611
Biochimica et Biophysica Acta, 4 2 8 ( 1 9 7 6 ) 6 1 1 - - 6 1 8 © Elsevier Scientific P u b l i s h i n g C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s
BBA 27880
R E G U L A T I O N OF 6-HYDROXY-2,4,5-TRIAMINOPYRIMIDINE SYNTHESIS BY R I B O F L A V I N AND I R O N IN R I B O F L A V I N - D E F I C I E N T MUTANTS OF PICHIA G U I L L I E R M O N D I I YEAST
G.M. S H A V L O V S K U , E.M. L O G V I N E N K O , D. S C H L E E * a n d L.V. K O L T U N
Institute of Biochemistry, The Ukrainian SSR Academy of Science Lvov Branch, 290005,
Lvov (USSR) (Received September 22nd, 1975)
Summary The effect of riboflavin and iron on 6-hydroxy-2,4,5-triaminopyrimidine synthesis rate was investigated in the cultures of the yeast Pichia guilliermondii (rib: mutants) with the blocked second reaction of flavinogenesis. It was shown that riboflavin inhibited the 6-hydroxy-2,4,5-triaminopyrimidine synthesis rate in iron-rich and iron-deficient cells of mutants with low riboflavin requirements. Cycloheximide did n o t prevent the stimulation of 6hydroxy-2,4,5-triaminopyrimidine synthesis caused by riboflavin starvation. 7-methyl-8-trifluoromethyl-10-(l'-D-ribityl)isoalloxazine strongly inhibited the 6-hydroxy-2,4,5-triaminopyrimidine synthesis, while 7-methyl-8-trifluoromethyl-10-(fl-hydroxyethyl)izoaUoxazine and galactoflavin exerted only a slight effect on this process. The 6-hydroxy-2,4,5-triaminopyrimidine synthesis rate in iron-deficient cells was significantly higher than in iron-rich cells. The 2,2'-dipyridyl treatment of iron-rich cells caused the stimulation of 6-hydroxy-2,4,5-triaminopyrimidine synthesis and cycloheximide abolished this effect. The results suggest that the activity of the first enzyme of flavinogenesis (guanylic cyclohydrolase) is under the control of feedback inhibition by flavins and the biosynthesis of this enzyme is regulated by iron.
Introduction
The biosynthesis of riboflavin in the flavinogenic yeast Pichia guilliermondii is under the control of two mechanisms, repression/dereliression in which iron b u t n o t flavins exerts a regulatory influence [1,2] and the feedback inhibition *
P r e s e n t address:
Martin-Luther-University, Halle-Wittenberg, G.D.R.
612 of the activity of early enzymes by flavins [3]. The target enzyme for the feedback control was suggested to be guanylic cyclohydrolase. As it is n o t yet possible to study this reaction in vitro, the hypothesis cannot be tested directly at the present time. The regulatory properties of guanylic cyclohydrolase may be investigated in vivo using yeast mutants with the blocked second reaction of flavinogenesis. Such mutants of Saccharomyces cerevisiae, the rib7 mutants which accumulated 6-hydroxy-2,4,5-triaminopyrimidine were isolated first by Lingens and Bacher [4]. Mutants with the similar property were also obtained in P. guiiliermondii yeast [5]. In the present communication, evidence is presented which is consistent with the regulatory role of riboflavin and iron in the 6-hydroxy-2,4,5-triaminopyrimidine biosynthesis by these mutants.
Materials and Methods
Organisms The riboflavin auxotrophs with the blocked second reaction of flavinogenesis (designated as rib: mutants) were isolated from wild-type strains P. guilliermondii ATCC-9058 and Candida guilliermondii, obtained from Martin-Luther University (Halle-Wittenberg). The latter were mated with P. guilliermondii CBS-2031 strain and therefore attributed to P. guilliermondii species. The mutants were isolated after ultraviolet treatment of the wild-type as described elsewhere [6]. Use was made of four mutants: strain R128 and R128/13 derived from P. guilliermondii ATCC-9058, HR45 and HR45/17, derived from C. guilliermondii. Cells were grown on a rotatory shaker at 30°C in iron-rich (0.2 mg Fe/1) or in iron-deficient {0.01 mg Fe/1) Burkholder media [7] supplemented with riboflavin (100--200 mg/1). Excess of iron was removed from media by shaking with 8-hydroxyquinoline [8]. The iron-rich cells were harvested by centrifugation at the logarithmic growth phase and the iron-deficient cells at the early stationary one. The cells were washed with 0.02 M potassium/sodium phosphate buffer (pH 7.0) and suspended in the fresh medium supplemented with riboflavin if necessary.
The 6-hydroxy-2, 4,5-triaminopyrimidine measurement 6-hydroxy-2,4,5-triaminopyrimidine was transformed into 2-amino-4-hydroxy-6,7-dimethylpteridine. For this purpose diacetyl (800 mg/1) and HC1 (at the final concentration of 0.1 M) were added to the yeast cultures (cells plus incubation fluid). The suspension was incubated for 2 h at 70 ° C. Subsequently it was centrifuged and the cells were discarded. The supernatant fluid (50-100 ml) was passed through a column of Dowex 50Wx4 (100--200 mesh, H ÷ form, 1.3 X 10 cm equilibrated with 0.1 M HC1) which was washed with deionized water (2--3 1) to remove flavins; all pteridines were eluted with 1 M NH4OH. The concentration of pteridines was measured fluorimetrically with synthetic 2-amino-4-hydroxy-6,7-dimethylpteridine as a standard.
