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On the mechanism of enzyme action. LIX. A relation between the structure of mold pigments and their interaction with enzymes
On the Mechanism of Enzyme Action. LIX. A Relation Between the Structure of Mold Pigments and Their Interaction with Enzymes Basilio Lopez-Ramosl and Walter J. Schubert2 From the Department
of Organic Chemistry and h’nzymology,’ University, New York, New York Received
October
Fotdham
8, 1954
INTRODUCTION
A consideration of the pigments identified in such diverse fungal genera as the Penicillia, AspergiUi, Helminthospora, or Fusaria reveals the fact that molds, in general, are capable of synthesizing a wide variety of colored compounds. Yet, with all the knowledge concerning the structures of these pigments, relatively little information is available on their possible interactions in the metabolic activities occurring within the living cell (1). Within the genus Fusarium, Nerd et al. demonstrated that certain xanthones affect the rate at which Fusarium Zini Bolley (FlB) dehydrogenates isopropyl alcohol (2). The observation that pigments affect dehydrogenating systems in Fusaria (3) prompted a consideration of their influence in fat formation. Thus, it was observed that when solanione, a pigment from Fusarium soZuni D2 purple (FsD) (4) was added to the growth medium of FIB not only is the quantity of fat produced affected by the presence of this pigment (5), but it.s qualitative composition is also changed (6). In distinction to the action of solanione, the presence of lycopersin in Fusarium lywpertii and Fusarium vasinfectum is accompanied by the 1 Holder of a Scholarship of the Government of the Commonwealth of Puerto Rico. * Procter and Gamble Fellow. 3 Communication No. 294. For the previous paper of this series see Arch. Riothem. and Riophys. 62, 464 (1954). This study was assisted by grants from the Office of Naval Research nnd the National Science Foundation. 566
PIGMENTS AND THEIR INTERACTION WITH ENZYMES
567
formation of a less desaturated fat than is formed by the unpigmented cells of these organisms (7). Furthermore, the pigment radicinin, from Stemphylium radicinum (Sterad), appreciably increased the rate of dehydrogenation of isopropyl alcohol by FlB (8). FsD and Sterad thus represent two typical, though unrelated, pigmented organisms, while Lentinus lepideus (Lelep) and Daedalea quercina (Daquer) are two wood-destroying Basidiomycetes of the “brown rot” type. The former decays softwoods, and the latter attacks hardwoods, but neither organism is pigmented. The selection of Lelep was furthermore motivated by the fact that it contains an enzyme system capable of effecting t)he transition from carbohydrates to a methoxylated aromatic compound (9). We are reporting here on the relation between the structure of solanione and its role in the enzymatic dehydrogenation of ethyl alcohol. EXPERIMENTAL The organisms employed in these experiments were FsD, Sterad, Lelep, and Daquer. FsD and Sterad were obtained from Dr. Wm. C. Snyder, Division of Plant Pathology, University of California. Lelep and Daquer were obtained from Dr. Wm. J. Robbins of the New York Botanical Garden. FsD was cultivated on a modified Czapek-Dox medium (10). Sterad was grown on a standard synthetic medium (8). Lelep and Daquer were both cultivated on a glucose-Neopeptonesalt-thiamin-containing medium (11). Alcohol dehydrogenase (twice recrystallized) and diphosphopyridine nucleotide (95% pure) were obtained from the Worthington Biochemical Corporation, Freehold, S. J.
Analytical Methods IZesidual Sugars. Glucose, galactose, and xylose were determined by precipitation of cuprous oxide (12a), and subsequent titration with potassium permanganate (12b). Ethyl alcohol was determined by a titrimetric method (13) after possible interfering substances had been removed (14). Acetaldehyde was determined spectrophotometrically, after conversion t,o iodoform by hypoiodite (15). pH of medium was determined using a Cambridge electron-ray pH meter.
RESULTS
When cultivated on media containing glucose as carbon source, all four organisms studied produced measurable quantities of ethyl alcohol (Fig. 1). FsD was by far the best alcohol former, producing over 300 mg. al-
568
B.
LOPEZ-RAMOS
AKD
W.
J. SCHUBERT 1
0 F. sdani 0 S. radicinum A L Iepideus 0 D. quwcina
cohol/lOO ml. medium after but 10 days of cultivation. The other three organisms produced lesser amounts. h significant obscrvat.ion was the sharp decrease in t.he alcohol level in t.he FsD media aft.er t.he maximum at 10 days. This seemed to indicate a dehydrogenat.ion of the alcohol during the course of the metabolic processes of t.he organism. Following t.he formation of alcohol from glucose by these organisms, their ability t.o ferment other sugars was investigated. For this purpose, the behavior of galactose (Table I) and xylose (Table II) was studied. TABLE Metabolism Organism
I
of Gulactose ‘be days
Residual
sugar
g.1100 ntl.
Alcohol mg.fIOO
F. solani
10 20 30
5.4 0.3 0.2
46.6 5.7 14.1
S. radicinum
10 20 30
3.4 2.3 1.9
16.2 13.2 15.2
L. lepideus
10 20 30
2.0 1.9 2.0
20.3 7.5 13.3
I). quercina
10 20 30
2.2 2.1 2.4
16.2 11.3 20.8
ml.
PIGMENTS
AND
THEIR
INTERACTION
TABLE Organism
WITH
ENZYMES
sugar g./IOO ml.
