Eur. J . Biochem. 88, 607-612 (1978)
Production of NADPH in the Mannitol Cycle and Its Relation to Polyketide Formation in Alternaria alternata Karl HULT and Sten GATENBECK Department of Pure and Applied Biochemistry, Royal Institute of Technology, Stockholm (Received February 4, 1978)
The enzymes mannitol-1-phosphate dehydrogenase, mannitol-I-phosphatase, mannitol dehydrogenase and hexokinase participate in an enzymatic cycle in the fungus Alternuria alternata. One turn of the cycle gives the net result: NADH
+ NADP' + ATP + NAD' + NADPH + A DP + P , .
The cycle alone can meet the total need of NADPH formation for fat synthesis in the organism. A polyketide producing strain of A . altrrnuta shows a lower mannitol oxidation as well as a lower fat synthesis than a nonproducing mutant, supporting the hypothesis that polyketide formation is favoured at limiting NADPH production. It is further suggested that the mannitol cycle is regulating the glycolytic flux by substrate withdrawal from phosphofructokinase.
The occurrence of polyols, particularly mannitol, in fungi has been known for several decades. The metabolism and the presence of mannitol in fungi are discussed in a recent review by Blumenthal [l]. Blumenthal concludes that mannitol serves as a reserve of carbon nutrient and perhaps also as a form of stored reducing power. The metabolic steps for niannitol synthesis and degradation seem to be uncertain mainly because of the manifold alternative pathways offered with the participation of the known enzymes. In this paper a new view on mannitol metabolism in the imperfect fungi is suggested. It is proposed that mannitol is involved in a cycle producing NADPH with the utilization of N A D H and ATP. The organism which is used in the described studies i s A l t e ~ ~ ualtcmatn. r~u This mould is known to produce the polyketide alternariol (Fig. 1) [2]. Alternariol is synthesized from acetyl-CoA and malonyl-CoA in a manner similar to the fatty acid synthesis but without the involvement of the NADPH-dependent reductions [3]. It has been suggested that the production of polyketides reflects a limiting availability of NADPH for fatty acid synthesis [4]. The proposed role of mannito1 in the NADPH production made it interesting to study the mannitol metabolism in relation to lipid synthesis and alternariol formation in an alternariolproducing strain of A . alternata as compared to a nonproducing mutant. f+qwws. Mannitol-I-phosphate dehydrogenase (EC 1.1.1.17); mannitol-I-phosphatase (EC 3.1.3.22); mannitol dehydrogenase (EC 1.1.1.138); hexokinase (EC 2.7.1 . I ) ; glucosephosphate isomerase (EC 5.3.1.9); phosphofructokinase (EC 2.7.1.11).
Fig. 1 . Alrci
MATERIALS AND METHODS
Cultural Conditions The alternariol-producing strain Alternuria alturnata (CMI 89343) was obtained from Commonwealth Mycological Institute (Kew, Surrey). The nonproducing strain was a spontaneous mutant isolated from a producing culture. Conical flasks of 500-in1 capacity containing 150 ml modified Czapek-Dox medium 151 were inoculated from agar slants and incubated on a rotary shaking table at 25 'C. Portions of the mycelium were harvested by filtration at appropriate times.
Incubations Portions of wet mycelium (0.70 g) were incubated in 25-ml conical flasks containing 7 ml modified nitrogen-free Czapek-Dox medium with 5 g glucose per 1000 ml. The flasks were incubated on a table rotary shaker at 20 "C. Two parallel incubations were run for each strain. After 1 h 2-ml samples were taken for analysis of glucose concentration in the medium and
The Mannitol Cycle in Alternaria alternata
608
of cellular mannitol. To the remaining 5 ml of the cultures labelled substrates were added and the incubations were continued for the time indicated. The following labelled compounds were used: 0.5 pCi [U-'4C]glucose in a separate incubation and 0.5 pCi [U-14C]mannitol in combination with 0.5 mCi 3 H 2 0 in a parallel incubation. Incorporation of Label
Lipids and alternariol were extracted and measured as described earlier [5]. The only modification done was the replacement of benzene with toluene in the thin-layer chromatography system. Mannitol was extracted from the cells after the lipid extraction with use of the water and water/methanol phases obtained from washing of the chloroform lipid extracts. Mannito1 was purified as the hexaacetate using a PerkinElmer F 21 preparative gas-liquid chromatograph with a 2.7-m column packed with 5 % SE-30 on 60180 Chromosorb P, temperature 190 "C. Mannitol hexaacetate was prepared in acetic anhydride with anhydrous sodium acetate 100 "C, 30 min. Remaining anhydride was hydrolyzed by the addition of ice and the mannitol hexaacetate was extracted with chloroform. [2,5-3H]Mannitol was added to the cell extracts as internal standard. The activity was counted in a Packard Tri-Carb Scintillation Spectrometer. Glucose was determined with the phenol-sulfuric acid method [6]. Total mannitol was assayed with periodate oxidation [7].
