Effect of Various Compounds on Isocitrate Lyase Formation in Candida tropicalist

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1 Agric. Biol. Chem., 41 (3), 503 `508, 1977 Effect of Various Compounds on Isocitrate Lyase Formation in Candida tropicalist Shigeyasu NABESHIMA, Masayoshi MISHINA, Atsuo TANAKA and Saburo FUKUI Laboratory of Industrial Biochemistry, Department of Industrial Chemistry, Faculty of Engineering, Kyoto University, Kyoto Received September 6, 1976 The synthesis of isocitrate lyase in Candida tropicalis, the growth of which was stimulated by exogenously added biotin, was released from repression by glucose under biotin-deficient conditions. Biotin deficiency reduced remarkably the levels of biotin-enzymes, pyruvate carboxylase and acetyl-coa carboxylase, in the glucose-utilizing cells of this yeast. A marked increase in intracellular level of pyruvate was observed in the biotin-deficient cells. Acetyl- CoA-donating compounds, such as pyruvate, acetate and alkanes, stimulated the formation of isocitrate lyase in the yeast regardless of the presence or absence of biotin. On the other hand, malate and succinate did not affect the enzyme synthesis. The isocitrate lyase synthesis under biotin-sufficient conditions was repressed by not only glucose but also glucosamine and 2-deoxyglucose. This repression by glucose was not eliminated by camp. The stimulat ed synthesis of isocitrate lyase under biotin-deficient conditions was also observed in C. albicans and C. guilliermondii growing on glucose. In the preceding paper of this series,1) we reported that the formation of isocitrate lyase in glucose-grown Candida tropicalis was af fected remarkably by the biotin concentration in the medium. That is, biotin-deficiency per mitted the synthesis of isocitrate lyase even when the yeast was grown on glucose, while the formation of the enzyme was repressed by glucose under the biotin-sufficient conditions. Such an effect of biotin on the regulation of isocitrate lyase has never been demonstrated. Many compounds have been reported to regulate the synthesis of isocitrate lyase: C3-compounds in Escherichia coli2) ; C4-compounds in Acinetobacter3) and in Neurospora crassa4); glucose or glucose-6-phosphate, rather than the catabolites of glucose, in Saccharo myces cerevisiae5) ; and thiamine in Candida lipolytica.6) However, the compound which regulate the synthesis of isocitrate lyase in hydrocarbon-utilizable yeasts has not been studied extensively, except for biotin1) and thiamine.6) This paper deals with the effect of several õ Studies on the Physiology and Metabolism of Hydrocarbon-utilizing Microorganisms. Part XVI. compounds, in connection with that of biotin, on the formation of isocitrate lyase in hydrocarbon-utilizable Candida tropicalis. MATERIALS AND METHODS Cultivation of yeasts. Yeast strains, media and the methods of cultivation were the same as those described in the previous papers.1.7) Assay methods. Cell-free extracts of the yeast cells were prepared by the same method as described in the preceding paper.1) The assay method of isocitrate lyase activity was reported previously.7) The activity of pyruvate carboxylase was measured by the method of Young et al.8) with a slight modification. The reaction mixture was composed of 100 ƒêmoles of Tris-HC1 buffer (ph 8.0), 10 ƒêmoles of MgCl2, 10 ƒêmoles of Na-pyruvate, 0.2 unit of malate dehydrogenase (Sigma), 0.2 ƒêmole of NADH, 15 ƒêmoles of KHCO3, 0.15 ƒêmole of acetyl-coa, 5 ƒêmoles of ATP and enzyme source in a final volume of 1.5 ml. The reaction was carried out at 30 Ž, and the decrease of NADH was followed by measuring absorbance at 340 nm. The reaction mixture without ATP was employed in the control run. The activity of acetyl-coa carboxylase was determined by the H14CO3-fixation method described by Mishina et al.9) The unit of the enzyme activities is defined as one nmole of the substrates converted per min. Protein was assayed by the method of Lowry et al.10)