Alcohol mg./lOO ml
II
Metabolism of Xylose A@- Residual days
569
F. solani
10 20 30
5.4 0.2 0.2
80.1 5.7 9.5
S. radicinum
10 20 30
3.7 2.5 2.5
32.4 13.2 26.5
L. lepideus
10 20 30
2.0 1.9 1.9
28.3 5.7 10.3
D. quercina
10 20 30
2.0 2.0 0.8
12.2 15.2 19.0
Again, all four organisms were able to produce measurable amounts of alcohol from the two sugars. As in the case of the glucose experiments, FsD was still the most efficient fermenter. However, the relat’ive amounts of alcohol formed by this organism from both galactose (46.6 mg.) and xylose (80.1 mg.) were quite inferior to the amount’ produced from glucose (321.4 mg.). Indeed, in no instance was any of the four organisms able to give rise to as much ethyl alcohol from either galactose or xylose as it had from glucose. However, in the case of FsD, Sterad, and Lelep, when developing on both galactose and xylose, the maximum concent’ration of ethyl alcohol was invariably reached at 10 days (with some slight upturn occurring at 30 days). Since control experiments have shown that this could not, have been due to evaporation, this established that the formed alcohol was being further utilized by the three organisms. In view of t,his, a dehydrogenation was considered to be the introductory step. To investigate this possibility, an experiment was performed in which the effect of added alcohol dehydrogenase was studied on the ability of resting cells of the pigmented organisms, FsD and Sterad, to dehydrogenate ethyl alcohol. Thus, cultures of these two organisms were developed on their standard liquid media. After 2 weeks of cultivation, the fermenting medium was replaced by sterile distilled water, and the cultures were allowed to in-
570
B.
LOPEZ-RAMOS
AND
W.
J. SCHUBERT
cubate thus for 48 hr., in order to metabolize completely any residual glucose. This solution was then again replaced by the basal (carbohydrate-free) media for each organism, to which were aseptically added graded amounts of alcohol dehydrogenase (2.6, 5.2, and 10.4 pg./50 ml.), diphosphopyridine nucleotide (0.12,0.24, and 0.48 mg./50 ml.), and ethyl alcohol (1570 mg./50 ml.). After incubation at 27”C., aliquots of the resulting media were analyzed at the end of 72, 144, and 216 hr. for their residual alcohol and acetaldehyde contents. In all cases,an uninoculated control (referred to in the Tables as Blank) was analyzed simultaneously with the experimental flasks (referred to as Inoc.). The results obtained with FsD are recorded in Table III, those with Sterad in Table IV. In the caseof both organisms, with any amount of added enzyme, and at all time intervals, the concentration of residual alcohol in the inoculated flasks is lower than that in the blanks, indicating that the organisms were dehydrogenating alcohol at a faster rate than was the enzyme itself. In relation t,o the formed acetaldehyde, the blanks show an accumulation of this product, while in the case of the flasks containing the St.erad mats, the acetaldehyde concentration diminishes with time, indicating the utilization of this substance by the organism. FsD consumed TABLE Dehydrogenation Concentration of enzyme added pg./so ml.
Ace hr.
of Alcohol
III by F. solani DI purple
Residual alcohol Blank IIXOC. mg./SO ml. mg.l50 ml.
Acetaldehyde IDOC. Blank n.g./50 ml. mg./SO ntl.
0.0
72 144 216
1259 1146 965
1273 427 39
7.40 6.85 6.85
0.0 0.0 17.5
2.6
72 144 216
1268 998 879
1098 553 97
10.75 13.60 9.30
0.0 75.0 25.0
6.2
72 144 216
1171 1008 658
1069 737 19
10.25 14.40 9.25
0.0 131.5 16.0
10.4
72 144 216
1116 918 817
1046 388 126
9.35 12.85 9.65
0.0 72.5 30.0
PIGMENTS
AND
THEIR
INTERACTION
TABLE Dehydrogenation Concentration of enzyme added pg.150 ml.
Age hr.
WITH
ENZYMES
571
IV
of Alcohol
by S. radicinum
Residual alcohol Blank IXX. ?ng./50 ml. tng.jiio ml.
Acetaldehyde Blank IilOC. mg.fSO ml. mg./so ml.
0.0
72 144 216
1203 1146 1015
1177 1068 823
7.10 7.85 7.40
12.30 9.65 9.30
2.6
72 144 216
1289 1063 840
1160 982 879
11.25 12.25 9.35
11.85 9.25 9.15
5.2
72 144 216
1289 1062 976
1186 953 760
10.65 13.05 9.75
10.85 9.40 9.35
10.4
72 144 216
1250 1007 -
1090 908 664
10.65 12.50 -
10.25 8.80 9.10
its formed acetaldehyde so rapidly at the early stages of incubation that none was detected at 72 hr. The lack of any noticeable effect of higher concentrations of alcohol dehydrogenase seems to indicate an enzyme saturation with respect to the experimental systems. Since Lelep is an unpigmented organism, it afforded an opportunity to investigate the possible effect that a natural mold pigment might have on the enzymatic dehydrogenation of ethyl alcohol to acetaldehyde. This could be accomplished by adding the pigment directly to the medium of a “resting cell” experiment before inoculation, and comparing analytical values with controls without added pigment. For this purpose, solanione, the pigment of FsD, was selected and was added to the Lelep medium in graded amounts (0.0, 0.05, 0.10, and 0.20 mg./50 ml.). To the media were also added graded amounts of alcohol dehydrogenase and diphosphopyridine nucleotide, and the standard amount of ethyl alcohol, as in the previously described resting-cell experiments with FsD and Sterad. Again, uninoculated controls were set up simultaneously with the experimental flasks. After 72 hr. of incubation, aliquots of the media were analyzed for their residual alcohol, and for acetaldehyde. The results are listed in Table V.
572
B.
LOPEZ-RAMOS
AND
W.
J.