613 Previously it had been shown [9] that in iron-deficient cultures of rib2 mutants, three other pteridines and probably the 6-hydroxy-2,4,5-triaminopyrimidine dimer were accumulated. These pigments were not detected in the culture of rib1 m u t a n t with blocked guanylic cyclohydrolase but were synthesized during the incubation of iron-deficient cells of this m u t a n t with exogenous 6hydroxy-2,4,5-triaminopyrimidine; therefore, these compounds in rib2 mutants were obviously derived from 6-hydroxy-2,4,5-triaminopyrimidine. Pteridines and 6-hydroxy-2,4,5-triaminopyrimidine dimer which were eluted together with 2-amino-4-hydroxy-6,7-dimethylpteridine formed approx. 25% of total fluorescence of the samples. O t h e r assays
The flavin c o n t e n t of iron-rich cells was determined as described by Burch et al. [10]. The flavins from iron-deficient cells were extracted according to Yagi [11] ; FAD was h y d r o l y z e d in 0.1 M HC1 at 37°C for 24 h. Other fluorescent pigments were separated from flavins by passing the extracts through a column of Dowex 50Wx4 (100--200 mesh, H ÷ form, 1.3 × 10 cm). The flavin concentration was measured fluorimetrically. The cell dry weight was determined by measuring optical density or after drying the cells on membrane filters. Chemicals
6-hydroxy-2,4,5-triaminopyrimidine, 7 methyl-8-trifluoromethyl-10-(l'-Dribityl}isoalloxazine and 7-methyl-8-trifluoromethyl-10(fl-hydroxyethyl)isoalloxazine were gifts of Professor Z.V. Pushkareva, the Ural Technological Institute. 2-amino-4-hydroxy-6,7-dimethylpteridine was synthesized from.6-hydroxy2,4,5-triaminopyrimidine by means of its reaction with diacetyl and purified on Dowex 50Wx4. Results The synthesis of 6-hydroxy-2,4,5-triaminopyrimidine by the two rib2 mutants R128 and HR45 was studied. Those auxotrophs had different properties: the strain R128 satisfied its requirements for half-maximal growth at 14 mg riboflavin/l and the strain HR45 at 45 mg riboflavin/1. The yeasts were previously cultivated in the iron-deficient (0.01 mg Fe/1) or iron-rich (0.2 mg Fe/l) media; the washed cells w,ere incubated with riboflavin (200 mg/1) or w i t h o u t it. Table I shows that after the incubation of iron-rich cells in the presence or absence of riboflavin the two-fold increase of the cell dry weight was observed. The flavin c o n t e n t of the cells incubated without riboflavin was reduced by 50% as compared with the c o n t e n t of the cells incubated in the medium supplemented with riboflavin. The 6-hydroxy-2,4,5-triaminopyrimidine synthesis rate in the cells of m u t a n t R128 in the media lacking riboflavin was five-times higher than the one in the cells incubated with riboflavin. This was n o t observed in the cells of m u t a n t HR45. The iron-deficient cells of mutants exhibited poor growth during the incubation and were characterized by a higher flavin content as compared with the
614 TABLE I EFFECT OF RIBOFLAVIN AND IRON ON 6-HYDROXY-2,4,5-TRIAMINOPYRIMIDINE R A T E S I N T H E C E L L S O F r i h 2 - M U T A N T S O F P. G U I L L I E R M O N D I I M u t a n t ceils w e r e g r o w n in iron-rich a n d i r o n - d e f i c i e n t m e d i a s u p p l e m e n t e d d e s c r i b e d in Material a n d M e t h o d s , h a r v e s t e d a n d i n c u b a t e d 10 h. Strain
SYNTHESIS
w i t h 2 0 0 m g riboflavin/1 as
Concentration in m e d i u m , ( m g / l )
Cell d r y w t . / m l
Flavins in t h e cells ( p m o l / g d r y w t . )
iron
riboflavin
before incubation
after incubation
before incubation
after incubation
R128
0.20 0.20 0.01 0.01
0 200 0 200
2.29 2.23 2.23 2.00
5.40 6.36 2.72 2.85
0.09 0.09 0.140 0.140
0.035 0.086 0.107 0.135
0.10 0.02 0.60 0.16
HR45
0.20 0.20 0.01 0.01
0 200 0 200
1.75 1.85 1.52 1.52
3.91 4.76 2.45 2.37
0.095 0.095 0.158 0.158
0.048 0.096 0.138 0.161
0.13 0.09 1.03 0.68
HR45/17
0.20 0.20
0 200
1.93 1.99
5.65 6.06
0.151 0.151
0.061 O.153
0.10 0.01
6-hydroxy~ 2,4,5-triaminopyrimidine synthesis rate, (pmol/h per g dry wt.)
iron-rich cells. After the incubation in vitamin B2-free medium, the flavin content of the yeast was somewhat lower than in the cells incubated with riboflavin, but this difference was n o t so significant as in iron-rich cells. The 6-hydroxy-2,4,5-triaminopyrimidine synthesis rate in iron-deficient cells of both mutants was 6--8 times higher than the one in iron-rich cells. Exogenous riboflavin strongly inhibited the 6-hydroxy-2,4,5-triaminopyrimidine synthesis in the mutant R 1 2 8 cells while it had only a negligible effect upon this process in the mutant HR45 cells. We supposed that low sensitivity of 6-hydroxy-2,4,5-triaminopyrimidine synthesis to the inhibitory action of riboflavin in the cells of mutant HR45 might be s o m e h o w c o n n e c t e d with its requirement in the high riboflavin concentrations for the optimal growth. To test this hypothesis a mutant H R 4 5 / 1 7 which required 4 mg riboflavin/1 for the half-maximal growth (Fig. 1) was selected from the strain HR45. Table I shows that 6-hydroxy-2,4,5-triaminopyrimidine synthesis in this strain is as sensitive to the inhibitory action of riboflavin as that in the strain R128. Riboflavin-auxotroph R 1 2 8 / 1 3 was isolated f r o m the R 1 2 8 strain with an ability of half-maximal growth at a riboflavin concentration of 1.7 mg/1. The dependence o f the 6-hydroxy-2,4,5-triaminopyrimidine synthesis rate in the cells of this mutant on different riboflavin concentrations was further investigated; it appeared (Fig. 2) that 10 mg riboflavin/1 of the medium exerted a halving of the biosynthetic rate.
