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Lysine biosynthesis in algae
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
Lysine MORTON From the Laboratory
373-377 (1963)
101,
Biosynthesis
ROTHSTEIN
in Algae’
AND ELEANOR
of Comparative Bioloyy, Kaiser Richmond, California Received
February
11. SAFFRAN
Foundution
Research Institute,
25, 1963
Both aminoadipic acid-B-C4 and pipecolic acid-6-Cl4 are converted almost exclusively to radioactive lysine in Euglena gracilis. These findings strongly support the idea that pipecolic acid is an intermediate in organisms utilizing the aminoadipic acid pathway of lysine biosynthesis. INTRODUCTION
The biosynthesis of lysine appears to follow two distinct pathways: One, based on the decarboxylation of diaminopimelic acid (DAP), is found in most bacteria and in plants (1, 2). The other, involving a-aminoadipic acid (AAA), is, in general, utilized by yeasts and molds (3-5). In a series of short papers, Vogel (6-S) used this metabolic dichotomy as a phylogenetic marker by testing a variety of organisms for their ability to utilize labeled aspartate for lysine synthesis. Where the protein-bound lysine had little activity compared to the incorporated aspartate, a non-DAP pathway (presumably involving AAA) was inferred. The DAP pathway has been well delineated (9-11). By contrast, the pathway of lysine synthesis via AAA is poorly understood and few intermediates have been unequivocally identified. The genesis of AAA itself is unknown, although Strassman and Weinhouse (12) have proposed a mechanism for its formation to explain the labeling pattern found in lysine isolated from yeast grown on isotopic acetate. In the area between A,4A and lysine, AAA-semialdehyde has been identified as an intermediate by Sagisaka and Shimura (4) and recently by Larson et al. (13). Aspen and Meister (14) reported that AAA gives rise to pipecolic acid in mutants of Aspergillus 1 Supported in part by grant National Science Foundation.
G-19807 from the 373
nidulans and point out that the latter may be a lysine precursor in this organism. If pipecolic acid is indeed an intermediate, lysine biosynthesis from AAA may involve the same intermediates as lysine breakdown in mammalian systems. In the latter case it has been established that lysine gives rise to AAA via pipecolic acid (15, 16). Vogel (7) found that Chlorella vulgaris utilizes the DAP pathway, whereas Euglena gracilis does not. These organisms were selected for the present study so that, providing Euglena does utilize the AAA pathway, comparative studies of lysine catabolism could be made in species utilizing the respective biosynthetic routes. This paper presents evidence to show that pipecolic acid is converted to lysine by Euglena gl-acilis and that this organism uses exclusively the A,4A pathway of lysine biosynthesis. MATERIALS
AND
METHOT
ORGAK-ISMS Chlorella vulgaris (Columbia strain) was grown at 25°C. on an illuminated shaker, using the medium of Shrift (17), except that 3 X lo-$ M K2S04 was used. Cultures were used for the experiments 4-6 days after inoculation from 6-Sday-old agar slants. Euglena gracilis Z. strain (Laboratory of Comparative Biology No. E-1.1.2) was grown in cotton-plugged Erlenmeyer flasks on an illuminated glass shelf at 25°C. The medium used was based on t.hat of Cramer and Myers (18) except that the combination of trace metals was changed to that of Allen and Arnon (19).
374
ROTHSTEIN
CO, diffusing from carbon source.
the laboratory
STERILITY Microscopic examination incubation in brain-heart carried out routinely.
air
AND
was the
tive isotopic substrates were added directly to the medium, and the organisms were allowed to grow for an additional 3 days. The cells were collected by centrifugation, washed twice with distilled water, then suspended in 80% ethanol and heated in a boiling water bath for I min. The precipitate was removed by centrifugation, washed with 807~ ethanol, and hydrolyzed in a sealed tube with 6 K HCl for 6 hr. at 105110°C. (In one experiment, one half of the alcohol precipitate was extracted thoroughly with hot SC/e trichloroacetic acid, and the precipitate was washed with ether.) The hydrolyzate was filtered and the HCl removed by evaporation of the warmed solution in a stream of air. The residue was dissolved in water and treated by ion-exchange chromatography and paper chromatography as described below.