2 504 S. NABESHIMA, M. MISHINA, A. TANAKA and S. FUKUI Metabolic levels of pyruvate and oxalacetate were determined in neutralized perchloric acid extracts of the cell suspensions in accordance with the procedures described by Bucher et al.11) and Hohorst and Reim,12) respectively. RESULTS Effect of biotin on isocitrate lyase formation in various yeasts As described in the preceding paper,'' the synthesis of isocitrate lyase in glucose-grown cells of C. tropicalis was markedly enhanced under biotin-deficient conditions. Such a phenomenon, however, was not observed in C. lipolytica. In the cases of C. albicans and C. guilliermondii, similar enhancement in the level of isocitrate lyase under biotin-deficient conditions was also observed (Table I). These results indicate that the effect of biotin on the isocitrate lyase formation is not specific for C. tropicalis, but rather common to various strains of yeasts. On the other hand, the enzyme level in C. intermedia, as well as C. lipolytica, was low irrespective of the presence or absence of biotin added exogenously (Table I). TABLE 1. EFFECT OF BIOTIN ON ISOCITRATE LYASE FORMATION IN DIFFERENT STRAINS OF Candida The yeasts were cultivated glucose medium. YEASTS GROWN ON GLUCOSE for 12 hr in a synthetic Effect of biotin on isocitrate lyase formation in Candida tropicalis growing on various carbon sources As shown in Table II, the cells grown on alkane and acetate contained high levels of isocitrate lyase regardless of the presence or absence of biotin. On the other hand, exo genous addition of biotin reduced the enzyme synthesis in the cells grown on fructose and mannose, as in the cells grown on glucose. These results suggest that the change in the isocitrate lyase level caused by biotin is closely related to the metabolism of sugars in the yeast. TABLE II. EFFECT OF BIOTIN ON ISOCITRATE LYASE FORMATION IN Candida tropicalis GROWN ON DIFFERENT CARBON SOURCES The yeast was cultivated for 12 hr in synthetic media. Effect of biotin on levels of isocitrate lyase, pyruvate carboxylase and acetyl-coa carboxylase As mentioned above, the effect of biotin on the isocitrate lyase synthesis is related to the metabolism of sugars. Biotin is the coenzyme for pyruvate carboxylase and acetyl-coa carboxylase, which play important roles in the sugar metabolism. Hence, the levels of these enzymes were compared in the presence and absence of biotin in glucose-grown C. lipolytica and C. tropicalis. In C. tropicalis, biotin-deficiency resulted in decreased levels of both biotin-enzymes, pyru vate carboxylase and acetyl-coa carboxylase, and an enhanced level of isocitrate lyase (Table III). A reduced level of pyruvate car boxylase under biotin-deficiency was also demonstrated in S. cerevisiae.13) Although the levels of both carboxylases were also reduc ed by biotin-deficiency in C. lipolytica, the

3 Isocitrate Lyase of Candida tropicalis 505 TABLE III. EFFECT OF BIOTIN ON FORMATION OF PYRUVATE CARBOXYLASE, ISOCITRATE LYASE AND ACETYL-COA CARBOXYLASE in Candida lipolytica AND Candida tropicalis GROWN ON GLUCOSE The yeasts were cultivated for 12 hr in a synthetic glucose medium. TABLE IV. EFFECT OF ORGANIC ACIDS ON ISOCITRATE LYASE FORMATION IN Candida tropicalis The yeast was cultivated in semisynthetic media. levels of these enzymes were significantly higher in C. lipolytica than in C. tropicalis (Table III). The isocitrate lyase level in C. lipolytica was the same regardless of the pre sence or absence of biotin. Effect of metabolic intermediates on isocitrate lyase formation To obtain further information concerning the regulation of the isocitrate lyase synthesis in C. tropicalis, the effect of several compounds on the enzyme formation was inves tigated. When glucose-grown cells of C. tropicalis were transferred into the synthetic media containing different carboxylic acids as the sole carbon source and cultivated, the formation of isocitrate lyase was not observed on malate or succinate, while lactate, pyruvate and citrate stimulated the enzyme synthesis (Fig. 1). The stimulatory effect of pyruvate and citrate (lactate not tested) was also confirmed when the yeast was grown in the semisynthetic media containing these acids as the sole carbon source (Table N). Malate and succinate supple mented to the synthetic acetate medium did not exhibit any inhibitory effect on the enzyme FIG. 1. Isocitrate Lyase Formation in Candida tropicalis Growing on Carboxylic Acids. Cells grown on glucose for 12 hr were diluted and transferred into synthetic media containing the following carbon sources: (A) glucose (1.65%); (B) pyruvate (0.1 M); (C) lactate (0.1 M); (D) citrate (0.1 M); (E) succinate (0.1 M), and (F) malate (0.1 M). Cultivation was aerobically carried out at 30 Ž for 6 hr. FIG. 2. Effect of Carboxylic Acids on Isocitrate Lyase Formation in Candida tropicalis Growing on Acetate. The yeast was aerobically cultivated in a synthetic acetate medium and the following compounds were added at 10 hr (arrow): (A) none; (B) pyruvate (0.1 M); (C) succinate (0.1 M), and (D) malate (0.1 M).