615
9.0
E 7.0
S
5.0
o /
"u3.C
2O
\ I I
I I I I
~ 2.C 1,0 I
10 20
30 4 0
I
50 6 0 70
I
I
i
8 0 9 0 100
10
0
Riboflavin (~g/rnl)
I
I
I
10
20
30
Riboflavin (}.Jg/mL)
Fig. 1. G r o w t h o f m u t a n t s H R 4 5 (1) a n d H R 4 5 / 1 7 (2) in m e d i a s u p p l e m e n t e d w i t h d i f f e r e n t r i b o f l a v i n concentrations. Fig. 2. T h e d e p e n d e n c e o f 6 - h y d r o x y - 2 , 4 , 5 - t r i a m i n o p y r i m i d i n e s y n t h e s i s r a t e b y the cells of m u t a n t R 1 2 8 / 1 3 on different riboflavin concentrations. 100% velocity corresponds to 0.17 p m o l / h per g dry wt.: initial cell d r y w t . was 2.0 m g / m l .
When the cells incubated for 4 h in the presence of riboflavin (100 mg/1) were transferred to the medium lacking riboflavin the increase of the 6-hydroxy-2,4,5-triaminopyrimidine synthesis rate from 0.005 p m o l / h per g dry wt. up to 0.17 mmol/h per g dry wt. occurred; cycloheximide (10'ml/1) did n o t inhibit this process (Table II). Obviously, the high rate of the 6-hydroxy-2,4,5-triaminopyrimidine synthesis in the cells incubated without riboflavin was not due to derepression of the first enzyme of flavinogenesis. The effect of three riboflavin analogues on the 6-hydroxy-2,4,5-triaminopyr-
T A B L E II E F F E C T OF R I B O F L A V I N , ITS A N A L O G U E S AND C Y C L O H E X I M I D E ON 6-HYDROXY-2,4,5-TRIA M I N O P Y R I M I D I N E S Y N T H E S I S R A T E S IN T H E M U T A N T R 1 2 8 / 1 3 CELLS Cells w e r e p r e v i o u s l y g r o w n i n , i r o n - r i c h m e d i u m s u p p l e m e n t e d w i t h 1 0 0 m g riboflavin/1 (see Materials a n d M e t h o d s ) , h a r v e s t e d a n d i n c u b a t e d in m e d i a w i t h d i f f e r e n t a d d i t i o n s : c o n c e n t r a t i o n o f flavins in t h e i n c u b a t i o n m i x t u r e was 2 0 0 m g f l ; c y c l o h e x i m i d e , 10 m g / l . Initial cell d r y w t . was 2.0 m g / m l ; 6 - h y d r o x y 2 , 4 , 5 - t r i a m i n o p y r i m i d i n e s y n t h e s i s r a t e in u n s u p p l e m e n t e d m e d i u m w a s 0.17 ~zmol/h p e r g d r y w t . Additions to incubation mixture
Time of incubation (h)
Per c e n t of i n h i b i t i o n
Riboflavin 7-methyl-8-trifluoromethyl10- / 1 - D - t r i b i t y l / i s o a l l o x a z i n e 7-methyl-8-trifluoromethyl10-(~-hy d r o x y e t h y l ) i s o a l l o x a z i n e Galactoflavine Cycloheximide
6 6
97 87
6
11 "
6 3
6 0
616 []
without additions
[]
DP
[]
DP+C
0.3 R3 m o~ 0.2
-5 E =L 0.1 o >
A
B
Fig. 3. E f f e c t of 2 , 2 ' - d i p y r i d y l (DP) a n d c y c l o h e x i m i d e (C) o n 6 - h y d r o x y - 2 , 4 , 5 4 r i a m l n o p y r i m i d i n e synthesis r a t e s in t h e cells of m u t a n t s R 1 2 S ( A ) a n d H R 4 5 (B). T h e cells were p r e v i o u s l y g r o w n in the m e d i u m s u p p l e m e n t e d w i t h 0.1 m g Fe/1 a n d 2 0 0 m g r i b o f l a v i n / l f o r 24 h, h a r v e s t e d a n d i n c u b a t e d for 4 h in i r o n - d e f i c i e n t m e d i u m w i t h 2 , 2 r - d i p y r i d y l ( 1 8 0 mg/1); 1 h a f t e r b e g i n n i n g of i n c u b a t i o n , c y c l o h e x i m i d e ( 1 0 rag/l) was a d d e d ; initial cell d r y wt. was 1.0 m g / m l .
