JOURNAL

OF

BACTERIOLOGY, OCt. 1990, p. 6145-6147

Vol. 172, No. 10

0021-9193/90/106145-03$02.00/0 Copyright © 1990, American Society for Microbiology

Regulation of Thiamine Biosynthesis in Saccharomyces cerevisiae YUKO

KAWASAKI,'*

Department

KAZUTO NOSAKA,1 YOSHINOBU KANEKO,2 HIROSHI NISHIMURA.' AND AKIO IWASHIMA'

of Biochemistry,

Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto 602,' and Institute for Fermentation, Osaka, Yodogawa-ku, Osaka 532,2 Japan Received 18 January 1990/Accepted 13 July 1990

A pho6 mutant of Saccharomyces cerevisiae, lacking a regulatory gene for the synthesis of periplasmic thiamine-repressible acid phosphatase activity, was found to be auxotrophic for thiamine. The activities of four enzymes involved in the synthesis of thiamine monophosphate were hardly detectable in the crude extract from the pho6 mutant. On the other hand, the activities of these enzymes and thiamine-repressible acid phosphatase in a wild-type strain of S. cerevisiae, H42, decreased with the increase in the concentration of thiamine in yeast cells. These results suggest that thiamine synthesis in S. cerevisiae is subject to a positive regulatory gene, PHO6, whereas it is controlled negatively by the intracellular thiamine level.

Two species of acid phosphatase present in the periplasmic space of Saccharomyces cerevisiae are repressible by Pi and thiamine, respectively (14, 16). The thiamine-repressible acid phosphatase (T-rAPase) is coded by the PH03 gene (16) and requires two additional complementary genes, PH06 and PHO7, for its synthesis (15). During the study on a possible role for T-rAPase we found that the pho6 mutant is auxotrophic for thiamine. In this communication we present evidence indicating that the PH06 gene is involved in the regulation of the synthesis not only of T-rAPase but also of enzymes synthesizing thiamine monophosphate from

Assay of overall thiamine-synthesizing activity from hydroxymethylpyrimidine and hydroxyethylthiazole. The reaction mixture contained 0.05 M Tris hydrochloride buffer (pH 7.5), 10 ,uM hydroxymethylpyrimidine, 10 p.M hydroxyethylthiazole, 10 mM ATP, 20 mM MgCl2, and the crude extract (2 mg of protein) in a final volume of 5 ml. After incubation at 37°C for 30 min, the reaction was stopped by adding 2.5 ml of 10% metaphosphoric acid and then centrifuged at 2,000 x g for 10 min to remove denatured protein. After the pH of the supernatant was adjusted to 4.5 with 4 M sodium acetate, thiamine phosphates were hydrolyzed to free thiamine by incubation for 1 h at 45°C after 0.5 ml of 1% takadiastase was added. Thiamine was determined fluorometrically by the thiochrome method (5). The enzyme activity is expressed as nanomoles of thiamine formed per milligram of enzyme protein. Hydroxymethylpyrimidine kinase assay. The reaction mixture contained 0.05 M Tris hydrochloride buffer (pH 7.5), 10 puM [3H]hydroxymethylpyrimidine (6.5 Ci/mol) prepared as previously described (9), 10 mM ATP, 10 mM MgCl2, and the crude extract (50 to 70 pLg of protein) in a final volume of 0.5 ml. After incubation at 37°C for 30 min, the reaction was terminated by heating at 90°C for 5 min, followed by centrifugation at 2,000 x g for 10 min to remove denatured protein. Samples (20 [lI) of deproteinized reaction mixture were chromatographed (ascending) with an authentic hydroxymethylpyrimidine monophosphate standard on Toyo filter paper (no. 50, 2 by 40 cm) with 2-propanol-0.5 M sodium acetate buffer (pH 4.5)-water (65:15:20, by volume) as the solvent system. After development, the UV (254 nm)-absorbing spots of hydroxymethylpyrimidine monophosphate (Rf, 0.33) were cut out, dried, and put into 10 ml of scintillation fluid. The radioactivity was measured in a Packard Tri-Carb liquid scintillation spectrometer. The enzyme activity is expressed as nanomoles of hydroxymethylpyrimidine monophosphate formed per milligram of enzyme protein. Phosphomethylpyrimidine kinase assay. The reaction mixture contained 0.05 M Tris hydrochloride buffer (pH 7.5), 10 ,uM hydroxymethylpyrimidine monophosphate, 10 mM ATP, 20 mM MgCl2, and the crude extract (0.3 mg of protein) in a final volume of 5 ml. After incubation at 37°C for 30 min, the reaction was terminated by heating at 90°C for 5 min, followed by centrifugation at 2,000 x g for 10 min to remove denatured protein. The amount of hydroxymeth-