SCHUBERT
TABLE V Effect of Solanione on Dehydrogenation by L. lepideus ConcentraConcentration of tion of igResidual alcohol Blank enzyme added ment aBded hoc. pg./so ml. mg.jSO ml. mg.lSO ml. mg.150 ml.
Acetaldehyde Blank IllOC. mg./so ml. mg.fSO ml.
0.0
0.00 0.05
1225 1425
1130 663
8.40 15.15
10.65 10.35
2.6
0.00 0.05
1326 1445
998 997
10.25 20.75
11.25 10.40
5.2
0.00 0.10
1202 1284
998 1010
9.60 19.50
11.50 10.15
10.4
0.00 0.20
1172 1249
919 987
9.10 17.15
11.15 10.25
Again, at all concentrations of enzyme, in the presence or absence of pigment, the concentrations of residual alcohol in the inoculated flasks is always lower than that in the blanks, indicating that the organism was still dehydrogenating alcohol at a faster rate than was the enzyme (with or without added pigment). In relation to the formed acetaldehyde, the flasks containing the Lelep mats maintain an almost constant concentration of this product, suggesting that the organism is utilizing for its further metabolism all additional amounts of t.his substance as it is being formed. However, in the absence of the organism, there is an accumulation of this substance, and, most significantly, in all cases the presence of the pigment has the effect of increasing this accumulation over and above the values of the corresponding flasks without pigment. Furthermore, even in the absence of added enzyme, higher concentrations of pigment caused increased accumulation of acetaldehyde, presumably catalyzing an autoxidation of the ethyl alcohol. As in the previous case, however, increasing concentrations of enzyme have no apparent effect on the reaction, suggesting an enzyme saturation with respect to the experimental system. Alcohol dehydrogenase cat.alyzes the reversible dehydrogenation of ethyl alcohol to acetaldehydc, with the intervention of diphosphopyridine nucleotide. (DPK+) according to the equation : &H&H
+ DPN+ = CH,CHO
Solanione, 2-methyl-3-methoxy-5
+ DPNH
,%dihydroxy-6
+ II+
(or 7)-acetonyl-1,4-
PIGMENTS
naphthoquinone
AND
THEIR
INTERACTION
WITH
ENZYMES
(4)) is reversibly reducible to a hydroquinone OH
0
I
II
OH
0
OH
OH
OH
OH
573 as follows :
It would appear then that the effect of solanione on the rate of dehydrogenation of ethyl alcohol by Lelep may be by intervention in the enzymic transfer of hydrogen from ethyl alcohol to its ult’imate accept’or, molecular oxygen : CzHsOH BDPNH solsnione
+ DPN+
ti
CH,CHO
+ solanione
*
2DPll’
+ DPNH + solanione
[2H] + [0] + solanione
+ Hf [ZH]
(la) (lb)
+ H?O
UC)
Or alternatively, CzHhOH solanione
+ solanione
e
CH&HO
+ solanione
[2H] + DPN+
ti
solanione
+ DPNH
BDPNH
+ [O]
+ 2DPN
+ H20
[2H]
@a)
+ H+
(21)) (2c)
The choice between these two reaction sequences should depend on the relative redox potentials of diphosphopyridine nucleotide and solanione. The potential for DPN has been reported (16) as 320 mv., while that of solanione (6) is 291 mv. Hence, the proximity of these values does not allow a preference for either sequence. Equations (lb) and (2b) above were verified by means of spectrophotometric analyses. While it is known that reduced diphosphopyridine nucleotide (DPNH) exhibits an absorption maximum at 340 ml*, the oxidized form (DPN+) does not (17). Furthermore, solanione possesses an absorption maximum at approximately 500 rnp, while its hydroquinone (solanione [2H]) shows no visible absorption (4). Reduced diphosphopyridine nucleotide (DPNH) was prepared according to Ohlmeyer (18) and its absorption spectrum determined (Fig. 2, curve A). Addition of a solution of solanione caused oxidation to DPN+, as indicated by the diminution of the peak at 340 rnp concomitant with the reduction of solanione to its hydroquinone, as evidenced by the lack of its visible spectrum (curve B).
574
B.
LOPEZ-BA?dOS
AND
W.
J.
SCHUBERT
The absorption spectrum of a standard solution of solanione was likewise determined (Fig. 3, curve A), and the solut.ion was treated with an excess of sodium hydrosulfite. That the solanione was reduced is apparent from the loss of its visible absorption (curve B). The solution
P. D.
l.ooO 0.750 t
::fi 350
400
_
f
450
500
A :mrl
-O-Cl-
-0---Cl----A-
Fro. 2. Curve A: DP?JH (30 mg. DPN+ reduced with 40 mg. Na&O,; in final volume of 25 ml. HZO). Curve B: One milliliter above solution treated with 4 pg. solanione in 2 ml. HIO.
Fro. 3. Curve A: Solanione (3 X 10-r moles/ml. H,O). Curve B: One milliliter above solution reduced with 1.5 ml. 3 X 10-T M Na&O, . Curve C: One milliliter above solution oxidized with 2 ml. 1.6 X 1O-a M DPN+.