imidine synthesis was also investigated. One of them, 7-methyl-8-trifluoromethyl-10-(l'-D-ribityl)isoalloxazine was actively phosphorylated by riboflavin kinase of P. guilliermondii, the other two, 7-methyl-8-trifluoromethyl-10-(~-hydroxyethyl)iso-alloxazine and galactoflavin were not phosphorylated at all or phosphorylated very slowly [12]. It appeared that only 7-methyl-8-trifluoromethyl-10-(l'-D-ribityl)isoalloxazine exhibitied a strong inhibitory action on the 6-hydroxy-2,4,5-triaminopyrimidine synthesis (Table II). As it had been previously shown the rates of 6-hydroxy-2,4,5-triaminopyrimidine synthesis in iron-deficient cells of mutants R128 and H R 4 5 were essentially higher then that in the iron-rich cells. We have investigated the effect of 2,2'-dipyridyl on the 6-hydroxy-2,4,5-triaminopyrimidine synthesis rate in iron-rich cells. The chelator stimulates the riboflavin overproduction and causes the riboflavin=synthetase derepression in P. guilliermondii yeast [2]. It was shown (Fig. 3) that 2,2'-dipyridyl treatment of the iron-rich cells of mutants R 1 2 8 or H R 4 5 enhanced the 6-hydroxy-2,4,5-triaminopyrimidine synthesis and cycloheximide prevented this action. Therefore, the protein biosynthesis is involved in this phenomenon. Discussion
The major control of biosynthetic pathways operates through end-product regulation either by repression of enzyme synthesis or by feedback inhibition of the activity of early enzymes. In bacterial systems the repression of flavinogenesis enzymes was found in B. subtillis [13]. From our experiments with riboflavin-deficient mutants of P. guilliermondii
617
and P. ohmeri with blocked lumazine synthetase it appeared that riboflavin synthesis was under the control of feedback inhibition [3]. However, a point of effector action was obscure. The present results with rib: mutants of P. guilliermondii indicate that riboflavin inhibits the rate of the first reaction of flavinogenesis which is catalyzed by guanylic cyclohydrolase. As cycloheximide did not prevent the stimulation of 6-hydroxy-2,4,5-triaminopyrimidine synthesis in the media lacking riboflavin one may conclude that the cyclohydrolase was under the control of feedback inhibition by flavins but n o t of repression. It is interesting to note that riboflavin inhibits the 6-hydroxy-2,4,5-triaminopyrimidine synthesis only in the cells of mutants with the low riboflavin requirements and exerted only a negligible action on the process in the HE45 mutant. Earlier we had observed that riboflavin did not inhibit the synthesis of 6,7dimethyllumazine in the cells of P. guilliermondii m u t a n t R14 with blocked riboflavin synthetase [2]. It seems possible that such strains have a low rate of the riboflavin transport to the cells or {and) a slow conversion of exogenous riboflavin to flavin nucleotides which may appear to be the true effectors of cyclohydrolase. Of interest in this regard is our finding that only 7-methyl-8-trifluoromethyl10-(l'-D-ribityl)isoalloxazine which is phosphorylated by riboflavin kinase in P. guilliermondii [12] and thus converted into FMN analogue strongly inhibits the 6-hydroxy-2,4,5-triaminopyrimidine synthesis. Furthermore, iron-deficient cells in which the flavinogenesis is also regulated by the negative feedback control, synthesized a great a m o u n t of riboflavin but not of flavin nucleotides. Thus, the experimental data appear to support the suggestion that flavin coenzymes are true cyclohydrolase inhibitors. Early in vivo studies with riboflavin-overproducers indicated that the iron deficiency caused the derepression of riboflavin-synthetase [1]. The stimulation of riboflavin precursor synthesis in the cells of riboflavin-deficient mutants of P. guilliermondii was also observed [2]. The data presented show that the first enzyme of flavinogenesis guanylic cyclohydrolase is obviously derepressed also in the iron-deficient cells of P. guilliermondii. The final resolution of the problem must come from the isolation and studying the properties of guanylic cyclohydrolase of flavinogenic yeasts in vitro. References
1 S h a v l o v s k y , G . M . , L o g v i n e n k o , E.M. a n d T r a c h , V.M. ( 1 9 7 2 ) P r o c . A c a d . Sci. U S S R 2 0 4 , 2 4 1 - - 2 4 4 2 S h a v l o v s k y , G . M . , L o g v i n e n k o , E . M . , T r a c h , V.M. a n d K o l t u n , L . V . ( 1 9 7 3 ) P r o c . T h i r d I n t . S p e c . Syrup. Yeast Metabolism and Regulation of Cellular Processes, Otaniemi Helsinki, Finland, 4--8 June, pp. 70--71 3 S h a v l o v s k y , G . M . , L o g v i n c n k o , E.M. a n d K o l t u n , L . V . ( 1 9 7 5 ) M i c r o b i o l o g y a ( U S S R ) 4 4 , 1 7 1 - - 1 7 3 4 B a e h e r , A. a n d L i n g e n s , F. ( 1 9 7 1 ) J. Biol. C h e m . 2 4 6 , 7 0 1 8 - - 7 0 2 2 5 L o g v i n e n k o , E.M., S h a v l o v s k y , G . M . , K o l t u n , L . V . a n d Ksheminskaya, G.P. ( 1 9 7 5 ) M i c r o b i o l o g y a (USSR) 44, 48--54 6 L o g v i n e n k o , E . M . , S h a v l o v s k y , G.M. a n d K o l t u n , L . V . ( 1 9 7 2 ) M i c r o b i o l o g y a 4 2 , 1 1 0 3 - - 1 1 0 4 7 B u r k h o l d e r , P. ( 1 9 4 3 ) A r c h . B i o c h e m . 3 , 1 2 1 - - 1 3 0 S W a r i n g , W.S. a n d W e r k m a n , C.H. ( 1 9 4 3 ) A r c h . B i o c h e m . 1 , 4 2 5 - - 4 3 3