2-methyl-4-amino-5-hydroxymethylpyrimidine(hydroxymethylpyrimidine) and 4-methyl-5-p-hydroxyethylthiazole (hydroxyethylthiazole) in S. cerevisiae (Fig. 1) (2, 3, 12, 13). We also report that the activities of these enzymes are coordinately repressed by exogenous thiamine, suggesting that both positive and negative regulatory mechanisms are involved in thiamine biosynthesis in S. cerevisiae. S. cerevisiae was grown at 30°C in Wickerham synthetic medium (17) with or without thiamine. After harvesting, yeast cells were washed once with cold water and then suspended in 0.05 M potassium phosphate buffer (pH 7.5) containing 1 mM 2-mercaptoethanol and 1 mM EDTA. The cell suspensions were sonicated at 9 KHz for 20 min below 4°C. The supernatant after centrifugation at 28,000 x g for 30 min was used immediately as a crude extract, or it was stored for several weeks at -80°C without any loss of enzyme activity. Measurement of intracellular contents of thiamine and thiamine phosphates was carried out by highpressure liquid chromatography as previously reported (10). T-rAPase activity with p-nitrophenyl phosphate as a substrate was determined from the amount of p-nitrophenol produced as previously described (14). Protein was determined by the method of Lowry et al. (8). Hydroxymethylpyrimidine and hydroxyethylthiazole were kind gifts from the late S. Yurugi, Takeda Pharmaceutical Industries Ltd. (Osaka, Japan). Hydroxymethylpyrimidine monophosphate and pyrophosphate were synthesized as reported previously (1). Hydroxyethylthiazole monophosphate was prepared by the sulfite cleavage of thiamine monophosphate as reported previously (6). All other chemicals were purchased from commercial suppliers. *

Corresponding author. 6145

6146

J. BACTERIOL.

NOTES

NH2

N

NH2

ATP

yCH20H

ATP

NH2

N%CH:O®Y(II)

Hfr

H3C-N'7

H3C

ADP

OMP-P

ADP

OMP-PP

ppi ATP I AkHII HS

R~

H,

HiCH20H

ADP

H3CN

H,3:H2CH20-(

H,C19

HH2-

Hz

HXCH20-

Thiamin nonophosphate Th Th-P FIG. 1. Pathway of thiamine monophosphate synthesis from hydroxymethylpyrimidine and hydroxyethylthiazole in S. cerevisiae. (I) Hydroxymethylpyrimidine kinase, (II) phosphomethylpyrimidine kinase, (III) hydroxyethylthiazole kinase, (IV) thiamine-phosphate pyrophosphorylase. OMP, Hydroxymethylpyrimidine; OMP-P, hydroxymethylpyrimidine monophosphate; OMP-PP, hydroxymethylpyrimidine pyrophosphate; Th, hydroxyethylthiazole; Th-P, hydroxyethylthiazole monophosphate.