4 506 S. NABESH1MA, M. MISFIINA, A. TANAKA and S. FUKUI formation, whereas pyruvate remarkably stimu lated the enzyme synthesis (Fig. 2). The effect of these acids on the isocitrate lyase synthesis in C. lipolytica was essentially the same as that in C. tropicalis. Effect of biotin on intracellular levels of pyruvate and oxalacetate Table V shows the metabolic levels of pyru vate and oxalacetate in C. tropicalis cells grown on glucose in the presence and absence of biotin. The level of pyruvate was higher in the biotin-deficient cells than in the biotin sufficient cells, while that of oxalacetate was reverse, indicating the accumulation of pyru vate at the low level of pyruvate carboxylase under biotin-deficient conditions. TABLE V. EFFECT OF BIOTIN ON METABOLIC LEVELS OF PYRUVATE AND OXALACETATE IN Candida tropicalis GROWN ON GLUCOSE Pyruvate and oxalacetate were extracted by perchloric acid from 100 mg of dry cells, and assayed with lactate dehydrogenase and malate dehydrogenase, respectively.11.12) Effect of camp on isocitrate lyase formation In spite of the inductive nature of the iso- citrate lyase synthesis in C. tropicalis by acetyl CoA-donating substrates, the enzyme for mation was completely repressed by glucose under biotin-sufficient conditions. This repres sion by glucose was not eliminated by camp or its dibutyryl ester under the conditions em ployed (Fig. 3). Effect of glucose analogs on isocitrate lyase formation Witt et al.5) described that not only glucose but also glucosamine and 2-deoxyglucose repressed the synthesis of isocitrate lyase in S. cerevisiae, suggesting that the enzyme synthe sis would be regulated by glucose itself or glucose-6-phosphate rather than its catabolites, or the ratio of ATP to ADP. The formation of isocitrate lyase in C. tropicalis and C. lipoly tica growing on acetate was also inhibited by 2-deoxyglucose and glucosamine as well as glucose (Fig. 4). Since 2-deoxyglucose did not serve as growth substrate for C. tropicalis or C. lipolytica, these sugars, not being altered or after phosphorylated, are supposed to affect the enzyme synthesis in these yeasts. However, the effect of the sugars on the regulation of isocitrate lyase may be indirect, since biotin deficiency permitted the synthesis of the enzyme in glucose-assimilating cells of C. tropicalis. FIG. 3. Effects of camp on Isocitrate Lyase Formation and Growth of Candida tropicalis. To the 10-hr cultures aerobically growing in a synthetic acetate medium was added none (A and B), camp (2 mm) (C) or dibutyryl-camp (0.5 mm) (D), and then glucose (0.5 %) after incubation for 30 min (B, C and D). Cultivation was further continued for 5.5 hr. FIG. 4. Effect of Glucose and Its Analogs on Iso citrate Lyase Formation in Candida tropicalis and Candida lipolytica Growing on Acetate. The yeasts were aerobically cultivated in a synthetic acetate medium and the following compounds were added at 10 hr (arrow): (A) none; (B) glucose (0.5%); (C) 2-deoxyglucose (0.5%), and (D) glucosamine (0.5%).