618 9 Shavlovsky, G.M., Logvinenko, E.M. and Koltun, L.V. Proc. Third All-Union Biochem. Congress, Abstracts,Vol. 1, Riga, p. 211 10 Butch, H.B., Bessey, O.A. and Lowry, O.R. (1948) J. Biol. Chem. 175, 457 11 Yagi, K. (1957) Bull. Soc. Chim. France 11/12, 1543--1550 12 Shavlovsky, G.M., Strugovshchikova, L.P., Kashchenko, V.E. and Sibirny, A.A. (1974) Proc. Fourth Int. Syrup. Yeasts, Part 1 Paper Sessions Abstracts (Klaushofer, H. and Sleytr. U.B., eds.),Wien, p. 3 13 Bresler,S.E., Tcherepenko, D.A. and Perumov, D.A. (1970), Genetyka (USSR) 6,126--139
Biochimica et Biophysica Acta, 4 2 8 ( 1 9 7 6 ) 6 1 1 - - 6 1 8 © Elsevier Scientific P u b l i s h i n g C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s
BBA 27880
R E G U L A T I O N OF 6-HYDROXY-2,4,5-TRIAMINOPYRIMIDINE SYNTHESIS BY R I B O F L A V I N AND I R O N IN R I B O F L A V I N - D E F I C I E N T MUTANTS OF PICHIA G U I L L I E R M O N D I I YEAST
G.M. S H A V L O V S K U , E.M. L O G V I N E N K O , D. S C H L E E * a n d L.V. K O L T U N
Institute of Biochemistry, The Ukrainian SSR Academy of Science Lvov Branch, 290005,
Lvov (USSR) (Received September 22nd, 1975)
Summary The effect of riboflavin and iron on 6-hydroxy-2,4,5-triaminopyrimidine synthesis rate was investigated in the cultures of the yeast Pichia guilliermondii (rib: mutants) with the blocked second reaction of flavinogenesis. It was shown that riboflavin inhibited the 6-hydroxy-2,4,5-triaminopyrimidine synthesis rate in iron-rich and iron-deficient cells of mutants with low riboflavin requirements. Cycloheximide did n o t prevent the stimulation of 6hydroxy-2,4,5-triaminopyrimidine synthesis caused by riboflavin starvation. 7-methyl-8-trifluoromethyl-10-(l'-D-ribityl)isoalloxazine strongly inhibited the 6-hydroxy-2,4,5-triaminopyrimidine synthesis, while 7-methyl-8-trifluoromethyl-10-(fl-hydroxyethyl)izoaUoxazine and galactoflavin exerted only a slight effect on this process. The 6-hydroxy-2,4,5-triaminopyrimidine synthesis rate in iron-deficient cells was significantly higher than in iron-rich cells. The 2,2'-dipyridyl treatment of iron-rich cells caused the stimulation of 6-hydroxy-2,4,5-triaminopyrimidine synthesis and cycloheximide abolished this effect. The results suggest that the activity of the first enzyme of flavinogenesis (guanylic cyclohydrolase) is under the control of feedback inhibition by flavins and the biosynthesis of this enzyme is regulated by iron.
Introduction
The biosynthesis of riboflavin in the flavinogenic yeast Pichia guilliermondii is under the control of two mechanisms, repression/dereliression in which iron b u t n o t flavins exerts a regulatory influence [1,2] and the feedback inhibition *
P r e s e n t address:
Martin-Luther-University, Halle-Wittenberg, G.D.R.
612 of the activity of early enzymes by flavins [3]. The target enzyme for the feedback control was suggested to be guanylic cyclohydrolase. As it is n o t yet possible to study this reaction in vitro, the hypothesis cannot be tested directly at the present time. The regulatory properties of guanylic cyclohydrolase may be investigated in vivo using yeast mutants with the blocked second reaction of flavinogenesis. Such mutants of Saccharomyces cerevisiae, the rib7 mutants which accumulated 6-hydroxy-2,4,5-triaminopyrimidine were isolated first by Lingens and Bacher [4]. Mutants with the similar property were also obtained in P. guiiliermondii yeast [5]. In the present communication, evidence is presented which is consistent with the regulatory role of riboflavin and iron in the 6-hydroxy-2,4,5-triaminopyrimidine biosynthesis by these mutants.
Materials and Methods
Organisms The riboflavin auxotrophs with the blocked second reaction of flavinogenesis (designated as rib: mutants) were isolated from wild-type strains P. guilliermondii ATCC-9058 and Candida guilliermondii, obtained from Martin-Luther University (Halle-Wittenberg). The latter were mated with P. guilliermondii CBS-2031 strain and therefore attributed to P. guilliermondii species. The mutants were isolated after ultraviolet treatment of the wild-type as described elsewhere [6]. Use was made of four mutants: strain R128 and R128/13 derived from P. guilliermondii ATCC-9058, HR45 and HR45/17, derived from C. guilliermondii. Cells were grown on a rotatory shaker at 30°C in iron-rich (0.2 mg Fe/1) or in iron-deficient {0.01 mg Fe/1) Burkholder media [7] supplemented with riboflavin (100--200 mg/1). Excess of iron was removed from media by shaking with 8-hydroxyquinoline [8]. The iron-rich cells were harvested by centrifugation at the logarithmic growth phase and the iron-deficient cells at the early stationary one. The cells were washed with 0.02 M potassium/sodium phosphate buffer (pH 7.0) and suspended in the fresh medium supplemented with riboflavin if necessary.
The 6-hydroxy-2, 4,5-triaminopyrimidine measurement 6-hydroxy-2,4,5-triaminopyrimidine was transformed into 2-amino-4-hydroxy-6,7-dimethylpteridine. For this purpose diacetyl (800 mg/1) and HC1 (at the final concentration of 0.1 M) were added to the yeast cultures (cells plus incubation fluid). The suspension was incubated for 2 h at 70 ° C. Subsequently it was centrifuged and the cells were discarded. The supernatant fluid (50-100 ml) was passed through a column of Dowex 50Wx4 (100--200 mesh, H ÷ form, 1.3 X 10 cm equilibrated with 0.1 M HC1) which was washed with deionized water (2--3 1) to remove flavins; all pteridines were eluted with 1 M NH4OH. The concentration of pteridines was measured fluorimetrically with synthetic 2-amino-4-hydroxy-6,7-dimethylpteridine as a standard.