ylpyrimidine pyrophosphate formed was then determined as follows. A 1-ml sample of the supernatant with or without 1 ,uM hydroxymethylpyrimidine pyrophosphate as a standard sample was added to the second reaction mixture, containing 0.05 M Tris hydrochloride buffer (pH 7.5), 0.1 ml of a sonic extract (1.5 mg of protein) of commercial baker's yeast (Oriental Yeast Co.) in which thiamine-phosphate pyrophosphorylase was fully active but phosphomethylpyrimidine kinase was completely inactivated by the previous heat treatment at 55°C (7) for 30 min, and 10 puM hydroxyethylthiazole monophosphate in a final volume of 5 ml. After incubation at 37°C for 30 min, the thiamine formed was determined as described above. The amount of hydroxymethylpyrimidine pyrophosphate formed at the first reaction was calculated in proportion to the amount of thiamine formed from a standard sample. The enzyme activity is expressed as nanomoles of hydroxymethylpyrimidine pyrophosphate equivalent to thiamine formed per milligram of enzyme protein. Hydroxyethylthiazole kinase assay. The reaction mixture contained 0.05 M Tris hydrochloride buffer (pH 7.5), 10 FM hydroxyethyl[2-'4C]thiazole (24.1 Ci/mol) prepared from thiamine[thiazole-2-14C]hydrochloride (Amersham Corp.) as previously described (4), 10 mM ATP, 10 mM MgCl2, and the crude extract (50 to 70 ,ug of protein) in a final volume of 0.5 ml. The incubation and the analysis of hydroxyethylthiazole monophosphate formed were carried out by the same procedure for hydroxymethylpyrimidine kinase assay with an authentic standard (Rf, 0.72). The enzyme activity is expressed as nanomoles of hydroxyethylthiazole monophosphate formed per milligram of enzyme protein. Thiamine-phosphate pyrophosphorylase assay. The reaction mixture contained 0.05 M Tris hydrochloride buffer (pH 7.5), 10 ,uM hydroxymethylpyrimidine pyrophosphate, 10 ,uM hydroxyethylthiazole monophosphate, 20 mM MgCl2, and the crude extract (0.2 mg of protein) in a final volume of 5 ml. The amount of thiamine formed after incubation at 37°C for 30 min was determined as described above. The enzyme activity is expressed as nanomoles of thiamine formed per milligram of enzyme protein. The pho6 mutant, 058-M5 (MATa pho6-1 gal4), isolated from a haploid strain of S. cerevisiae, H42 (MATa gal4) (15), was kindly supplied by Y. Oshima (Department of Fermentation Technology, Osaka University, Osaka, Japan). The mutant did not grow in Wickerham minimal medium in the absence of thiamine but grew in the presence of 0.02 ,uM thiamine as well as the parent strain does without thiamine.

We therefore analyzed the segregation of the phenotype of the thiamine auxotroph to determine whether the defect of thiamine biosynthesis was caused by the mutation of PH06 gene. The pho6 mutants with three different alleles were crossed with the wild-type strain, and the resultant diploids were subjected to tetrad analysis. The defect of the TrAPase and the thiamine auxotroph were cosegregated in all 136 tetrads, showing 2+ :2- segregation. These findings indicate that the pho6 mutant is impaired in the de novo synthesis of thiamine in addition to the synthesis of periplasmic T-rAPase encoded by PHO3. Thus, we concluded that the syntheses of T-rAPase and of enzyme(s) involved in thiamine biosynthesis are regulated by the same protein encoded by PH06 gene. The biosynthesis of thiamine in S. cerevisiae involves the independent formation of the pyrimidine and thiazole moiety of thiamine and their subsequent condensation to thiamine monophosphate (Fig. 1). Although the biosynthetic pathway for the formation of hydroxymethylpyrimidine and hydroxyethylthiazole has not yet been completely elucidated, four reactions leading to the synthesis of thiamine monophosphate from the two moieties have been demonstrated in S. cerevisiae (2, 3, 12, 13) and Escherichia coli (11). The overall enzyme activity for thiamine synthesis from hydroxymethylpyrimidine and hydroxyethylthiazole in the presence of ATP and Mg2+ was not detectable in the crude extract from the pho6 mutant cells (Table 1). Therefore, the activities of four enzymes involved in the formation of thiamine monophosphate from hydroxymethylpyrimidine and hydroxyethylthiazole, namely, hydroxymethylpyrimidine kinase (EC TABLE 1. Enzyme activities involved in thiamine monophosphate synthesis from hydroxymethylpyrimidine and hydroxyethylthiazole in a wild strain, H42, and a pho6 mutant Enzyme or reaction