RESULTS AND DISCUSSION Primary Incorporation Results
Table 1 shows the incorporation of label from [U-'4C]glucose into alternariol and fat. The specific activity of glucose was calculated individually for every incubation from the actual glucose concentration and the activity obtained in the media after addition of label. In Table 2 the incorporation in fat from 3H20 is shown. The specific activity of water was measured in the medium after isotope addition. The amount of fat synthesized was calculated using the isotope effect factor 0.87 according to [8]. No correction was made for the glycerol content in fat. Incorporation of [U-'4C]inannitol into alternariol and fat is shown in Table 3. The specific activity of mannitol was calculated from the added amount of radioactivity and the mannitol contents of the cells. The 14C activity in the medium was measured at the end of the incubations to check that all activity was taken up by the cells. Mannitol contents of the cells as measured by periodate oxidation are shown in Table 4. The synthesis of mannitol from glucose was estimated from the incorporation of [U-'4C]glucose into mannitol (Table 5). [2,5-3H]Mannitol was added to the cell extracts as internal standard. Recovery of mannitol in the derivatization step was 70-100 %. The preparative gas liquid chromatography procedure had a recovery of 9 - 15 The uptake of glucose (Table 6) was calculated from the glucose concentration in the medium before and after incubation. The two values for each strain and day are from the two parallel incubations with [U-I4C]glucose and with [U-'4C]niannitol in combination with 3H20 respectively. The uptake of glucose agreed well with the loss of radioactivity in the medium containing [U-'4C]glucose.
x.
Materials
[U-14C]Mannitol and [2,5-3H]mannitol were synthesized from [U-14C]mannose and [2-3H]mannose respectively by reduction with NaBH4. [U-'4C]Glucose and [2-3H]mannose were obtained from The Radiochemical Centre (Amersham, England) and [U-'4C]mannose and 3Hz0 from New England Nuclear (Dreieichenhain, F .R.G .).
Table 1. Incorporation of [U-'4C]glucose info alternariol and fat The mycelium was washed and incubated in 5 ml modified nitrogen-free Czapek-Dox medium supplemented with 0.5 pCi [U-'4C]glucose Strain
Nonproducing
Producing
Age
Incubation time
Cell dry weight
Specific activity of glucose
Alternariol
days
h
mg
dis x inin x(nmolC)-'
dis./min
4
~
Fat
Alternariol
Fat
nmol C x (mg cell dry wt) x h-'
~~
6 8
3.33 4.00 4.10
57.4 65.3 66.9
0.99 0.87 0.95
0 0 0
18785 3580 2769
4 6 8
3.33 4.00 4.10
37.8 70.2 67.4
0.98 0.76 0.81
0 641 797
2909 1816 1727
0 0 0
99 16 11
0 3.0 3.6
24 8.5 7.7
'
609
K. Hult and S. Gatenbeck
Table 2. Incorporation q f 3 H z 0 intofat The mycelium was washed and incubated in 5 ml modified nitrogen-free Czapek-Dox medium supplemented with 0.5 mCi 3H20 and 0.5 pCi [U-14C]mannitol Age
Incubation time
Cell dry weight
Specific activity of " 2 0
Fat
Fat
days
h
mg
dis. x min-' x (nmol H ) - '
dis./min
nmol C x (mg cell dry wtj-' x h
Nonproducing
4 6 8
3.33 4.00 4.10
60.3 67.3 72.0
3.13 3.16 3.12
126030 33214 14824
23 1 45
Producing
4 6 8
3.33 4.00 4.10
33.5 76.1 92.4
3.08 2.94 2.30
7311 6155 6 238
Strain
19
25 8.7 8.2
Table 3. Incorporalion of / U-14C/~annitol into a l t e ~ ~ a ~and.fat iffl The mycelium was washed and incubated in 5 i d modified nitrogen-free Czapek-Dox medium supplemented with 0.5 mCi 0.5 pCi [U-'4C]mannitol Strain
Nonproducing
Producing
Age
Incubation time
Cell dry weight
Specific actlvlty of nlanilltol
Alternariol
days
h
mg
dis. x inin-' x (nmol C)-'
Fat
3.33 4.00 4.10
60.3 67.3 72.0
4.62 5.65 6.50
0 0 0
58810 392Xh
4 6 8
3.33 4.00 4.10
33.5 76.1 92.4
8.66 4.86 2.89
0 903 3847
2939 1512 7 200
Strain
Age
Mannitol
days
nmol C / mg cell dry wt
of cell dry weight
Nonproducing
4 6 8
4840 3400 2820
15 10 8.6
Producing
4 6 8
4850 3750 3080
15 11 9.3
Synthesis of Fat Table 7 summarizes the fat syntheses from glucose and mannitol respectively and compares the total fat synthesis with the sum of fat syntheses from glucose and mannitol. It should be expected that during the incubation the amount of fat formed from glucose by Alternuria alternata, when using glucose as the sole carbon source, is equal to the total amount of fat synthesized. As can
20 1 7 6
0 0 0
and
Fat
_______
~-
4 6 8
Table 4. Munnitol contents of the cells The mycelium was washed and incubated in modified nitrogen-free Czapek-Dox medium for 1 h before extraction of mannitol
Alternariol
3 H 2 0
nmol C x (mg cell dry wt)
dis.jmin
I
'xh
~~
63 26 11
0 06 35
30 10 66
be seen from Table 7 the amount of fat derived from glucose is always smaller than the total amount. As A . alternata contains large quantities of mannito1 (Table 4) unlabelled fructose 6-phosphate can be formed from mannitol, thus diluting the radioactivity of fructose 6-phosphate as compared to that of glucose. The decrease in specific activity would then give a lower estimate of fat synthesis from glucose than the true amount of total fat synthesis. To test this hypothesis, [U-14C]mannitol was added to the medium and its incorporations into fat and alternariol were measured. The quantity of fat synthesized from endogenous mannitol agrees well with the difference of total fat and fat from glucose (Table 7). The conclusion is that mannitol is the endogenous carbon source responsible for the additional fat synthesis. Carbon Flux fkom Glucose and Fructose to Fructose 6-Phosphate The flux from mannitol to fructose 6-phosphate compared to the flux from glucose to fructose 6-phosphate is calculated from the ratio of fat formed from mannitol and glucose respectively.