613 Previously it had been shown [9] that in iron-deficient cultures of rib2 mutants, three other pteridines and probably the 6-hydroxy-2,4,5-triaminopyrimidine dimer were accumulated. These pigments were not detected in the culture of rib1 m u t a n t with blocked guanylic cyclohydrolase but were synthesized during the incubation of iron-deficient cells of this m u t a n t with exogenous 6hydroxy-2,4,5-triaminopyrimidine; therefore, these compounds in rib2 mutants were obviously derived from 6-hydroxy-2,4,5-triaminopyrimidine. Pteridines and 6-hydroxy-2,4,5-triaminopyrimidine dimer which were eluted together with 2-amino-4-hydroxy-6,7-dimethylpteridine formed approx. 25% of total fluorescence of the samples. O t h e r assays
The flavin c o n t e n t of iron-rich cells was determined as described by Burch et al. [10]. The flavins from iron-deficient cells were extracted according to Yagi [11] ; FAD was h y d r o l y z e d in 0.1 M HC1 at 37°C for 24 h. Other fluorescent pigments were separated from flavins by passing the extracts through a column of Dowex 50Wx4 (100--200 mesh, H ÷ form, 1.3 × 10 cm). The flavin concentration was measured fluorimetrically. The cell dry weight was determined by measuring optical density or after drying the cells on membrane filters. Chemicals
6-hydroxy-2,4,5-triaminopyrimidine, 7 methyl-8-trifluoromethyl-10-(l'-Dribityl}isoalloxazine and 7-methyl-8-trifluoromethyl-10(fl-hydroxyethyl)isoalloxazine were gifts of Professor Z.V. Pushkareva, the Ural Technological Institute. 2-amino-4-hydroxy-6,7-dimethylpteridine was synthesized from.6-hydroxy2,4,5-triaminopyrimidine by means of its reaction with diacetyl and purified on Dowex 50Wx4. Results The synthesis of 6-hydroxy-2,4,5-triaminopyrimidine by the two rib2 mutants R128 and HR45 was studied. Those auxotrophs had different properties: the strain R128 satisfied its requirements for half-maximal growth at 14 mg riboflavin/l and the strain HR45 at 45 mg riboflavin/1. The yeasts were previously cultivated in the iron-deficient (0.01 mg Fe/1) or iron-rich (0.2 mg Fe/l) media; the washed cells w,ere incubated with riboflavin (200 mg/1) or w i t h o u t it. Table I shows that after the incubation of iron-rich cells in the presence or absence of riboflavin the two-fold increase of the cell dry weight was observed. The flavin c o n t e n t of the cells incubated without riboflavin was reduced by 50% as compared with the c o n t e n t of the cells incubated in the medium supplemented with riboflavin. The 6-hydroxy-2,4,5-triaminopyrimidine synthesis rate in the cells of m u t a n t R128 in the media lacking riboflavin was five-times higher than the one in the cells incubated with riboflavin. This was n o t observed in the cells of m u t a n t HR45. The iron-deficient cells of mutants exhibited poor growth during the incubation and were characterized by a higher flavin content as compared with the
614 TABLE I EFFECT OF RIBOFLAVIN AND IRON ON 6-HYDROXY-2,4,5-TRIAMINOPYRIMIDINE R A T E S I N T H E C E L L S O F r i h 2 - M U T A N T S O F P. G U I L L I E R M O N D I I M u t a n t ceils w e r e g r o w n in iron-rich a n d i r o n - d e f i c i e n t m e d i a s u p p l e m e n t e d d e s c r i b e d in Material a n d M e t h o d s , h a r v e s t e d a n d i n c u b a t e d 10 h. Strain
SYNTHESIS
w i t h 2 0 0 m g riboflavin/1 as
Concentration in m e d i u m , ( m g / l )
Cell d r y w t . / m l
Flavins in t h e cells ( p m o l / g d r y w t . )
iron
riboflavin
before incubation
after incubation
before incubation
after incubation
R128
0.20 0.20 0.01 0.01
0 200 0 200
2.29 2.23 2.23 2.00
5.40 6.36 2.72 2.85
0.09 0.09 0.140 0.140
0.035 0.086 0.107 0.135
0.10 0.02 0.60 0.16
HR45
0.20 0.20 0.01 0.01
0 200 0 200
1.75 1.85 1.52 1.52
3.91 4.76 2.45 2.37
0.095 0.095 0.158 0.158
0.048 0.096 0.138 0.161
0.13 0.09 1.03 0.68
HR45/17
0.20 0.20
0 200
1.93 1.99
5.65 6.06
0.151 0.151
0.061 O.153
0.10 0.01
6-hydroxy~ 2,4,5-triaminopyrimidine synthesis rate, (pmol/h per g dry wt.)
iron-rich cells. After the incubation in vitamin B2-free medium, the flavin content of the yeast was somewhat lower than in the cells incubated with riboflavin, but this difference was n o t so significant as in iron-rich cells. The 6-hydroxy-2,4,5-triaminopyrimidine synthesis rate in iron-deficient cells of both mutants was 6--8 times higher than the one in iron-rich cells. Exogenous riboflavin strongly inhibited the 6-hydroxy-2,4,5-triaminopyrimidine synthesis in the mutant R 1 2 8 cells while it had only a negligible effect upon this process in the mutant HR45 cells. We supposed that low sensitivity of 6-hydroxy-2,4,5-triaminopyrimidine synthesis to the inhibitory action of riboflavin in the cells of mutant HR45 might be s o m e h o w c o n n e c t e d with its requirement in the high riboflavin concentrations for the optimal growth. To test this hypothesis a mutant H R 4 5 / 1 7 which required 4 mg riboflavin/1 for the half-maximal growth (Fig. 1) was selected from the strain HR45. Table I shows that 6-hydroxy-2,4,5-triaminopyrimidine synthesis in this strain is as sensitive to the inhibitory action of riboflavin as that in the strain R128. Riboflavin-auxotroph R 1 2 8 / 1 3 was isolated f r o m the R 1 2 8 strain with an ability of half-maximal growth at a riboflavin concentration of 1.7 mg/1. The dependence o f the 6-hydroxy-2,4,5-triaminopyrimidine synthesis rate in the cells of this mutant on different riboflavin concentrations was further investigated; it appeared (Fig. 2) that 10 mg riboflavin/1 of the medium exerted a halving of the biosynthetic rate.