PIGMENTS
AND
THEIR
INTERACTION
WITH
ENZYMES
575
was then reoxidized (with the exclusion of atmospheric oxygen) by the addition of DPN+ (curve C). The instantaneous recoloration of the solution demonstrated that the DPN+ had oxidized the dihydrosolanione, and in turn, was reduced to DPNH by this reaction. Thus, it has been demonstrated experimentally that diphosphopyridine nucleotide and the pigment solanione are interchangeable in their action. DISCUSSION
Although yeasts can tolerate high concentrations of formed ethyl alcohol, molds are usually inhibited when this compound is present to a great extent. However, molds, unlike yeasts, utilize their produced alcohol further as a carbon source. Earlier studies by Nord and co-workers (19) showed that certain fungi contain powerful dehydrogenases, and, as a consequence, the alcohol initially formed by the molds, instead of being the terminal product (as in yeast), was found to undergo a series of dehydrogenations and oxidations (1). This consideration has now been amply demonstrated in the case of three organisms studied in the present investigations. Thus, FsD, Sterad, and Lelep possess the ability of dehydrogenating ethyl aclohol to acetaldehyde, and of using the latter as a carbon source. Furthermore, in many mold fermentations, a dissimilation of the substrate usually occurs prior to the formation of the terminal compounds. Often, it is found that various carbon sources can be employed to yield the same end product, suggesting that certain Cz or Ca fragments such as acetaldehyde, acetic acid, or pyruvic acid might be intermediates in such a series of reactions. For example, pyruvic acid acts as a key compound in the metabolism of Fusaria. That acetaldehyde could act as an intermediate in the synthesis of various metabolic products was recognized in 1919 (20) and has subsequently been repeatedly confirmed. Thus, acetaldehyde has been shown to be a key intermediate in the conversion of glucose to fats (7) and pigments (4) in Fusaria, and in the synthesis of methyl p-methoxycinnamate (21) and sterol (11) by Lelep. Regarding the effect of the pigment solanione on the dehydrogenation of ethyl alcohol to acetaldehyde by Lelep, it has been noted that this compound is also instrumental in increasing the rate of certain dehydrogenating processes in FlB, as shown by the formation of a fat containing more unsaturated acids than a control (5). On the other hand, it may inhibit other reactions, as indicated by the decrease in the rate of
576
B. LOPEZ-RAMOS AND
W.
J. SCHUBERT
dehydrogenation of isopropyl alcohol by FIB (3). It is also possible that other enzyme systems in the organism are affected by t.he pigment,, since the addition of naphthoquinones serves to lower the carbohydrate conversion factor, decrease the mycelial weight, and inhibit the synthetic activities of FIB (22). ACKNOWLEDGMEKTS The authors wish to express their appreciation to hlr. D. D. Clarke of this Laboratory for valuable discussions and suggestions. In addition, one of us (U. L.-R.) wishes to express his thanks for the hospitality extended to him by this
Department. SUMMARY
1. Fusarium soluni D, purple, Slemphylium radicinum, Lentinus lepi&us, and Daedalea qwrcina form ethyl alcohol from glucose, galactose, and xylose. FsD is the most efficient alcohol fermenter from all three sugars. Upon attaining a maximum alcohol concentration after about 10 days of cultivation, t,he concentration of alcohol diminishes in t,he casesof FsD, Sterad, and I&p. 2. FsD, Sterad, and Lelep dehydrogenate ethyl alcohol to acetaldehyde. All three organisms have the ability of utilizing the formed acet,aldehyde in their further metabolic pathways. 3. Solanione, the pigment produced by FsD, causes increased accumulation of acetaldehyde in the dehydrogenation of ethyl alcohol by added alcohol dehydrogenase. 4. Mechanisms for the effect of solanione in t.he enzymatic dehydrogenation of et,hyl alcohol were studied. REFERENCES 1. 2. 3.
4. 5. 6. 7. 8.
9. 10. 11. 12.
F. F., AND WEISS, S., in “The Enzymes” (J. 13.Sumnerand K. Myrback, eds.), Vol. II, Part I, p. 684. Academic Press, New York, 1951. MULL, R. P., AND NORD, F. F., Arch. Hiochem. 4, 422 (1944). SORD, F. F., FIORE, J. V., AND WEISS, S., Arch. Biochem. 17, 345 (1948). WEISS, S., ASD NORD, F. F., Arch. Biochem. 22, 2% (1949). DESCHAMPS, I., Arch. Rio&em. 20, 457 (1949). WEISS, S., FIORE, J. V., AND NORD, F. F., Arch. Biochem. 22, 314 (1949). KREITMAN, G., SEBEK, 0. K., AND I~ORD, F. F., Arch. Biochem. 28, 77 (1950). CLARKE, D. D., ANI) NORD, F. F., Arch. Biochem. and Biophys. 46, 469 (1953). DESTEVESS, G., AND I~ORD, F. F., Forfschr. them. Forsch. 3, 101 (1954). FIORE, J. V., Arch. Biochem. 16. 161 (1948). SCHUBERT, W. J., AND SORD, F. F., Arch. Riochem.. 20, 466 (1949). Association of Official Agricultural Chemists, “Official Methods of Analysis,” 7th cd. Washington, D. C., 1950: (a) pp. 512, 513; (h) ibid., pp. 508, 509. NORD,
PIGMENTS
13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
AND
THEIR
INTERACTION
WITH
ENZYMl3S
577
