elaborated pairs of enzymes for hydroxylation, dioxygenation, and delactonization. One enzyme of each pair was more active against its methyl-substituted substrate, and the other was more active against its unsubstituted substrate. Column chromatography was used to separate two hydroxylase activities and also 1,2-dioxygenase activities; the catechol 1,2-dioxygenases were further purified to electrophoretic homogeneity. Extracts of phenol-grown cells contained only those enzymes in this group that were more active against unsubstituted substrates. In contrast, whether cells were grown with phenol or p-cresol, only one muconate cycloisomerase (lactonizing enzyme) was elaborated which was more active against 3-methyl-cis,cis-muconate than against cis,cis-muconate; in this respect it differed from a cycloisomerase of another strain of T. cutaneum which has been characterized. The cycloisomerase was purified from both phenol-grown and p-cresol-grown cells, and some characteristics were determined.     

Transport and hydrolysis of disaccharides by Trichosporon cutaneum.

Trichosporon cutaneum is shown to utilize six disaccharides, cellobiose, maltose, lactose, sucrose, melibiose, and trehalose. T. cutaneum can thus be counted with the rather restricted group of yeasts (11 to 12% of all investigated) which can utilize lactose and melibiose. The half-saturation constants for uptake were 10 +/- 3 mM sucrose or lactose and 5 +/- 1 mM maltose, which is of the same order of magnitude as those reported for Saccharomyces cerevisiae. Our results indicate that maltose shares a common transport system with sucrose and that there may be some interaction between the uptake systems for lactose, cellobiose, and glucose. Lactose, cellobiose, and melibiose are hydrolyzed by cell wall-bound glycosidase(s), suggesting hydrolysis before or in connection with uptake. In contrast, maltose, sucrose, and trehalose seem to be taken up as such. The uptake of sucrose and lactose is dependent on a proton gradient across the cell membrane. In contrast, there were no indications of the involvement of gradients of H+, K+, or Na+ in the uptake of maltose. The uptake of lactose is to a large extent inducible, as is the corresponding glycosidase. Also the glycosidases for cellobiose, trehalose, and melibiose are inducible. In contrast, the uptake of sucrose and maltose and the corresponding glycosidases is constitutive.     

Purification and properties of two lactose hydrolases from Trichosporon cutaneum

Two enzymes that hydrolysed lactose were purified essentially to homogeneity from cell extracts of the oleaginous yeast Trichosporon cutaneum. One enzyme of Mr 120,000 had properties typical of a beta-galactosidase (EC 3.2.1.23). It hydrolysed lactose, lactulose and nitrophenyl-beta-D-galactosides. The enzyme required K+ or Rb+ for activity, and other monovalent cations tested were not effective. Enzyme activity was abolished by EDTA and stimulated by Mg2+, Mn2+ and Ca2+. The beta-galactosidase was induced by lactose, galactose, lactulose and lactobionic acid. The other enzyme, a beta-glycosidase (EC 3.2.1.21) of Mr 52,000 showed no ionic requirements and it hydrolysed lactose, nitrophenyl-beta-D-galactosides, 4-nitrophenyl-beta-D-glucoside, cellobiose, laminaribiose, laminaritriose and sophorose, but not gentiobiose, 4-nitrophenyl-beta-D-mannoside or sucrose. This enzyme was induced by lactose, galactose and lactulose, and also by cellobiose.     

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

Trichosporon cutaneum degraded L-tryptophan by a reaction sequence that included L-kynurenine, anthranilate, 2,3-dihydroxybenzoate, catechol, and beta-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 18O, derived from either 18O2 or H2(18)O, into 2,3-dihydroxybenzoic acid.     