Overall reaction

Hydroxymethylpyrimidine kinase Phosphomethylpyrimidine kinase Hydroxyethylthiazole kinase Thiamine-phosphate pyrophosphorylase "

Sp act" (nmol/mg of protein per 30 min) in: H42

pho6b

2.61 7.77 38.01 45.40 11.29

0 0 0 1.27 0.12

Each value is the mean of two experiments. The pho6 mutant was cultured in Wickerham synthetic medium supplemented with 10-8 M thiamine. b

VOL. 172, 1990

NOTES

.0-4.

E

50

0.5

LU~~~~~~~~~~

thiamine in the crude extract of pho6 mutant was approximately 4% of that of the parent cells, and the phosphatase activities of yeast cells grown with 5 x 10-8 and 2 x 10-7 M thiamine were 55 and 5% of that from control cells, respectively. Although these results suggest that there is a regulatory phosphatase for hydrolysis of thiamine monophosphate, it is unknown whether the enzyme is specific for thiamine monophosphate. We finally concluded that the expression of structural genes for T-rAPase and enzymes involved in thiamine biosynthesis in S. cerevisiae is regulated positively by PHO6, whereas the expression of these genes is controlled negatively by the thiamine (probably thiamine pyrophosphate) level in S. cerevisiae. LITERATURE CITED 1. Brown, G. M. 1970. Preparation of the mono- and pyrophosphate esters of 2-methyl-4-amino-5-hydroxymethylpyrimidine for thiamine biosynthesis. Methods Enzymol. 18A:162-163. 2. Camiener, G. W., and G. M. Brown. 1959. The biosynthesis of

3. 02

5

20

Thiamin Conc.

6147

50

(xlO8M)

FIG. 2. Activities of thiamine-synthesizing enzymes and TrAPase and intracellular concentrations of total thiamine and thiamine pyrophosphate in wild strain H42 cells grown in media supplemented with various concentration of thiamine. Symbols: 0, hydroxymethylpyrimidine kinase; 0, phosphomethylpyrimidine kinase; A, hydroxyethylthiazole kinase; A, thiamine-phosphate pyrophosphorylase; OJ, T-rAPase; O, intracellular total thiamine; *, thiamine pyrophosphate. Each value is the mean of two experi-

4.

5. 6. 7.

ments.

2.7.1.49), phosphomethylpyrimidine kinase (EC 2.7.4.7),

8.

hydroxyethylthiazole kinase (EC 2.7.1.50), and thiamine-

phosphate pyrophosphorylase (EC 2.5.1.3), were determined. All of the four enzyme activities were markedly low in the extract from pho6 mutant cells, which was less than 3% of the corresponding activities in the parent cells (Table 1). The activity of T-rAPase of the pho6 mutant cells was also undetectable (data not shown). These results suggest that the PH06 gene positively regulates not only the expression of PH03 but the expression of at least four genes coding for the enzymes synthesizing thiamine monophosphate indicated above. However, the physiological significance of the presence of a common positive regulatory mechanism for periplasmic T-rAPase and intracellular thiamin-synthesizing enzymes is unknown at this stage. In this connection, we further determined the activities of the four enzymes described above in the crude extract from wild-type strain H42 cells grown in minimal medium supplemented with various concentrations of thiamine. The activities of the four enzymes and T-rAPase were decreased in correlation with the increase in the concentration of intracellular total thiamine and thiamine pyrophosphate in yeast cells (Fig. 2). At a concentration of 0.5,uM, exogenous thiamine caused almost complete depression of all of the enzyme activities tested. These results suggest that there exists a common negative regulatory gene(s) for thiamine monophosphate synthesis and T-rAPase that functions to control these enzyme activities depending on the intracellular thiamine level. From a preliminary experiment, the activity of a phosphatase involved in the conversion of thiamine monophosphate to

9.