,JANKE, A., AND KROPACSY, S., Biochem. 2. 278, 30 (1935). Vas SLYKE, D. D., J. BioZ. Chem. 32, 455 (1917). XOG~RE, S. D., NORRIS, T. O., AND MITCHELL, J., Anal. Chem. 23, 1473 (1951) BURTOX, K., AND WILSON, T. H., Biochem. J. 64, 86 (1953). WARBURG, O., CHRISTIAN, W., AND GRIESE, .4., Biochem. Z. 282, 157 (1935). OHLMEYER, P., Biochem. Z. 297, 66 (1938). ROTISI, 0. T., DBMMANN, E., ASD NORD, F. F., Biochem. Z. 288, 414 (1936). NORI), F. F., Naturwissenschaften ‘7, 685 (1919). XORD, F. F., AND VITUCCI, J. C., Arch. Biochem. 16, 465 (1947). MASELLI, J. A., AND NORD, F. F., Arch. Rio&m. and Biophys. 39, 406 (1952)
of Organic Chemistry and h’nzymology,’ University, New York, New York Received
October
Fotdham
8, 1954
INTRODUCTION
A consideration of the pigments identified in such diverse fungal genera as the Penicillia, AspergiUi, Helminthospora, or Fusaria reveals the fact that molds, in general, are capable of synthesizing a wide variety of colored compounds. Yet, with all the knowledge concerning the structures of these pigments, relatively little information is available on their possible interactions in the metabolic activities occurring within the living cell (1). Within the genus Fusarium, Nerd et al. demonstrated that certain xanthones affect the rate at which Fusarium Zini Bolley (FlB) dehydrogenates isopropyl alcohol (2). The observation that pigments affect dehydrogenating systems in Fusaria (3) prompted a consideration of their influence in fat formation. Thus, it was observed that when solanione, a pigment from Fusarium soZuni D2 purple (FsD) (4) was added to the growth medium of FIB not only is the quantity of fat produced affected by the presence of this pigment (5), but it.s qualitative composition is also changed (6). In distinction to the action of solanione, the presence of lycopersin in Fusarium lywpertii and Fusarium vasinfectum is accompanied by the 1 Holder of a Scholarship of the Government of the Commonwealth of Puerto Rico. * Procter and Gamble Fellow. 3 Communication No. 294. For the previous paper of this series see Arch. Riothem. and Riophys. 62, 464 (1954). This study was assisted by grants from the Office of Naval Research nnd the National Science Foundation. 566
PIGMENTS AND THEIR INTERACTION WITH ENZYMES
567
formation of a less desaturated fat than is formed by the unpigmented cells of these organisms (7). Furthermore, the pigment radicinin, from Stemphylium radicinum (Sterad), appreciably increased the rate of dehydrogenation of isopropyl alcohol by FlB (8). FsD and Sterad thus represent two typical, though unrelated, pigmented organisms, while Lentinus lepideus (Lelep) and Daedalea quercina (Daquer) are two wood-destroying Basidiomycetes of the “brown rot” type. The former decays softwoods, and the latter attacks hardwoods, but neither organism is pigmented. The selection of Lelep was furthermore motivated by the fact that it contains an enzyme system capable of effecting t)he transition from carbohydrates to a methoxylated aromatic compound (9). We are reporting here on the relation between the structure of solanione and its role in the enzymatic dehydrogenation of ethyl alcohol. EXPERIMENTAL The organisms employed in these experiments were FsD, Sterad, Lelep, and Daquer. FsD and Sterad were obtained from Dr. Wm. C. Snyder, Division of Plant Pathology, University of California. Lelep and Daquer were obtained from Dr. Wm. J. Robbins of the New York Botanical Garden. FsD was cultivated on a modified Czapek-Dox medium (10). Sterad was grown on a standard synthetic medium (8). Lelep and Daquer were both cultivated on a glucose-Neopeptonesalt-thiamin-containing medium (11). Alcohol dehydrogenase (twice recrystallized) and diphosphopyridine nucleotide (95% pure) were obtained from the Worthington Biochemical Corporation, Freehold, S. J.
Analytical Methods IZesidual Sugars. Glucose, galactose, and xylose were determined by precipitation of cuprous oxide (12a), and subsequent titration with potassium permanganate (12b). Ethyl alcohol was determined by a titrimetric method (13) after possible interfering substances had been removed (14). Acetaldehyde was determined spectrophotometrically, after conversion t,o iodoform by hypoiodite (15). pH of medium was determined using a Cambridge electron-ray pH meter.
RESULTS
When cultivated on media containing glucose as carbon source, all four organisms studied produced measurable quantities of ethyl alcohol (Fig. 1). FsD was by far the best alcohol former, producing over 300 mg. al-
568
B.
LOPEZ-RAMOS
AKD
W.
J. SCHUBERT 1
0 F. sdani 0 S. radicinum A L Iepideus 0 D. quwcina
cohol/lOO ml. medium after but 10 days of cultivation. The other three organisms produced lesser amounts. h significant obscrvat.ion was the sharp decrease in t.he alcohol level in t.he FsD media aft.er t.he maximum at 10 days. This seemed to indicate a dehydrogenat.ion of the alcohol during the course of the metabolic processes of t.he organism. Following t.he formation of alcohol from glucose by these organisms, their ability t.o ferment other sugars was investigated. For this purpose, the behavior of galactose (Table I) and xylose (Table II) was studied. TABLE Metabolism Organism
I
of Gulactose ‘be days
Residual
sugar
g.1100 ntl.
Alcohol mg.fIOO
F. solani
10 20 30
5.4 0.3 0.2
46.6 5.7 14.1
S. radicinum
10 20 30
3.4 2.3 1.9
16.2 13.2 15.2
L. lepideus
10 20 30
2.0 1.9 2.0
20.3 7.5 13.3
I). quercina
10 20 30
2.2 2.1 2.4
16.2 11.3 20.8
ml.
PIGMENTS
AND
THEIR
INTERACTION
TABLE Organism
WITH
ENZYMES
sugar g./IOO ml.