The Mannitol Cycle in Altrrnaria alternata
610
Table 5. Mannitol synthesisfr-om glucose Incubation conditions were as stated in the legend of Table 1, Synthesized ['4C]mannitol was measured by liquid scintillation technique after purification by preparative gas-liquid chromatography with [2,5-'H]mannitol as internal standard Strain
Age
Incubdtion time
Cell dry weight
h
days
Specific activity of glucose
mg
14C
Added [2,5-%]mannitol
dis x m i n x(nmo1C)
dis /min
Recovered mannitol ~
3H __
~
Synthesized mannitol
~~
I4C
~~~
nmol C/mg cell dry wt
~
Nonproducing
4 6 8
3 33 4 00 4 10
57 4 65 3 66 9
0 99 0 87 0 95
290000 145 000 145 000
5 787 9032 I6322
1985 2784 5644
530 200 190
Producing
4 6 8
3 33 4 00 4 10
37 8 70 2 67 4
0 98 0 76 0 81
290000 I45 000 145000
16197 6 672 3317
3276 2661 1276
480 270 250
Table 6 Uptuke of glucose [ncubation conditions were as stated in the legcnds of Tables 1 and 2 Str'iin
Incubdtion time
Age
days
h
Cell diY weight
Glucose concenttation
n'g
mM
.~-
_.
before incubation
Producing
57.4 60.3 65.3 67.3 66.9 72.0
23.4 20.1 29.0 28.2 26.9 27.6
15.6 13.5 23.5 21.6 24.9 25.8
1224 986 632 724 219 183
4 4 6 6 8 8
3.33 3.33 4.00 4.00 4.10 4.10
37.8 33.5 70.2 76.1 67.4 92.4
25.1 24.3 30.0 30.8 27.9 27.9
20.2 19.9 25.7 26.7 25.5 25.0
1168 1183 460 404 260 229
Fat synthes15 from __ glucose mannitol
T o t d fat
~
Nonproducing
Producing
days
nmol C x (mg cell dry wt)-' x h - '
4 6 8
99 16
63 26
231 45
11
11
19
4 6 8
24 85 77
30 10 66
25 87 82
The total carbon flux from glucose to glucose 6-phosphate is taken as being equal to the glucose uptake from the medium (Table 6). Glucose 6-phosphate and fructose 6-phosphate are rapidly equilibrated by the glucosephosphate isomerase which
~.
~
~~~~
3.33 3.33 4.00 4.00 4.10 4.10
Age
~-
mean
4 4 6 6 8 8
Table 7. Fut .syntlie.~is See legend of Tables 1 and 2 Strain
~~
after incubation
niiiol c ' x (ins CCIIdr! w t ) __
Nonproducing
Glucose uptake
' x 11 '
~-
1 110
680 200 1 I80
430 250
means that the two compounds have the same specific radioactivity. The total carbon flux from glucose to fructose 6-phosphate and the ratio of fat formed from mannitol and glucose discussed above permit the calculation of the total flux of mannitol to fructose 6-phosphaie (Table 8).
Synthesis of Mannitol Mannitol synthesis from glucose is shown in Table 5. The figures represent, as in the case with fat, only the synthesis from glucose and are not equal to the total amount of mannitol formed. Glucose, when incorporated into mannitol, passes through fructose 6-phosphate and the specific activity of glucose is thus diluted by unlabelled mannitol as in the fat synthesis. The total carbon flux to mannitol is hence calculated from the glucose incorporation data (Table 5) and from the incorporation ratio of mannitol and glucose into fat. The results are shown in Table 8.
K Hult dnd S Gatenbeck
61 1
Table 8 Metcrhoht fluxes The metabolic fluxes were calculated from data of Tables 5,6 and 7 taking into account the dilution of specific activities of glucose 6-phosphdte nnd fructose 6-phosphate Strain
P'ithway ,md enzyme _ _ _ _ __ Glucose-. Mdnnitol-t Glu-6- P, fi uctose-t Fru-6-P, hexokinase mannitol dehydrogendse + hcxokinnse
Age
days
nmol C x (mg cell dry wt)
~-
~
Glucosc+Glu-h-P fructose+Fiu-6-P, hexokinase
+
~-
____
-
Nonproducing
4 6 8
1110 680 200
700 1110 200
1810 1790 400
860 520 180
Producing
4 6 8
1180 430 250
1 50 50 210
1330 480 460
540 300 460
Table 9. Alrerncrriol synthesis See legend ol' Tables 1 and 2
Glucose _____
Strain
Age
Alternariol synthesis from ___. __ __ glucose mmnitol glucose + innnnitol ~~
Nonproducing
PI oducn1g
days
iimol C x (mg cell dry wt)-' x
4 6 8
0 0
4 6 8
0 0 30
36
~
Fru-6-P+mdnnitol-l -P-+ mannitol, mannitol-1-P dehydrogendw + mannitol-1-pliosphntdse
xh -__
~
~
0 0 0
0 0 0
0 06 35
0 3.6 7.1
11-I
Alternariol Production Alternariol productions from glucose and mannitol are summarized in Table 9. The total synthesis of alternariol cannot be determined from 3H20 incorporation but is taken as the sum of the synthesis from glucose and mannitol respectively.