CHECKS of the cultures or infusion (Difco) was
ISOTOPIC SUBSTRATES Aspartic acid-4-Cl4 was purchased from commercial sources. DL-AAA-6-V was synthesized by the method of Rothstein and Claus (20) and purified on a column of Dowex l-acetate with 0.5 A’ acetic acid as eluant. The product was sterilized by filtration (Millipore), since autoclaving caused nearly half of the AAA-B-Cl4 to cyclize to piperidonecarboxylic acid. The product used in the experiments reported in Table I was free of this contaminant. nn-Pipecolic acid-6.Cl4 was synthesized as reported for nn-pipecolic acid204 (21).
(B) CHLORELLA A %-day-old culture (100 ml.) was collected by centrifugation under sterile conditions and resuspended in sterile potassium phosphate buffer (0.2 fi!l pH 4.6). Sterile AAA-6-Cl4 (Table I) was added, and the cells were shaken in the light for 24 hr. After being harvested by centrifugation, the cells were washed twice with water and then suspended in cold 5’7, TCA. The suspension was centrifuged after one half hour, and the residue
EXPERIMENTAL
(A) EUGLENA In typical experiments, Euglena was grown for 3 days in 250.ml. cotton-plugged Erlenmeyer flasks containing 100 ml. R medium. The respecTABLE RADIOACTIVITY Expt.
lc
2 3” 4
Organism
Euglena Euglena Euglena Chlorella
OF PRODUCTS
SAFFRAN
I
RECOVERED
FROM ALQAL
Counts/min.”
Substrateb
DL-AAA-~-C’~ DL-Pip-6-Cl4
HYDROLYZATES
GIU
A&
0 Trace”
0 Tracee 20,500 32,800
DI,-As~-~-C’”
19,200
DI,-AAA-~-C~~
66,300
Lysine
27,100 29 ) OOOf 2,730 0
Soluble
12,8W 62, OOOe 2,250 67,000’
a Except as noted, values are based on aliquots of fractions from Dowex 50 columns. * The amounts of radioactivesubstrateare as follows: Expt. 1, 15,~c., 2pmoles; Expt. 2,5rc., 5pmoles; Expt.3,2~~.,25~moles;Expt.4,5rc.,5pmole.s. c Three experiments were performed with varying amounts of AAA-6-Ci4. Paper chromatograms of protein hydrolyzates showed detectable activity only in the lysine area in each case. d Alcohol extract; on paper chromatograms, only lysine appeared to be radioactive. e A total of 250 counts/min. was obtained in the combined glutamic acid, AAA, and aspartic acid. f Radioactivity in the protein hydrolyzate. Only the lysine area contained detectable radioactivity on paper chromatograms in two sets of solvents and on high-voltage electrophoretograms. Samples were removed for chromatography before counting so that the actual figure should be several thousand counts/min. larger. g All the activity appeared to be present as pipecolic acid. h A second experiment yielded similar results, but labeling was lower in all categories. i Trichloroacetic acid extract (5%); most of the activity was in AAA. None was detectable in the lysine area of paper chromatograms.
LYSINE was washed with ether material was hydrolyzed ISOL.~TIOS
BIOSYNTHESIS
IK
ALGAE
375
and dried. The resulting with 6 S HCl as above.