Alcohol mg./lOO ml
II
Metabolism of Xylose A@- Residual days
569
F. solani
10 20 30
5.4 0.2 0.2
80.1 5.7 9.5
S. radicinum
10 20 30
3.7 2.5 2.5
32.4 13.2 26.5
L. lepideus
10 20 30
2.0 1.9 1.9
28.3 5.7 10.3
D. quercina
10 20 30
2.0 2.0 0.8
12.2 15.2 19.0
Again, all four organisms were able to produce measurable amounts of alcohol from the two sugars. As in the case of the glucose experiments, FsD was still the most efficient fermenter. However, the relat’ive amounts of alcohol formed by this organism from both galactose (46.6 mg.) and xylose (80.1 mg.) were quite inferior to the amount’ produced from glucose (321.4 mg.). Indeed, in no instance was any of the four organisms able to give rise to as much ethyl alcohol from either galactose or xylose as it had from glucose. However, in the case of FsD, Sterad, and Lelep, when developing on both galactose and xylose, the maximum concent’ration of ethyl alcohol was invariably reached at 10 days (with some slight upturn occurring at 30 days). Since control experiments have shown that this could not, have been due to evaporation, this established that the formed alcohol was being further utilized by the three organisms. In view of t,his, a dehydrogenation was considered to be the introductory step. To investigate this possibility, an experiment was performed in which the effect of added alcohol dehydrogenase was studied on the ability of resting cells of the pigmented organisms, FsD and Sterad, to dehydrogenate ethyl alcohol. Thus, cultures of these two organisms were developed on their standard liquid media. After 2 weeks of cultivation, the fermenting medium was replaced by sterile distilled water, and the cultures were allowed to in-
570
B.
LOPEZ-RAMOS
AND
W.
J. SCHUBERT
cubate thus for 48 hr., in order to metabolize completely any residual glucose. This solution was then again replaced by the basal (carbohydrate-free) media for each organism, to which were aseptically added graded amounts of alcohol dehydrogenase (2.6, 5.2, and 10.4 pg./50 ml.), diphosphopyridine nucleotide (0.12,0.24, and 0.48 mg./50 ml.), and ethyl alcohol (1570 mg./50 ml.). After incubation at 27”C., aliquots of the resulting media were analyzed at the end of 72, 144, and 216 hr. for their residual alcohol and acetaldehyde contents. In all cases,an uninoculated control (referred to in the Tables as Blank) was analyzed simultaneously with the experimental flasks (referred to as Inoc.). The results obtained with FsD are recorded in Table III, those with Sterad in Table IV. In the caseof both organisms, with any amount of added enzyme, and at all time intervals, the concentration of residual alcohol in the inoculated flasks is lower than that in the blanks, indicating that the organisms were dehydrogenating alcohol at a faster rate than was the enzyme itself. In relation t,o the formed acetaldehyde, the blanks show an accumulation of this product, while in the case of the flasks containing the St.erad mats, the acetaldehyde concentration diminishes with time, indicating the utilization of this substance by the organism. FsD consumed TABLE Dehydrogenation Concentration of enzyme added pg./so ml.
Ace hr.
of Alcohol
III by F. solani DI purple
Residual alcohol Blank IIXOC. mg./SO ml. mg.l50 ml.
Acetaldehyde IDOC. Blank n.g./50 ml. mg./SO ntl.
0.0
72 144 216
1259 1146 965
1273 427 39
7.40 6.85 6.85
0.0 0.0 17.5
2.6
72 144 216
1268 998 879
1098 553 97
10.75 13.60 9.30
0.0 75.0 25.0
6.2
72 144 216
1171 1008 658
1069 737 19
10.25 14.40 9.25
0.0 131.5 16.0
10.4
72 144 216
1116 918 817
1046 388 126
9.35 12.85 9.65
0.0 72.5 30.0
PIGMENTS
AND
THEIR
INTERACTION
TABLE Dehydrogenation Concentration of enzyme added pg.150 ml.
Age hr.
WITH
ENZYMES
571
IV
of Alcohol
by S. radicinum
Residual alcohol Blank IXX. ?ng./50 ml. tng.jiio ml.
Acetaldehyde Blank IilOC. mg.fSO ml. mg./so ml.
0.0
72 144 216
1203 1146 1015
1177 1068 823
7.10 7.85 7.40
12.30 9.65 9.30
2.6
72 144 216
1289 1063 840
1160 982 879
11.25 12.25 9.35
11.85 9.25 9.15
5.2
72 144 216
1289 1062 976
1186 953 760
10.65 13.05 9.75
10.85 9.40 9.35
10.4
72 144 216
1250 1007 -
1090 908 664
10.65 12.50 -
10.25 8.80 9.10
its formed acetaldehyde so rapidly at the early stages of incubation that none was detected at 72 hr. The lack of any noticeable effect of higher concentrations of alcohol dehydrogenase seems to indicate an enzyme saturation with respect to the experimental systems. Since Lelep is an unpigmented organism, it afforded an opportunity to investigate the possible effect that a natural mold pigment might have on the enzymatic dehydrogenation of ethyl alcohol to acetaldehyde. This could be accomplished by adding the pigment directly to the medium of a “resting cell” experiment before inoculation, and comparing analytical values with controls without added pigment. For this purpose, solanione, the pigment of FsD, was selected and was added to the Lelep medium in graded amounts (0.0, 0.05, 0.10, and 0.20 mg./50 ml.). To the media were also added graded amounts of alcohol dehydrogenase and diphosphopyridine nucleotide, and the standard amount of ethyl alcohol, as in the previously described resting-cell experiments with FsD and Sterad. Again, uninoculated controls were set up simultaneously with the experimental flasks. After 72 hr. of incubation, aliquots of the media were analyzed for their residual alcohol, and for acetaldehyde. The results are listed in Table V.
572
B.
LOPEZ-RAMOS
AND
W.
J.
SCHUBERT
TABLE V Effect of Solanione on Dehydrogenation by L. lepideus ConcentraConcentration of tion of igResidual alcohol Blank enzyme added ment aBded hoc. pg./so ml. mg.jSO ml. mg.lSO ml. mg.150 ml.