Conclusions Fig.2 shows the enzymic reactions involved in mannitol metabolism in A . alternuta (Hult, unpublished results). The enzymes matinitol-1-phosphate dehydrogenase and mannitol-1 -phosphatase are involved in the biosynthesis of mannitol as the reaction catalyzed by mannitol-1-phosphatase is irreversible. This leaves mannitol dehydrogenase to the utilization of mannitol. The high mannitol concentration in the cells and the lack of a fructose source in fungi, as shown by Strandberg in Aspergillus candidus [9] are compatible with a flux from mannitol to fructose catalyzed by mannitol dehydrogenase. The enzymes in Asp. candidus dealing with mannitol metabolism,
I-
NADPH
A TP
ATP]
Glucose &-phosphate
Fructose
Fructose 6-phosphate
I+
NAD
4 1 -
Mannitol
Mannitol I-phosphate
4 7
/ ~ / i~A / . allcrFig. 2. E~?:j,micrraclions i n i d v e d in ? ~ t ~ r / l tnw/trho/isru nata. The following enzymes participate in the m;innilol cycle: mannitol-1-phosphate dehydrogenase. maniiitol-1-phosphatase, mannitol dehydrogenase and hexokinase. The cycle produces NADPH at the expense of N A D H and ATP
as described by Strandberg [9], are the same as in A . alternata. It is shown in this paper (Table 8) that mannitol simultaneously is formed and utilized. Mannitol is hence not only a reserve carbon source but is also metabolically very active. The experimental results suggest that there exists in fungi a mannitol cycle with the participation of the following enzymes : mannitol-1-phosphate dehydrogenase, mannitol-lphosphatase, mannitol dehydrogenase and hexokinase. One turn of the cycle gives the net result: NADH + NADP' + ATP-NAD' + ADP + P i . The importance of the cycle for the NADPH production in the metabolism is evident from the high fluxes through mannitol-1-phosphatase and rnannjtol dehydrogenase as compared to the flux through hexokinase. The total flow through hexokinase is the sum of glucose and mannitol consumption as the fructose formed from mannitol is used by hexokinase. Hexokinase from A . alternatu is active with fructose as substrate (Hult, unpublished results).
612
K. Hult and S. Gatenbeck: The Mannitol Cycle in Alternaria alternata
Table 10. Relative metaholic,fluxes compared to hexokinase The figures were calculated from data in Table 8
Table 11. N A D P H balance The figures were calculated from data in Table 8 and Table 2
Strain
Strain
Age
days
Hexokinase total
Mannitol dehydrogenase
%
Producing
Age
Production by mannitol oxidation
days
nmol NADPH x (mg cell dry wt)-' x h - ' -~ 117 231 185 45 33 19
~-
~~
Nonproducing
Mannitol-lphosphatase
4 6 8
100 100 100
39 62 50
48 29 95
4 6 8
100 100 100
11 10 46
41 63 100
Table 10 shows the relative metabolic fluxes in the mannitol cycle compared with the total flux through hexokinase. On average more than 50% of the flux through hexokinase is passed through mannitol-lphosphatase. The high flux through mannitol-l-phosphate dehydrogenase and mannitol-1-phosphatase is remarkable and it may indicate the site of a regulation mechanism of the glycolysis by fructose 6-phosphate withdrawal from phosphofructokinase monitored by the NADH availability. The importance of the mannitol cycle for the NADPH production in the organism is evident as the mannitol oxidation alone can meet the total need of NADPH for fat synthesis (Table 11). Variations in mannitol oxidation will thus indicate the availability of NADPH in the cells. The alternariol-producing strain of A . altevnata has a lower mannitol oxidation and a lower fat synthesis than the nonproducing strain, which could imply that a shortage of NADPH in the cell could contribute to the explanation of why the polyketide
~~
Nonproducing
4 6 8
Producing
4 6 8
25 8.5 35
Consumption in fat synthesis
~
-
25 87 82
alternariol is formed. On the other hand, the low mannitol oxidation in the producing strain could be the consequence of an adaption to a reduced fat synthesis. This work was supported by grants from the Swedish Natural Science Research Council.
REFERENCES 1. Blumenthal, H. J. (1976) Tile Filamentous Fungi, Volume 11, Biosynthesis and Metabolism (Smith, J. E. and Berry, D. R., eds) pp. 292- 307, Edward Arnold (Publishers) Ltd, London. 2. Raistrick, H., Stickings, C. E. & Thomas, R. (1953) Biochem. J . 55, 421 -433. 3. Gatenbeck, S. & Hermodsson, S. (1965) Acta Cbem. Scand. 19, 65-72. 4. Mosbach, K. & Bivertoft, I. (1971) Acta Ckem. Scand. 25, 1931 - 1936. 5. Hult, K. & Gatenbeck, S. (1976) Acta Chem. Scand. B 3 0 , 283- 286. 6. Ashwell, G. (1966) Methods Enzymol. 8, 85-95. 7. Lewis, D. H. & Smith, D. C. (1967) New Phytol. 66, 185-204. 8. Jungas, R. L. (1965) Biochemistry, 7, 3708-3717. 9. Strandberg, G . W. (1969) J . Bacferiol. 97, 1305-1309.