Isocitrate Lyase of Candida tropicalis 507 DISCUSSION The stimulated synthesis of isocitrate lyase under biotin-deficient conditions was observed in glucose-grown cells of not only C.

5 Isocitrate Lyase of Candida tropicalis 507 DISCUSSION The stimulated synthesis of isocitrate lyase under biotin-deficient conditions was observed in glucose-grown cells of not only C. tropicalis but also C. albicans and C. guilliermondii. However, the enzyme level in C. lipolytica and C. intermedia was low regardless of the pre sence or absence of exogenous biotin when these yeasts were grown on glucose (Table 1). This discrepancy would be explained by the difference in biotin-synthesizing ability among the yeasts tested. The growth of these yeasts, except for C. lipolytica, was stimulated by exogenous biotin. Especially, that of C. intermedia was absolutely dependent on the presence of biotin. Such an effect of biotin on the isocitrate lyase formation in C. tropicalis was observed only when the yeast was grown on sugars (Table II). Common metabolite(s) of these sugars tested may be the regulating factor(s) in the formation of the enzyme. Biotin-deficiency reduced the levels of the biotin-enzymes, pyruvate carboxylase and acetyl-coa carboxylase in C, lipolytica and C. tropicalis. However, the level of pyruvate carboxylase in C. lipolytica was significantly higher than that in C. tropicalis (Table III). This level of the enzyme seemed to be sufficient to supply oxalacetate in C. lipolytica. Pyru vate carboxylase, as well as isocitrate lyase, has an anaplerotic role where oxalacetate should be supplied.14) Thus, the carboxylase is an important enzyme in glycolytic system. When biotin is deficient in C. tropicalis metabolizing glucose, the synthesis of pyruvate carboxylase is reduced, and subsequently the supply of oxalacetate will decrease. Under these cir cumstances, an alternative pathway, e.g. gly oxylate cycle, must serve to produce oxal acetate or malate. The anaplerotic role of isocitrate lyase has been reported in Arthrobacter atrocyaneus growing on glucose, which lacks the ability to convert pyruvate to C4- carboxylic acid directly.15 The following two mechanisms will be pre sumed for the stimulated synthesis of iso citrate lyase in glucose-grown C. tropicalis under biotin-deficient conditions. (1) Induc tion of the enzyme synthesis by pyruvate or its metabolite, which accumulates under a decreas ed level of pyruvate carboxylase induction by C,-compounds,2) (2) Release of the re pression of the enzyme synthesis by limited supply of oxalacetate or malate under a decreased level of the carboxylase-----repression by C4-compounds.3,4) The results shown in Fig. 1 and Table IV indicate that the synthesis of isocitrate lyase is regulated by the C3-compound induction mechanism rather than the C4-compound repression mechanism. The most probable inducer in the isocitrate lyase synthesis might be acetyl-coa, the common metabolite of pyruvate, citrate (cleaved by citrate lyase), acetate and n-alkane, which were the substrates stimulating the enzyme synthesis. Accumula tion of a high level of pyruvate under biotin deficient conditions (Table V) is consistent with the concept that pyruvate or its metabolite (probably acetyl-coa) induces the synthesis of isocitrate lyase in C. tropicalis. In spite of the fact that acetyl-coa-donating substrates stimulated the isocitrate lyase synthesis in C. tropicalis, sugars, such as glucose, fructose, mannose, glucosamine and 2-deoxyglucose, inhibited the enzyme synthesis under biotin-sufficient conditions irrespective of the presence or absence of acetyl-coa-donating substrates, such as acetate and n-alkane (Table II and Fig. 4). Inhibition of acetate or alkane uptake by glucose is not plausible, since the morphological change in C. tropicalis due to alkane assimilation was observed in the medium containing glucose and alkane as carbon sources."' On the contrary, the sugars, added as the sole carbon source, permitted the synthe sis of isocitrate lyase under biotin-deficient conditions (Table II). These facts suggest that the role of sugars themselves or phosphorylated sugars in the regulation of the isocitrate layse synthesis would be indirect, probably through the induction of the synthesis of the biotin enzyme, pyruvate carboxylase. This type of regulation might overcome the inductive synthesis of isocitrate lyase in the yeasts. Glucose repression of the isocitrate lyase