615
9.0
E 7.0
S
5.0
o /
"u3.C
2O
\ I I
I I I I
~ 2.C 1,0 I
10 20
30 4 0
I
50 6 0 70
I
I
i
8 0 9 0 100
10
0
Riboflavin (~g/rnl)
I
I
I
10
20
30
Riboflavin (}.Jg/mL)
Fig. 1. G r o w t h o f m u t a n t s H R 4 5 (1) a n d H R 4 5 / 1 7 (2) in m e d i a s u p p l e m e n t e d w i t h d i f f e r e n t r i b o f l a v i n concentrations. Fig. 2. T h e d e p e n d e n c e o f 6 - h y d r o x y - 2 , 4 , 5 - t r i a m i n o p y r i m i d i n e s y n t h e s i s r a t e b y the cells of m u t a n t R 1 2 8 / 1 3 on different riboflavin concentrations. 100% velocity corresponds to 0.17 p m o l / h per g dry wt.: initial cell d r y w t . was 2.0 m g / m l .
When the cells incubated for 4 h in the presence of riboflavin (100 mg/1) were transferred to the medium lacking riboflavin the increase of the 6-hydroxy-2,4,5-triaminopyrimidine synthesis rate from 0.005 p m o l / h per g dry wt. up to 0.17 mmol/h per g dry wt. occurred; cycloheximide (10'ml/1) did n o t inhibit this process (Table II). Obviously, the high rate of the 6-hydroxy-2,4,5-triaminopyrimidine synthesis in the cells incubated without riboflavin was not due to derepression of the first enzyme of flavinogenesis. The effect of three riboflavin analogues on the 6-hydroxy-2,4,5-triaminopyr-
T A B L E II E F F E C T OF R I B O F L A V I N , ITS A N A L O G U E S AND C Y C L O H E X I M I D E ON 6-HYDROXY-2,4,5-TRIA M I N O P Y R I M I D I N E S Y N T H E S I S R A T E S IN T H E M U T A N T R 1 2 8 / 1 3 CELLS Cells w e r e p r e v i o u s l y g r o w n i n , i r o n - r i c h m e d i u m s u p p l e m e n t e d w i t h 1 0 0 m g riboflavin/1 (see Materials a n d M e t h o d s ) , h a r v e s t e d a n d i n c u b a t e d in m e d i a w i t h d i f f e r e n t a d d i t i o n s : c o n c e n t r a t i o n o f flavins in t h e i n c u b a t i o n m i x t u r e was 2 0 0 m g f l ; c y c l o h e x i m i d e , 10 m g / l . Initial cell d r y w t . was 2.0 m g / m l ; 6 - h y d r o x y 2 , 4 , 5 - t r i a m i n o p y r i m i d i n e s y n t h e s i s r a t e in u n s u p p l e m e n t e d m e d i u m w a s 0.17 ~zmol/h p e r g d r y w t . Additions to incubation mixture
Time of incubation (h)
Per c e n t of i n h i b i t i o n
Riboflavin 7-methyl-8-trifluoromethyl10- / 1 - D - t r i b i t y l / i s o a l l o x a z i n e 7-methyl-8-trifluoromethyl10-(~-hy d r o x y e t h y l ) i s o a l l o x a z i n e Galactoflavine Cycloheximide
6 6
97 87
6
11 "
6 3
6 0
616 []
without additions
[]
DP
[]
DP+C
0.3 R3 m o~ 0.2
-5 E =L 0.1 o >
A
B
Fig. 3. E f f e c t of 2 , 2 ' - d i p y r i d y l (DP) a n d c y c l o h e x i m i d e (C) o n 6 - h y d r o x y - 2 , 4 , 5 4 r i a m l n o p y r i m i d i n e synthesis r a t e s in t h e cells of m u t a n t s R 1 2 S ( A ) a n d H R 4 5 (B). T h e cells were p r e v i o u s l y g r o w n in the m e d i u m s u p p l e m e n t e d w i t h 0.1 m g Fe/1 a n d 2 0 0 m g r i b o f l a v i n / l f o r 24 h, h a r v e s t e d a n d i n c u b a t e d for 4 h in i r o n - d e f i c i e n t m e d i u m w i t h 2 , 2 r - d i p y r i d y l ( 1 8 0 mg/1); 1 h a f t e r b e g i n n i n g of i n c u b a t i o n , c y c l o h e x i m i d e ( 1 0 rag/l) was a d d e d ; initial cell d r y wt. was 1.0 m g / m l .