K . Hult and S. Gatenbeck, Institutionen for Biokemi och Biokemisk Teknologi, Kungliga Telmiska Hogskolan, S-100 44 Stockholm 70, Sweden
Production of NADPH in the Mannitol Cycle and Its Relation to Polyketide Formation in Alternaria alternata Karl HULT and Sten GATENBECK Department of Pure and Applied Biochemistry, Royal Institute of Technology, Stockholm (Received February 4, 1978)
The enzymes mannitol-1-phosphate dehydrogenase, mannitol-I-phosphatase, mannitol dehydrogenase and hexokinase participate in an enzymatic cycle in the fungus Alternuria alternata. One turn of the cycle gives the net result: NADH
+ NADP' + ATP + NAD' + NADPH + A DP + P , .
The cycle alone can meet the total need of NADPH formation for fat synthesis in the organism. A polyketide producing strain of A . altrrnuta shows a lower mannitol oxidation as well as a lower fat synthesis than a nonproducing mutant, supporting the hypothesis that polyketide formation is favoured at limiting NADPH production. It is further suggested that the mannitol cycle is regulating the glycolytic flux by substrate withdrawal from phosphofructokinase.
The occurrence of polyols, particularly mannitol, in fungi has been known for several decades. The metabolism and the presence of mannitol in fungi are discussed in a recent review by Blumenthal [l]. Blumenthal concludes that mannitol serves as a reserve of carbon nutrient and perhaps also as a form of stored reducing power. The metabolic steps for niannitol synthesis and degradation seem to be uncertain mainly because of the manifold alternative pathways offered with the participation of the known enzymes. In this paper a new view on mannitol metabolism in the imperfect fungi is suggested. It is proposed that mannitol is involved in a cycle producing NADPH with the utilization of N A D H and ATP. The organism which is used in the described studies i s A l t e ~ ~ ualtcmatn. r~u This mould is known to produce the polyketide alternariol (Fig. 1) [2]. Alternariol is synthesized from acetyl-CoA and malonyl-CoA in a manner similar to the fatty acid synthesis but without the involvement of the NADPH-dependent reductions [3]. It has been suggested that the production of polyketides reflects a limiting availability of NADPH for fatty acid synthesis [4]. The proposed role of mannito1 in the NADPH production made it interesting to study the mannitol metabolism in relation to lipid synthesis and alternariol formation in an alternariolproducing strain of A . alternata as compared to a nonproducing mutant. f+qwws. Mannitol-I-phosphate dehydrogenase (EC 1.1.1.17); mannitol-I-phosphatase (EC 3.1.3.22); mannitol dehydrogenase (EC 1.1.1.138); hexokinase (EC 2.7.1 . I ) ; glucosephosphate isomerase (EC 5.3.1.9); phosphofructokinase (EC 2.7.1.11).
Fig. 1 . Alrci
MATERIALS AND METHODS
Cultural Conditions The alternariol-producing strain Alternuria alturnata (CMI 89343) was obtained from Commonwealth Mycological Institute (Kew, Surrey). The nonproducing strain was a spontaneous mutant isolated from a producing culture. Conical flasks of 500-in1 capacity containing 150 ml modified Czapek-Dox medium 151 were inoculated from agar slants and incubated on a rotary shaking table at 25 'C. Portions of the mycelium were harvested by filtration at appropriate times.
Incubations Portions of wet mycelium (0.70 g) were incubated in 25-ml conical flasks containing 7 ml modified nitrogen-free Czapek-Dox medium with 5 g glucose per 1000 ml. The flasks were incubated on a table rotary shaker at 20 "C. Two parallel incubations were run for each strain. After 1 h 2-ml samples were taken for analysis of glucose concentration in the medium and
The Mannitol Cycle in Alternaria alternata
608
of cellular mannitol. To the remaining 5 ml of the cultures labelled substrates were added and the incubations were continued for the time indicated. The following labelled compounds were used: 0.5 pCi [U-'4C]glucose in a separate incubation and 0.5 pCi [U-14C]mannitol in combination with 0.5 mCi 3 H 2 0 in a parallel incubation. Incorporation of Label
Lipids and alternariol were extracted and measured as described earlier [5]. The only modification done was the replacement of benzene with toluene in the thin-layer chromatography system. Mannitol was extracted from the cells after the lipid extraction with use of the water and water/methanol phases obtained from washing of the chloroform lipid extracts. Mannito1 was purified as the hexaacetate using a PerkinElmer F 21 preparative gas-liquid chromatograph with a 2.7-m column packed with 5 % SE-30 on 60180 Chromosorb P, temperature 190 "C. Mannitol hexaacetate was prepared in acetic anhydride with anhydrous sodium acetate 100 "C, 30 min. Remaining anhydride was hydrolyzed by the addition of ice and the mannitol hexaacetate was extracted with chloroform. [2,5-3H]Mannitol was added to the cell extracts as internal standard. The activity was counted in a Packard Tri-Carb Scintillation Spectrometer. Glucose was determined with the phenol-sulfuric acid method [6]. Total mannitol was assayed with periodate oxidation [7].