OF AMINO
ACIDS
The Eziglena hydrolysate from one of the AA.4C” incubations (Expt. 1, Table I) was chromatographed on Dowex l-acetate and Dowex 50 (H+) by the method of Hirs et al. (22). In one case, 5 Hmoles of carrier lysine was added before treatment on the columns. The Euglena hydrolyzate from the aspartic acid-J-C’” and the ChloreUa hydrolyzates were treated as above, but the Dowex 50 (H+) column was shortened to 18.5 cm. (“short column”). This was treated first with 1 S HCl (65 ml.) and then with 2.5 A’ HCl at a flow rate of approximately 0.2 ml./min. Fractions of -i ml. were collected. This procedure yielded lysine in a discrete peak at approximately 140-150 ml. of effluent (including the 1 A- HCI). IDEKTIFICATIOK
OF LYSINE
Portions of the fractions containing lysine were chromatographed on paper in butanol-acetic acid-water, -1: 1:l (BAW), ethanol-ammoniawater, 18: 1: 1 (EAW), and butanol-pyridinewater, 1: 1: 1 (BPW), and the chromatograms were scanned for radioactivit,y. After chromatography on Dowex 50, part of the lysine peak from the Euglena experiment with AAA-B-Cl4 (Table I, Expt. 1) was used to prepare the phenylthiohydantoin derivative after addition of 200 mg. of carrier nL-lysine. The product was recrystallized to constant specific activity. PREP~R~TIOS
OF LYSIKE
“ARTIFACT”
A 3-day-old culture of Euglena was collected and treated with ethanol as described above. To the washed precipitate was added 20,000 counts/ min. of nL-lysine-6-C14, which had been shown to yield one radioactive spot coinciding with the position of authentic lysine in BPW (as well as BAW). The mixt,ure was hydrolyzed in a sealed tube as described above. The hydrolyzate was dried, taken up in a little water, filtered, and chromatographed on paper in BPW. The remainder was treatted on a short column of Dowex 50 (H+) as described above. The lysine peak was collected, dried, and chromatographed on paper in BPW, BAW, and EAW. RESCLTS
Hydrolysis of Euglena protein yielded a radioactive “Iysine artifact” rather than lysine. This artifact could be distinguished from lysine on paper chromatograms de-
FIG. 1. Paper chromatography of radioactive lysine and “lysine artifact.” The chromatograms were scanned after development in butanolpyridine-water (1:l:l). d, B, Dr.-lysine-6-C11 and “lysine artifact” from Ezrglena protein hydrolyzate, respectively, run on the same chromatogram; C, “lysine” fraction after treatment of hydrolyzate on Dowex 50.
376
ROTHSTEIN
veloped in butanol-pyridine-water, 1: 1: 1 (A, B, Fig. l), but not in BAW or EAW. An identical product was obtained by subjecting a mixture of authentic DL-lysine-6-C14 and unlabeled Euglena protein to the hydrolysis procedure. From columns of Dowex 50, the material emerged as a peak in the lysine position. Paper chromatograms of this peak showed the presence of both lysine and “lysine artifact” (C, Fig. 1). The phenylthiohydantoin derivative prepared from this material after addition of carrier lysine, led to a product which, after four recrystallizations, showed a constant specific activity (23 counts/min./mg.) through three additional crystallizations. The “artifact” appears to be formed during the evaporation of the protein hydrolyzate, rather than during hydrolysis of the protein; preliminary experiments show that addition of nL-lysine-6-C’” to previously hydrolyzed Euglena protein, followed by evaporation of the mixture, leads to formation of the artifact. This may be analogous to the formation of artifacts from glutamic acid (23). The results of the isotopic experiments are given in Table I. DISCUSSION