Induction of phenol-metabolizing enzymes in Trichosporon cutaneum

Some aspects of the induction of enzymes participating in the metabolism of phenol and resorcinol in Trichosporon cutaneum were studied using intact cells and cell-free preparations.Activities of phenol hydroxylase (1.14.13.7), catechol 1,2-oxygenase (1.13.11.1), cis,cis-muconate cyclase (5.5.1.-), delactonizing enzyme(s) and maleolylacetate reductase were 50–400 times higher in fully induced cells than in noninduced cells.In addition to phenol and resorcinol, also catechol, cresols and fluorophenols could induce phenol hydroxylase.The induction was severely inhibited by phenol concentrations higher than 1 mM. Using optimum inducer concentrations (0.01–0.10 mM), it took more than 8 h to obtain full induction, whether in proliferating or in nonproliferating cells.Phenol hydroxylase, catechol 1,2-oxygenase and cis,cis-muconate cyclase were induced simultaneously. The synthesis of the de-lactonizing activity was delayed in relation to these three preceeding enzymes of the pathway.High glucose concentration (over 15 mM) inhibited completely the induction of phenol oxidation by nonproliferating cells. It also inhibited phenol oxidation by pre-induced cells.Among the NADPH-generating enzymes, the activity of iso-citrate dehydrogenase was elevated in cells grown on phenol and resorcinol instead of glucose.     

Phenol utilization by filamentous yeast Trichosporon cutaneum

The investigated strain Trichosporon cutaneum shows well expressed capability for metabolizing high concentrations of phenol, up to 1 g/l, utilizing it as the sole carbon source for the growth and development of the population. The data reported, prove the good perspectives for its application in protecting the environment from phenol pollution. No data about modelling the process of cultivation of Trichosporon cutaneum in phenol media is available in scientific literature up to now. The mathematical model, reported here, consists of two nonlinear differential equations, describing cell growth and substrate consumption. The unknown parameters are estimated following the method of Hooke and Jeeves. A number of simulation investigations are carried out. They prove the adequacy of the model and its applicability in further studies on the processes of growth and phenol uptake of Trichosporon cutaneum.     

beta-Ketoadipate pathway in Trichosporon cutaneum modified for methyl-substituted metabolites.

Trichosporon cutaneum, when grown with p-cresol, catalyzed intradiol fission of the benzene nucleus of 4-methylcatechol before the complete catabolism of these two substrates. Steps in their conversion to pyruvate and acetyl coenzyme A were investigated by using cell extracts, and some properties of various new microbial catabolites are also described. These included (-)-2,5-dihydro-3-methyl-5-oxofuran-2-acetic acid (beta-methylmuconolactone) and (-)-3-keto-4-methyladipic acid and its coenzyme A ester; the latter was degraded by an enzymatic reaction sequence that included the coenzyme A esters of methylsuccinic, itaconic, and citramalic acids. A notable feature of this sequence is the formation of beta-methylmuconolactone which can be readily metabolized, in contrast to the analogous reaction in bacteria that gives the "dead-end" compound gamma-methylmuconolactone; this compound cannot be enzymatically degraded and so renders the beta-ketoadipate pathway unavailable for methyl-substituted bacterial sources of carbon that are catabolized by way of 4-methylcatechol.     

关于皮状丝孢酵母（Trichosporon cutaneum）ST851破壁条件的研究

用脱壁酶（纤维素酶、半纤维素酶和蜗牛酶）作用于皮状丝孢酵母ＳＴ８５１制备原生质体。本文就酶的种类、酶液浓度、酶作用时间与温度、酶混合时间、混酶作用效果、酵母细胞的不同生长期诸因素对ＳＴ８５１原生质体形成的影响,进行了较为详尽的探讨。结果表明采用混酶作用（即先用纤维素酶和半纤维素酶预处理后再用蜗牛酶作用）是较理想的破壁条件,在ｐＨ５．８、３７℃条件下作用３．５－４ｈｒ,破壁率最高可达９５－９８％。     

Induction of high-affinity phenol uptake in glycerol-grown Trichosporon cutaneum.

Two uptake systems for phenol are identified in Trichosporon cutaneum. One is an inducible, high-affinity system, sensitive to protonophores. It is induced coordinately with phenol hydroxylase but can operate independently of phenol metabolism. The other is a constitutive, low-affinity system with different specificity and different pH optimum. It is not sensitive to protonophores.