imidine synthesis was also investigated. One of them, 7-methyl-8-trifluoromethyl-10-(l'-D-ribityl)isoalloxazine was actively phosphorylated by riboflavin kinase of P. guilliermondii, the other two, 7-methyl-8-trifluoromethyl-10-(~-hydroxyethyl)iso-alloxazine and galactoflavin were not phosphorylated at all or phosphorylated very slowly [12]. It appeared that only 7-methyl-8-trifluoromethyl-10-(l'-D-ribityl)isoalloxazine exhibitied a strong inhibitory action on the 6-hydroxy-2,4,5-triaminopyrimidine synthesis (Table II). As it had been previously shown the rates of 6-hydroxy-2,4,5-triaminopyrimidine synthesis in iron-deficient cells of mutants R128 and H R 4 5 were essentially higher then that in the iron-rich cells. We have investigated the effect of 2,2'-dipyridyl on the 6-hydroxy-2,4,5-triaminopyrimidine synthesis rate in iron-rich cells. The chelator stimulates the riboflavin overproduction and causes the riboflavin=synthetase derepression in P. guilliermondii yeast [2]. It was shown (Fig. 3) that 2,2'-dipyridyl treatment of the iron-rich cells of mutants R 1 2 8 or H R 4 5 enhanced the 6-hydroxy-2,4,5-triaminopyrimidine synthesis and cycloheximide prevented this action. Therefore, the protein biosynthesis is involved in this phenomenon. Discussion
The major control of biosynthetic pathways operates through end-product regulation either by repression of enzyme synthesis or by feedback inhibition of the activity of early enzymes. In bacterial systems the repression of flavinogenesis enzymes was found in B. subtillis [13]. From our experiments with riboflavin-deficient mutants of P. guilliermondii
617
and P. ohmeri with blocked lumazine synthetase it appeared that riboflavin synthesis was under the control of feedback inhibition [3]. However, a point of effector action was obscure. The present results with rib: mutants of P. guilliermondii indicate that riboflavin inhibits the rate of the first reaction of flavinogenesis which is catalyzed by guanylic cyclohydrolase. As cycloheximide did not prevent the stimulation of 6-hydroxy-2,4,5-triaminopyrimidine synthesis in the media lacking riboflavin one may conclude that the cyclohydrolase was under the control of feedback inhibition by flavins but n o t of repression. It is interesting to note that riboflavin inhibits the 6-hydroxy-2,4,5-triaminopyrimidine synthesis only in the cells of mutants with the low riboflavin requirements and exerted only a negligible action on the process in the HE45 mutant. Earlier we had observed that riboflavin did not inhibit the synthesis of 6,7dimethyllumazine in the cells of P. guilliermondii m u t a n t R14 with blocked riboflavin synthetase [2]. It seems possible that such strains have a low rate of the riboflavin transport to the cells or {and) a slow conversion of exogenous riboflavin to flavin nucleotides which may appear to be the true effectors of cyclohydrolase. Of interest in this regard is our finding that only 7-methyl-8-trifluoromethyl10-(l'-D-ribityl)isoalloxazine which is phosphorylated by riboflavin kinase in P. guilliermondii [12] and thus converted into FMN analogue strongly inhibits the 6-hydroxy-2,4,5-triaminopyrimidine synthesis. Furthermore, iron-deficient cells in which the flavinogenesis is also regulated by the negative feedback control, synthesized a great a m o u n t of riboflavin but not of flavin nucleotides. Thus, the experimental data appear to support the suggestion that flavin coenzymes are true cyclohydrolase inhibitors. Early in vivo studies with riboflavin-overproducers indicated that the iron deficiency caused the derepression of riboflavin-synthetase [1]. The stimulation of riboflavin precursor synthesis in the cells of riboflavin-deficient mutants of P. guilliermondii was also observed [2]. The data presented show that the first enzyme of flavinogenesis guanylic cyclohydrolase is obviously derepressed also in the iron-deficient cells of P. guilliermondii. The final resolution of the problem must come from the isolation and studying the properties of guanylic cyclohydrolase of flavinogenic yeasts in vitro. References
1 S h a v l o v s k y , G . M . , L o g v i n e n k o , E.M. a n d T r a c h , V.M. ( 1 9 7 2 ) P r o c . A c a d . Sci. U S S R 2 0 4 , 2 4 1 - - 2 4 4 2 S h a v l o v s k y , G . M . , L o g v i n e n k o , E . M . , T r a c h , V.M. a n d K o l t u n , L . V . ( 1 9 7 3 ) P r o c . T h i r d I n t . S p e c . Syrup. Yeast Metabolism and Regulation of Cellular Processes, Otaniemi Helsinki, Finland, 4--8 June, pp. 70--71 3 S h a v l o v s k y , G . M . , L o g v i n c n k o , E.M. a n d K o l t u n , L . V . ( 1 9 7 5 ) M i c r o b i o l o g y a ( U S S R ) 4 4 , 1 7 1 - - 1 7 3 4 B a e h e r , A. a n d L i n g e n s , F. ( 1 9 7 1 ) J. Biol. C h e m . 2 4 6 , 7 0 1 8 - - 7 0 2 2 5 L o g v i n e n k o , E.M., S h a v l o v s k y , G . M . , K o l t u n , L . V . a n d Ksheminskaya, G.P. ( 1 9 7 5 ) M i c r o b i o l o g y a (USSR) 44, 48--54 6 L o g v i n e n k o , E . M . , S h a v l o v s k y , G.M. a n d K o l t u n , L . V . ( 1 9 7 2 ) M i c r o b i o l o g y a 4 2 , 1 1 0 3 - - 1 1 0 4 7 B u r k h o l d e r , P. ( 1 9 4 3 ) A r c h . B i o c h e m . 3 , 1 2 1 - - 1 3 0 S W a r i n g , W.S. a n d W e r k m a n , C.H. ( 1 9 4 3 ) A r c h . B i o c h e m . 1 , 4 2 5 - - 4 3 3
618 9 Shavlovsky, G.M., Logvinenko, E.M. and Koltun, L.V. Proc. Third All-Union Biochem. Congress, Abstracts,Vol. 1, Riga, p. 211 10 Butch, H.B., Bessey, O.A. and Lowry, O.R. (1948) J. Biol. Chem. 175, 457 11 Yagi, K. (1957) Bull. Soc. Chim. France 11/12, 1543--1550 12 Shavlovsky, G.M., Strugovshchikova, L.P., Kashchenko, V.E. and Sibirny, A.A. (1974) Proc. Fourth Int. Syrup. Yeasts, Part 1 Paper Sessions Abstracts (Klaushofer, H. and Sleytr. U.B., eds.),Wien, p. 3 13 Bresler,S.E., Tcherepenko, D.A. and Perumov, D.A. (1970), Genetyka (USSR) 6,126--139