RESULTS AND DISCUSSION Primary Incorporation Results
Table 1 shows the incorporation of label from [U-'4C]glucose into alternariol and fat. The specific activity of glucose was calculated individually for every incubation from the actual glucose concentration and the activity obtained in the media after addition of label. In Table 2 the incorporation in fat from 3H20 is shown. The specific activity of water was measured in the medium after isotope addition. The amount of fat synthesized was calculated using the isotope effect factor 0.87 according to [8]. No correction was made for the glycerol content in fat. Incorporation of [U-'4C]inannitol into alternariol and fat is shown in Table 3. The specific activity of mannitol was calculated from the added amount of radioactivity and the mannitol contents of the cells. The 14C activity in the medium was measured at the end of the incubations to check that all activity was taken up by the cells. Mannitol contents of the cells as measured by periodate oxidation are shown in Table 4. The synthesis of mannitol from glucose was estimated from the incorporation of [U-'4C]glucose into mannitol (Table 5). [2,5-3H]Mannitol was added to the cell extracts as internal standard. Recovery of mannitol in the derivatization step was 70-100 %. The preparative gas liquid chromatography procedure had a recovery of 9 - 15 The uptake of glucose (Table 6) was calculated from the glucose concentration in the medium before and after incubation. The two values for each strain and day are from the two parallel incubations with [U-I4C]glucose and with [U-'4C]niannitol in combination with 3H20 respectively. The uptake of glucose agreed well with the loss of radioactivity in the medium containing [U-'4C]glucose.
x.
Materials
[U-14C]Mannitol and [2,5-3H]mannitol were synthesized from [U-14C]mannose and [2-3H]mannose respectively by reduction with NaBH4. [U-'4C]Glucose and [2-3H]mannose were obtained from The Radiochemical Centre (Amersham, England) and [U-'4C]mannose and 3Hz0 from New England Nuclear (Dreieichenhain, F .R.G .).
Table 1. Incorporation of [U-'4C]glucose info alternariol and fat The mycelium was washed and incubated in 5 ml modified nitrogen-free Czapek-Dox medium supplemented with 0.5 pCi [U-'4C]glucose Strain
Nonproducing
Producing
Age
Incubation time
Cell dry weight
Specific activity of glucose
Alternariol
days
h
mg
dis x inin x(nmolC)-'
dis./min
4
~
Fat
Alternariol
Fat
nmol C x (mg cell dry wt) x h-'
~~
6 8
3.33 4.00 4.10
57.4 65.3 66.9
0.99 0.87 0.95
0 0 0
18785 3580 2769
4 6 8
3.33 4.00 4.10
37.8 70.2 67.4
0.98 0.76 0.81
0 641 797
2909 1816 1727
0 0 0
99 16 11
0 3.0 3.6
24 8.5 7.7
'
609
K. Hult and S. Gatenbeck
Table 2. Incorporation q f 3 H z 0 intofat The mycelium was washed and incubated in 5 ml modified nitrogen-free Czapek-Dox medium supplemented with 0.5 mCi 3H20 and 0.5 pCi [U-14C]mannitol Age
Incubation time
Cell dry weight
Specific activity of " 2 0
Fat
Fat
days
h
mg
dis. x min-' x (nmol H ) - '
dis./min
nmol C x (mg cell dry wtj-' x h
Nonproducing
4 6 8
3.33 4.00 4.10
60.3 67.3 72.0
3.13 3.16 3.12
126030 33214 14824
23 1 45
Producing
4 6 8
3.33 4.00 4.10
33.5 76.1 92.4
3.08 2.94 2.30
7311 6155 6 238
Strain
19
25 8.7 8.2
Table 3. Incorporalion of / U-14C/~annitol into a l t e ~ ~ a ~and.fat iffl The mycelium was washed and incubated in 5 i d modified nitrogen-free Czapek-Dox medium supplemented with 0.5 mCi 0.5 pCi [U-'4C]mannitol Strain
Nonproducing
Producing
Age
Incubation time
Cell dry weight
Specific actlvlty of nlanilltol
Alternariol
days
h
mg
dis. x inin-' x (nmol C)-'
Fat
3.33 4.00 4.10
60.3 67.3 72.0
4.62 5.65 6.50
0 0 0
58810 392Xh
4 6 8
3.33 4.00 4.10
33.5 76.1 92.4
8.66 4.86 2.89
0 903 3847
2939 1512 7 200
Strain
Age
Mannitol
days
nmol C / mg cell dry wt
of cell dry weight
Nonproducing
4 6 8
4840 3400 2820
15 10 8.6
Producing
4 6 8
4850 3750 3080
15 11 9.3
Synthesis of Fat Table 7 summarizes the fat syntheses from glucose and mannitol respectively and compares the total fat synthesis with the sum of fat syntheses from glucose and mannitol. It should be expected that during the incubation the amount of fat formed from glucose by Alternuria alternata, when using glucose as the sole carbon source, is equal to the total amount of fat synthesized. As can
20 1 7 6
0 0 0
and
Fat
_______
~-
4 6 8
Table 4. Munnitol contents of the cells The mycelium was washed and incubated in modified nitrogen-free Czapek-Dox medium for 1 h before extraction of mannitol
Alternariol
3 H 2 0
nmol C x (mg cell dry wt)
dis.jmin
I
'xh
~~
63 26 11
0 06 35
30 10 66
be seen from Table 7 the amount of fat derived from glucose is always smaller than the total amount. As A . alternata contains large quantities of mannito1 (Table 4) unlabelled fructose 6-phosphate can be formed from mannitol, thus diluting the radioactivity of fructose 6-phosphate as compared to that of glucose. The decrease in specific activity would then give a lower estimate of fat synthesis from glucose than the true amount of total fat synthesis. To test this hypothesis, [U-14C]mannitol was added to the medium and its incorporations into fat and alternariol were measured. The quantity of fat synthesized from endogenous mannitol agrees well with the difference of total fat and fat from glucose (Table 7). The conclusion is that mannitol is the endogenous carbon source responsible for the additional fat synthesis. Carbon Flux fkom Glucose and Fructose to Fructose 6-Phosphate The flux from mannitol to fructose 6-phosphate compared to the flux from glucose to fructose 6-phosphate is calculated from the ratio of fat formed from mannitol and glucose respectively.