It is apparent from the data in Table I that Euglena uses the AAA pathway of lysine biosynthesis exclusively, whereas Chlorella uses only the DAP pathway shown by Vogel (7). Experiment 2 shows that pipecolic acid is converted almost exclusively to lysine in Euglena. The fact that no glutamic acid or, for that matter, any other intermediates are formed to any significant degree eliminates the possibility that lysine is being synthesized from carbon-chain fragments derived from the breakdown of the pipecolic acid. This finding supports the idea that pipecolic acid lies on the pathway of lysine biosynthesis. As to the order of formation of intermediates, Aspen and Meister (14) have implicated pipecolic acid in lysine biosynthesis by showing that mutants of AspeTgillus niger accumulate pipecolic acid in the medium after exposure to AAA. Thus, if pipecolic acid lies directly on the pathway, the mechanism of lysine
AND
SAFFRAN
biosynthesis should involve the steps AAA-+ pipecolic acid ---f lysine (pathway a below). pipecolic acid
La
allb AAASAAA-semialdehyde
lysine
7b
The above results do not exclude the possibility that AAA-semialdehyde plays a central role in the pathway, with pipecolic acid representing a side reaction (route b). AAA-semialdehyde has been reported by other investigat,ors to be an intermediate in lysine biosynthesis in yeast (4, 13). However, if AAA-semialdehyde is formed from pipecolic acid, one would expect to find at least a small amount of labeling in the isolated AAA. No such labeling could be detected either in the nonprotein cell extracts or the incubation medium. Of course, the reaction between AAA and the semialdehyde conceivably could be irreversible under the conditions of Expt. 2. The further steps of lysine synthesis are completely unknown. Pipecolic acid could be dehydrogenated to form Al-piperideine2-carboxylic acid e e-amino-cr-ketocaproic acid, and by transamination yield lysine. However, Aspen and Meister have reported that the latter compound does not support growth in lysine-requiring mutants of Aspergillus niger (14), although, an e-acyl derivative could be involved. A route such as this is essentially the reverse of the proposed breakdown pathway of lysine in mammalian species (15, 16, 21). Alternatively, the pathway may involve synthesis of the compound “saccharopine” (E-N-(L-glutaryl-2)-L-lysine) (13). REFERENCES 1. WORK, E., AND DEWEY, D. L., J. Gen. Microbiol. 9, 394 (1953). 2. VOGEL, H. J., Proc. Natl. Acad. Sci. U. S. 46, 1717 (1959). E., J. Biol. Chem. 192, 607 (1951). 3. WINDSOR, 4. SAGISAKA, S., AND SHIMURA, K., J. Biochem. (Tokyo) 49, 392 (1961). 5. BROQUIST, H. P., STIFFEY, A. V., AND ALBRECHT, A. M., Appl. Microbial. 9, 1 (1961). 6. VOGEL, H. J., Biochim. Biophys. Acta 41, 172 (1960). 7. VOGEL, H. J., Biochim. Biophys. ilcta 34, 282 (1959).
LYSINE
BIOSYXTHESIS
8. VOGEL, H. J., fiatwe 189, 1026 (1961). 9. (;II,VARG, C., J. Biol. Chem. 236, 1429 (1961). 10. PETERKOFSKY, B., AND GILVARG, C., J. Viol. Chem. 236, 1432 (1961). 11. RDELMAN, J. C., AND GILVARG, C., J. Biol. Chew. 236, 3295 (1961). 12. STRASSM.4N, M., AND WEINHOUSE, S., J. :17x. (*he,n. Sot. 75, 1680 (1953). 13. LARSON, It. L., SANDINE, W. I)., AND BROQCIST, H. P., J. Biol. Chem. 238, 275 (1963). 14. AWES, A. J., AND MEISTER, 8., Biochemistry 1, 606 (1962). 15. ROTHSTEIN, M., AND MILLER, L. L., J. Biol. Chem. 211, 851 (1954).
IN ALGAE
377
16. ROTHSTEIN,M.,COOKSEY,K. E., ANDGREENBERG, 1). M., J. Biol. Chew 237, 2828 (1962). 17. SHRIFT, A., dm. J. Botany, 41, 223 (1954). 18. CRAMER, M., AND MYERS, J., Arch. Mikrobiol. 17, 384 (1952). 19. ALI,EX, M. B., AND ARSON, 1). I., Planf Physiol. 30, 372 (1953). 20. ROTHSTEIN, &I., AND CICADAS,C. J., J. Sm. Chem. Sot. 76, 2981 (1953). 21. ROTHSTEIN, RI., AND GREESBERG, D. M., 1. Biol. Chem. 236, 714 (1960). 22. HIRS, C. H., MOORE, S., AND STEIN, W. H., J. Am. Chem. Sot. 76, 6063 (1954). 23. IKAIVA, M., AND RNELI., E. E., J. Biol. Chem. 236, 1955 (1961).