Acetaldehyde Blank IllOC. mg./so ml. mg.fSO ml.
0.0
0.00 0.05
1225 1425
1130 663
8.40 15.15
10.65 10.35
2.6
0.00 0.05
1326 1445
998 997
10.25 20.75
11.25 10.40
5.2
0.00 0.10
1202 1284
998 1010
9.60 19.50
11.50 10.15
10.4
0.00 0.20
1172 1249
919 987
9.10 17.15
11.15 10.25
Again, at all concentrations of enzyme, in the presence or absence of pigment, the concentrations of residual alcohol in the inoculated flasks is always lower than that in the blanks, indicating that the organism was still dehydrogenating alcohol at a faster rate than was the enzyme (with or without added pigment). In relation to the formed acetaldehyde, the flasks containing the Lelep mats maintain an almost constant concentration of this product, suggesting that the organism is utilizing for its further metabolism all additional amounts of t.his substance as it is being formed. However, in the absence of the organism, there is an accumulation of this substance, and, most significantly, in all cases the presence of the pigment has the effect of increasing this accumulation over and above the values of the corresponding flasks without pigment. Furthermore, even in the absence of added enzyme, higher concentrations of pigment caused increased accumulation of acetaldehyde, presumably catalyzing an autoxidation of the ethyl alcohol. As in the previous case, however, increasing concentrations of enzyme have no apparent effect on the reaction, suggesting an enzyme saturation with respect to the experimental system. Alcohol dehydrogenase cat.alyzes the reversible dehydrogenation of ethyl alcohol to acetaldehydc, with the intervention of diphosphopyridine nucleotide. (DPK+) according to the equation : &H&H
+ DPN+ = CH,CHO
Solanione, 2-methyl-3-methoxy-5
+ DPNH
,%dihydroxy-6
+ II+
(or 7)-acetonyl-1,4-
PIGMENTS
naphthoquinone
AND
THEIR
INTERACTION
WITH
ENZYMES
(4)) is reversibly reducible to a hydroquinone OH
0
I
II
OH
0
OH
OH
OH
OH
573 as follows :
It would appear then that the effect of solanione on the rate of dehydrogenation of ethyl alcohol by Lelep may be by intervention in the enzymic transfer of hydrogen from ethyl alcohol to its ult’imate accept’or, molecular oxygen : CzHsOH BDPNH solsnione
+ DPN+
ti
CH,CHO
+ solanione
*
2DPll’
+ DPNH + solanione
[2H] + [0] + solanione
+ Hf [ZH]
(la) (lb)
+ H?O
UC)
Or alternatively, CzHhOH solanione
+ solanione
e
CH&HO
+ solanione
[2H] + DPN+
ti
solanione
+ DPNH
BDPNH
+ [O]
+ 2DPN
+ H20
[2H]
@a)
+ H+
(21)) (2c)
The choice between these two reaction sequences should depend on the relative redox potentials of diphosphopyridine nucleotide and solanione. The potential for DPN has been reported (16) as 320 mv., while that of solanione (6) is 291 mv. Hence, the proximity of these values does not allow a preference for either sequence. Equations (lb) and (2b) above were verified by means of spectrophotometric analyses. While it is known that reduced diphosphopyridine nucleotide (DPNH) exhibits an absorption maximum at 340 ml*, the oxidized form (DPN+) does not (17). Furthermore, solanione possesses an absorption maximum at approximately 500 rnp, while its hydroquinone (solanione [2H]) shows no visible absorption (4). Reduced diphosphopyridine nucleotide (DPNH) was prepared according to Ohlmeyer (18) and its absorption spectrum determined (Fig. 2, curve A). Addition of a solution of solanione caused oxidation to DPN+, as indicated by the diminution of the peak at 340 rnp concomitant with the reduction of solanione to its hydroquinone, as evidenced by the lack of its visible spectrum (curve B).
574
B.
LOPEZ-BA?dOS
AND
W.
J.
SCHUBERT
The absorption spectrum of a standard solution of solanione was likewise determined (Fig. 3, curve A), and the solut.ion was treated with an excess of sodium hydrosulfite. That the solanione was reduced is apparent from the loss of its visible absorption (curve B). The solution
P. D.
l.ooO 0.750 t
::fi 350
400
_
f
450
500
A :mrl
-O-Cl-
-0---Cl----A-
Fro. 2. Curve A: DP?JH (30 mg. DPN+ reduced with 40 mg. Na&O,; in final volume of 25 ml. HZO). Curve B: One milliliter above solution treated with 4 pg. solanione in 2 ml. HIO.
Fro. 3. Curve A: Solanione (3 X 10-r moles/ml. H,O). Curve B: One milliliter above solution reduced with 1.5 ml. 3 X 10-T M Na&O, . Curve C: One milliliter above solution oxidized with 2 ml. 1.6 X 1O-a M DPN+.