The Mannitol Cycle in Altrrnaria alternata
610
Table 5. Mannitol synthesisfr-om glucose Incubation conditions were as stated in the legend of Table 1, Synthesized ['4C]mannitol was measured by liquid scintillation technique after purification by preparative gas-liquid chromatography with [2,5-'H]mannitol as internal standard Strain
Age
Incubdtion time
Cell dry weight
h
days
Specific activity of glucose
mg
14C
Added [2,5-%]mannitol
dis x m i n x(nmo1C)
dis /min
Recovered mannitol ~
3H __
~
Synthesized mannitol
~~
I4C
~~~
nmol C/mg cell dry wt
~
Nonproducing
4 6 8
3 33 4 00 4 10
57 4 65 3 66 9
0 99 0 87 0 95
290000 145 000 145 000
5 787 9032 I6322
1985 2784 5644
530 200 190
Producing
4 6 8
3 33 4 00 4 10
37 8 70 2 67 4
0 98 0 76 0 81
290000 I45 000 145000
16197 6 672 3317
3276 2661 1276
480 270 250
Table 6 Uptuke of glucose [ncubation conditions were as stated in the legcnds of Tables 1 and 2 Str'iin
Incubdtion time
Age
days
h
Cell diY weight
Glucose concenttation
n'g
mM
.~-
_.
before incubation
Producing
57.4 60.3 65.3 67.3 66.9 72.0
23.4 20.1 29.0 28.2 26.9 27.6
15.6 13.5 23.5 21.6 24.9 25.8
1224 986 632 724 219 183
4 4 6 6 8 8
3.33 3.33 4.00 4.00 4.10 4.10
37.8 33.5 70.2 76.1 67.4 92.4
25.1 24.3 30.0 30.8 27.9 27.9
20.2 19.9 25.7 26.7 25.5 25.0
1168 1183 460 404 260 229
Fat synthes15 from __ glucose mannitol
T o t d fat
~
Nonproducing
Producing
days
nmol C x (mg cell dry wt)-' x h - '
4 6 8
99 16
63 26
231 45
11
11
19
4 6 8
24 85 77
30 10 66
25 87 82
The total carbon flux from glucose to glucose 6-phosphate is taken as being equal to the glucose uptake from the medium (Table 6). Glucose 6-phosphate and fructose 6-phosphate are rapidly equilibrated by the glucosephosphate isomerase which
~.
~
~~~~
3.33 3.33 4.00 4.00 4.10 4.10
Age
~-
mean
4 4 6 6 8 8
Table 7. Fut .syntlie.~is See legend of Tables 1 and 2 Strain
~~
after incubation
niiiol c ' x (ins CCIIdr! w t ) __
Nonproducing
Glucose uptake
' x 11 '
~-
1 110
680 200 1 I80
430 250
means that the two compounds have the same specific radioactivity. The total carbon flux from glucose to fructose 6-phosphate and the ratio of fat formed from mannitol and glucose discussed above permit the calculation of the total flux of mannitol to fructose 6-phosphaie (Table 8).
Synthesis of Mannitol Mannitol synthesis from glucose is shown in Table 5. The figures represent, as in the case with fat, only the synthesis from glucose and are not equal to the total amount of mannitol formed. Glucose, when incorporated into mannitol, passes through fructose 6-phosphate and the specific activity of glucose is thus diluted by unlabelled mannitol as in the fat synthesis. The total carbon flux to mannitol is hence calculated from the glucose incorporation data (Table 5) and from the incorporation ratio of mannitol and glucose into fat. The results are shown in Table 8.
K Hult dnd S Gatenbeck
61 1
Table 8 Metcrhoht fluxes The metabolic fluxes were calculated from data of Tables 5,6 and 7 taking into account the dilution of specific activities of glucose 6-phosphdte nnd fructose 6-phosphate Strain
P'ithway ,md enzyme _ _ _ _ __ Glucose-. Mdnnitol-t Glu-6- P, fi uctose-t Fru-6-P, hexokinase mannitol dehydrogendse + hcxokinnse
Age
days
nmol C x (mg cell dry wt)
~-
~
Glucosc+Glu-h-P fructose+Fiu-6-P, hexokinase
+
~-
____
-
Nonproducing
4 6 8
1110 680 200
700 1110 200
1810 1790 400
860 520 180
Producing
4 6 8
1180 430 250
1 50 50 210
1330 480 460
540 300 460
Table 9. Alrerncrriol synthesis See legend ol' Tables 1 and 2
Glucose _____
Strain
Age
Alternariol synthesis from ___. __ __ glucose mmnitol glucose + innnnitol ~~
Nonproducing
PI oducn1g
days
iimol C x (mg cell dry wt)-' x
4 6 8
0 0
4 6 8
0 0 30
36
~
Fru-6-P+mdnnitol-l -P-+ mannitol, mannitol-1-P dehydrogendw + mannitol-1-pliosphntdse
xh -__
~
~
0 0 0
0 0 0
0 06 35
0 3.6 7.1
11-I
Alternariol Production Alternariol productions from glucose and mannitol are summarized in Table 9. The total synthesis of alternariol cannot be determined from 3H20 incorporation but is taken as the sum of the synthesis from glucose and mannitol respectively.