10. 11.

12. 13.

14.

15. 16. 17.

thiamine and thiamine phosphates by extracts of baker's yeast. J. Am. Chem. Soc. 81:3800. Camiener, G. W., and G. M. Brown. 1960. The biosynthesis of thiamine. II. Fractionation of enzyme system and identification of thiazole monophosphate and thiamine monophosphate as intermediates. J. Biol. Chem. 235:2411-2417. Iwashima, A., K. Nosaka, H. Nishimura, and Y. Kimura. 1986. Some properties of a Saccharomyces cerevisiae mutant resistant to 2-amino-4-methyl-5-p-hydroxyethylthiazole. J. Gen. Microbiol. 132:1541-1546. Fujita, A. 1955. Thiaminase. Methods Enzymol. 2:622-628. Leder, I. G. 1970. Preparation of ,3-(4-methyl-5-thiazolyl)-ethyl phosphate. Methods Enzymol. 18A:166-167. Lewin, L. M., and G. W. Brown. 1961. The biosynthesis of thiamine. lII. Mechanism of enzymatic formation of the pyrophosphate ester of 2-methyl-4-amino-5-hydroxymethylpyrimidine. J. Biol. Chem. 236:2768-2771. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. Nishimura, H., K. Sempuku, Y. Kawasaki, K. Nosaka, and A. Iwashima. 1989. Photoaffinity labeling of thiamine-binding component in yeast plasma membrane with [3H]4-azido-2-nitrobenzoylthiamin. FEBS Lett. 255:154-158. Nosaka, K., Y. Kaneko, H. Nishimura, and A. Iwashima. 1989. A possible role for acid phosphatase with thiamin-binding activity encoded by PH03 in yeast. FEMS Microbiol. Lett. 60:55-60. Nose, Y., M. Tokuda, M. Hirabayashi, and A. Iwashima. 1964. Thiamine biosynthesis from hydroxymethylpyrimidine and thiazole by washed cells and cell extracts of Escherichia coli and its mutants. J. Vitaminol. 10:105-110. Nose, Y., K. Ueda, and T. Kawasaki. 1959. Enzymic synthesis of thiamine. Biochim. Biophys. Acta 34:277-279. Nose, Y., K. Ueda, T. Kawasaki, A. Iwashima, and T. Fujita. 1961. Enzymatic synthesis of thiamine. II. The thiamine synthesis from pyrimidine and thiazole phosphates and the enzymatic synthesis of pyrimidine mono- and diphosphate and thiazole monophosphate. J. Vitaminol. 7:98-114. Schweingruber, M. E., R. Fluri, K. Maundrell, A.-M. Schweingruber, and E. Dumermuth. 1986. Identification and characterization of thiamin repressible acid phosphatase in yeast. J. Biol. Chem. 261:15877-15882. Toh-e, A., S. Kakimoto, and Y. Oshima. 1975. Two new genes controlling the constitutive acid phosphatase synthesis in Saccharomyces cerevisiae. Mol. Gen. Genet. 141:81-83. Toh-e, A., S. Kakimoto, and Y. Oshima. 1975. Genes coding for the structure of the acid phosphatase in Saccharomyces cerevisiae. Mol. Gen. Genet. 143:65-70. Wickerham, L. J. 1951. Taxonomy of yeast. I. Techniques of classification. II. A classification of the genus Hansenula. U.S. Dept. Agric. Tech. Bull. 1029:1-56.