6 508 S. NABESHIMA, M. MISHINA, A. TANAKA and S. Fukui synthesis in E. coli is demonstrated not to be released by camp17) unlike the so-called cata bolite repression described in the syntheses of the enzyme coded in lac operon of E. coli18) The repression observed in C. tropicalis might be similar to that of an unusual nature reported in E. coli, since the effect of glucose on the yeast was not eliminated by camp (Fig. 3). The synthesis of catalase in yeasts is also susceptible to glucose repression without the eliminating effect of camp,19,20) REFERENCES 1) S. Nabeshima, A. Tanaka and S. Fukui, Agric. Biol. Chem., 41, 281 (1977). 2) H. L. Kornberg, Biochem. J., 99, 1 (1966). 3) N. J. Herman and E. J. Bell, Can. J. Microbial., 9 16, 769 (1970). 4) R. E. Beever, J. Gen. Microbiol., 86, 197 (1975). 5) I. Witt, R. Kronau and H. Holzer, Biochem. Biophys. Acta, 118, 522 (1966). 6) I. T. Ermakova and T. V. Finogenova, Mikro biologiya, 40, 223 (1971). 7) S. Nabeshima, A. Tanaka and S. Fukui, Agric. Biol. Chem., 41, 275 (1977). 8) M.R. Young, B. Tolbert and M.F. Utter, Methods Enzymol., 13, 250 (1969). 9) M. Mishina, T. Kamiryo, A. Tanaka, S. Fukui and S. Numa, Eur. J. Biochem., 71, 295 (1976). 10) O. H. Lowry, N. J. Rosebrough, A. L. Farr and R. J. Randall, J. Biol. Chem., 193, 265 (1951). 11) T. Biicher, R. Czok, W. Lamprecht and E. Latzko, "Methods of Enzymatic Analysis," ed. by H. U. Bergmeyer, Verlag Chemie, Weinheim, 1963, p ) H. J. Hohorst and M. Reim, "Methods of Enzy matic Analysis," ed by H. U. Bergmeyer, Verlag Chemie, Weinheim, 1963, p ) J.J. Cazzulo, L.M. Claisse and A.O.M. Stoppani, J. Bacteriol., 96, 623 (1968). 14) H. L. Kornberg, "Essays in Biochemistry," Vol.2, ed. by P. N. Cambell and G. D. Greville, Acade mic Press, New York, 1966, p ) P. J. Wolfson and T. A. Krulwich, J. Bacteriol., 112,356 (1972). 16) M. Hirai, S. Shimizu, Y. Teranishi, A. Tanaka and S. Fukui, Agric. Biol. Chem., 36, 2335 (1972). 17) Y. Takahashi, J. Biochem., 78, 1097 (1975). 18) R. L. Perlman and I. Pastan, J. Biol. Chem., 243, 5420 (1968). 19) Y. Teranishi, S. Kawamoto, A. Tanaka, M. Osumi and S. Fukui, Agric. Biol. Chem., 38, 1221 (1974). 20) S. Yasuhara, S. Kawamoto, A. Tanaka, M. Osumi and S. Fukui, ibid., 40, 1771 (1976).

unlike the wild-type strain, cannot incorporate exogenous fatty of a second long-chain acyl-coa synthetase which occurs in the

unlike the wild-type strain, cannot incorporate exogenous fatty of a second long-chain acyl-coa synthetase which occurs in the Proc. Nati. Acad. Sci. USA Vol. 76, No. 9, pp. 4390-4394, September 1979 Biochemistry Involvement of long-chain acyl coenzyme A for lipid synthesis in repression of acetyl-coenzyme A carboxylase in Candida