Conclusions Fig.2 shows the enzymic reactions involved in mannitol metabolism in A . alternuta (Hult, unpublished results). The enzymes matinitol-1-phosphate dehydrogenase and mannitol-1 -phosphatase are involved in the biosynthesis of mannitol as the reaction catalyzed by mannitol-1-phosphatase is irreversible. This leaves mannitol dehydrogenase to the utilization of mannitol. The high mannitol concentration in the cells and the lack of a fructose source in fungi, as shown by Strandberg in Aspergillus candidus [9] are compatible with a flux from mannitol to fructose catalyzed by mannitol dehydrogenase. The enzymes in Asp. candidus dealing with mannitol metabolism,
I-
NADPH
A TP
ATP]
Glucose &-phosphate
Fructose
Fructose 6-phosphate
I+
NAD
4 1 -
Mannitol
Mannitol I-phosphate
4 7
/ ~ / i~A / . allcrFig. 2. E~?:j,micrraclions i n i d v e d in ? ~ t ~ r / l tnw/trho/isru nata. The following enzymes participate in the m;innilol cycle: mannitol-1-phosphate dehydrogenase. maniiitol-1-phosphatase, mannitol dehydrogenase and hexokinase. The cycle produces NADPH at the expense of N A D H and ATP
as described by Strandberg [9], are the same as in A . alternata. It is shown in this paper (Table 8) that mannitol simultaneously is formed and utilized. Mannitol is hence not only a reserve carbon source but is also metabolically very active. The experimental results suggest that there exists in fungi a mannitol cycle with the participation of the following enzymes : mannitol-1-phosphate dehydrogenase, mannitol-lphosphatase, mannitol dehydrogenase and hexokinase. One turn of the cycle gives the net result: NADH + NADP' + ATP-NAD' + ADP + P i . The importance of the cycle for the NADPH production in the metabolism is evident from the high fluxes through mannitol-1-phosphatase and rnannjtol dehydrogenase as compared to the flux through hexokinase. The total flow through hexokinase is the sum of glucose and mannitol consumption as the fructose formed from mannitol is used by hexokinase. Hexokinase from A . alternatu is active with fructose as substrate (Hult, unpublished results).
612
K. Hult and S. Gatenbeck: The Mannitol Cycle in Alternaria alternata
Table 10. Relative metaholic,fluxes compared to hexokinase The figures were calculated from data in Table 8
Table 11. N A D P H balance The figures were calculated from data in Table 8 and Table 2
Strain
Strain
Age
days
Hexokinase total
Mannitol dehydrogenase
%
Producing
Age
Production by mannitol oxidation
days
nmol NADPH x (mg cell dry wt)-' x h - ' -~ 117 231 185 45 33 19
~-
~~
Nonproducing
Mannitol-lphosphatase
4 6 8
100 100 100
39 62 50
48 29 95
4 6 8
100 100 100
11 10 46
41 63 100
Table 10 shows the relative metabolic fluxes in the mannitol cycle compared with the total flux through hexokinase. On average more than 50% of the flux through hexokinase is passed through mannitol-lphosphatase. The high flux through mannitol-l-phosphate dehydrogenase and mannitol-1-phosphatase is remarkable and it may indicate the site of a regulation mechanism of the glycolysis by fructose 6-phosphate withdrawal from phosphofructokinase monitored by the NADH availability. The importance of the mannitol cycle for the NADPH production in the organism is evident as the mannitol oxidation alone can meet the total need of NADPH for fat synthesis (Table 11). Variations in mannitol oxidation will thus indicate the availability of NADPH in the cells. The alternariol-producing strain of A . altevnata has a lower mannitol oxidation and a lower fat synthesis than the nonproducing strain, which could imply that a shortage of NADPH in the cell could contribute to the explanation of why the polyketide
~~
Nonproducing
4 6 8
Producing
4 6 8
25 8.5 35
Consumption in fat synthesis
~
-
25 87 82
alternariol is formed. On the other hand, the low mannitol oxidation in the producing strain could be the consequence of an adaption to a reduced fat synthesis. This work was supported by grants from the Swedish Natural Science Research Council.
REFERENCES 1. Blumenthal, H. J. (1976) Tile Filamentous Fungi, Volume 11, Biosynthesis and Metabolism (Smith, J. E. and Berry, D. R., eds) pp. 292- 307, Edward Arnold (Publishers) Ltd, London. 2. Raistrick, H., Stickings, C. E. & Thomas, R. (1953) Biochem. J . 55, 421 -433. 3. Gatenbeck, S. & Hermodsson, S. (1965) Acta Cbem. Scand. 19, 65-72. 4. Mosbach, K. & Bivertoft, I. (1971) Acta Ckem. Scand. 25, 1931 - 1936. 5. Hult, K. & Gatenbeck, S. (1976) Acta Chem. Scand. B 3 0 , 283- 286. 6. Ashwell, G. (1966) Methods Enzymol. 8, 85-95. 7. Lewis, D. H. & Smith, D. C. (1967) New Phytol. 66, 185-204. 8. Jungas, R. L. (1965) Biochemistry, 7, 3708-3717. 9. Strandberg, G . W. (1969) J . Bacferiol. 97, 1305-1309.
K . Hult and S. Gatenbeck, Institutionen for Biokemi och Biokemisk Teknologi, Kungliga Telmiska Hogskolan, S-100 44 Stockholm 70, Sweden