PIGMENTS
AND
THEIR
INTERACTION
WITH
ENZYMES
575
was then reoxidized (with the exclusion of atmospheric oxygen) by the addition of DPN+ (curve C). The instantaneous recoloration of the solution demonstrated that the DPN+ had oxidized the dihydrosolanione, and in turn, was reduced to DPNH by this reaction. Thus, it has been demonstrated experimentally that diphosphopyridine nucleotide and the pigment solanione are interchangeable in their action. DISCUSSION
Although yeasts can tolerate high concentrations of formed ethyl alcohol, molds are usually inhibited when this compound is present to a great extent. However, molds, unlike yeasts, utilize their produced alcohol further as a carbon source. Earlier studies by Nord and co-workers (19) showed that certain fungi contain powerful dehydrogenases, and, as a consequence, the alcohol initially formed by the molds, instead of being the terminal product (as in yeast), was found to undergo a series of dehydrogenations and oxidations (1). This consideration has now been amply demonstrated in the case of three organisms studied in the present investigations. Thus, FsD, Sterad, and Lelep possess the ability of dehydrogenating ethyl aclohol to acetaldehyde, and of using the latter as a carbon source. Furthermore, in many mold fermentations, a dissimilation of the substrate usually occurs prior to the formation of the terminal compounds. Often, it is found that various carbon sources can be employed to yield the same end product, suggesting that certain Cz or Ca fragments such as acetaldehyde, acetic acid, or pyruvic acid might be intermediates in such a series of reactions. For example, pyruvic acid acts as a key compound in the metabolism of Fusaria. That acetaldehyde could act as an intermediate in the synthesis of various metabolic products was recognized in 1919 (20) and has subsequently been repeatedly confirmed. Thus, acetaldehyde has been shown to be a key intermediate in the conversion of glucose to fats (7) and pigments (4) in Fusaria, and in the synthesis of methyl p-methoxycinnamate (21) and sterol (11) by Lelep. Regarding the effect of the pigment solanione on the dehydrogenation of ethyl alcohol to acetaldehyde by Lelep, it has been noted that this compound is also instrumental in increasing the rate of certain dehydrogenating processes in FlB, as shown by the formation of a fat containing more unsaturated acids than a control (5). On the other hand, it may inhibit other reactions, as indicated by the decrease in the rate of
576
B. LOPEZ-RAMOS AND
W.
J. SCHUBERT
dehydrogenation of isopropyl alcohol by FIB (3). It is also possible that other enzyme systems in the organism are affected by t.he pigment,, since the addition of naphthoquinones serves to lower the carbohydrate conversion factor, decrease the mycelial weight, and inhibit the synthetic activities of FIB (22). ACKNOWLEDGMEKTS The authors wish to express their appreciation to hlr. D. D. Clarke of this Laboratory for valuable discussions and suggestions. In addition, one of us (U. L.-R.) wishes to express his thanks for the hospitality extended to him by this
Department. SUMMARY
1. Fusarium soluni D, purple, Slemphylium radicinum, Lentinus lepi&us, and Daedalea qwrcina form ethyl alcohol from glucose, galactose, and xylose. FsD is the most efficient alcohol fermenter from all three sugars. Upon attaining a maximum alcohol concentration after about 10 days of cultivation, t,he concentration of alcohol diminishes in t,he casesof FsD, Sterad, and I&p. 2. FsD, Sterad, and Lelep dehydrogenate ethyl alcohol to acetaldehyde. All three organisms have the ability of utilizing the formed acet,aldehyde in their further metabolic pathways. 3. Solanione, the pigment produced by FsD, causes increased accumulation of acetaldehyde in the dehydrogenation of ethyl alcohol by added alcohol dehydrogenase. 4. Mechanisms for the effect of solanione in t.he enzymatic dehydrogenation of et,hyl alcohol were studied. REFERENCES 1. 2. 3.
4. 5. 6. 7. 8.
9. 10. 11. 12.
F. F., AND WEISS, S., in “The Enzymes” (J. 13.Sumnerand K. Myrback, eds.), Vol. II, Part I, p. 684. Academic Press, New York, 1951. MULL, R. P., AND NORD, F. F., Arch. Hiochem. 4, 422 (1944). SORD, F. F., FIORE, J. V., AND WEISS, S., Arch. Biochem. 17, 345 (1948). WEISS, S., ASD NORD, F. F., Arch. Biochem. 22, 2% (1949). DESCHAMPS, I., Arch. Rio&em. 20, 457 (1949). WEISS, S., FIORE, J. V., AND NORD, F. F., Arch. Biochem. 22, 314 (1949). KREITMAN, G., SEBEK, 0. K., AND I~ORD, F. F., Arch. Biochem. 28, 77 (1950). CLARKE, D. D., ANI) NORD, F. F., Arch. Biochem. and Biophys. 46, 469 (1953). DESTEVESS, G., AND I~ORD, F. F., Forfschr. them. Forsch. 3, 101 (1954). FIORE, J. V., Arch. Biochem. 16. 161 (1948). SCHUBERT, W. J., AND SORD, F. F., Arch. Riochem.. 20, 466 (1949). Association of Official Agricultural Chemists, “Official Methods of Analysis,” 7th cd. Washington, D. C., 1950: (a) pp. 512, 513; (h) ibid., pp. 508, 509. NORD,
PIGMENTS
13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
AND
THEIR
INTERACTION
WITH
ENZYMl3S
577
,JANKE, A., AND KROPACSY, S., Biochem. 2. 278, 30 (1935). Vas SLYKE, D. D., J. BioZ. Chem. 32, 455 (1917). XOG~RE, S. D., NORRIS, T. O., AND MITCHELL, J., Anal. Chem. 23, 1473 (1951) BURTOX, K., AND WILSON, T. H., Biochem. J. 64, 86 (1953). WARBURG, O., CHRISTIAN, W., AND GRIESE, .4., Biochem. Z. 282, 157 (1935). OHLMEYER, P., Biochem. Z. 297, 66 (1938). ROTISI, 0. T., DBMMANN, E., ASD NORD, F. F., Biochem. Z. 288, 414 (1936). NORI), F. F., Naturwissenschaften ‘7, 685 (1919). XORD, F. F., AND VITUCCI, J. C., Arch. Biochem. 16, 465 (1947). MASELLI, J. A., AND NORD, F. F., Arch. Rio&m. and Biophys. 39, 406 (1952)