OF
BIOCHEMISTRY
AND
BIOPHYSICS
Lysine MORTON From the Laboratory
373-377 (1963)
101,
Biosynthesis
ROTHSTEIN
in Algae’
AND ELEANOR
of Comparative Bioloyy, Kaiser Richmond, California Received
February
11. SAFFRAN
Foundution
Research Institute,
25, 1963
Both aminoadipic acid-B-C4 and pipecolic acid-6-Cl4 are converted almost exclusively to radioactive lysine in Euglena gracilis. These findings strongly support the idea that pipecolic acid is an intermediate in organisms utilizing the aminoadipic acid pathway of lysine biosynthesis. INTRODUCTION
The biosynthesis of lysine appears to follow two distinct pathways: One, based on the decarboxylation of diaminopimelic acid (DAP), is found in most bacteria and in plants (1, 2). The other, involving a-aminoadipic acid (AAA), is, in general, utilized by yeasts and molds (3-5). In a series of short papers, Vogel (6-S) used this metabolic dichotomy as a phylogenetic marker by testing a variety of organisms for their ability to utilize labeled aspartate for lysine synthesis. Where the protein-bound lysine had little activity compared to the incorporated aspartate, a non-DAP pathway (presumably involving AAA) was inferred. The DAP pathway has been well delineated (9-11). By contrast, the pathway of lysine synthesis via AAA is poorly understood and few intermediates have been unequivocally identified. The genesis of AAA itself is unknown, although Strassman and Weinhouse (12) have proposed a mechanism for its formation to explain the labeling pattern found in lysine isolated from yeast grown on isotopic acetate. In the area between A,4A and lysine, AAA-semialdehyde has been identified as an intermediate by Sagisaka and Shimura (4) and recently by Larson et al. (13). Aspen and Meister (14) reported that AAA gives rise to pipecolic acid in mutants of Aspergillus 1 Supported in part by grant National Science Foundation.
G-19807 from the 373
nidulans and point out that the latter may be a lysine precursor in this organism. If pipecolic acid is indeed an intermediate, lysine biosynthesis from AAA may involve the same intermediates as lysine breakdown in mammalian systems. In the latter case it has been established that lysine gives rise to AAA via pipecolic acid (15, 16). Vogel (7) found that Chlorella vulgaris utilizes the DAP pathway, whereas Euglena gracilis does not. These organisms were selected for the present study so that, providing Euglena does utilize the AAA pathway, comparative studies of lysine catabolism could be made in species utilizing the respective biosynthetic routes. This paper presents evidence to show that pipecolic acid is converted to lysine by Euglena gl-acilis and that this organism uses exclusively the A,4A pathway of lysine biosynthesis. MATERIALS
AND
METHOT
ORGAK-ISMS Chlorella vulgaris (Columbia strain) was grown at 25°C. on an illuminated shaker, using the medium of Shrift (17), except that 3 X lo-$ M K2S04 was used. Cultures were used for the experiments 4-6 days after inoculation from 6-Sday-old agar slants. Euglena gracilis Z. strain (Laboratory of Comparative Biology No. E-1.1.2) was grown in cotton-plugged Erlenmeyer flasks on an illuminated glass shelf at 25°C. The medium used was based on t.hat of Cramer and Myers (18) except that the combination of trace metals was changed to that of Allen and Arnon (19).
374
ROTHSTEIN
CO, diffusing from carbon source.
the laboratory
STERILITY Microscopic examination incubation in brain-heart carried out routinely.
air
AND
was the
tive isotopic substrates were added directly to the medium, and the organisms were allowed to grow for an additional 3 days. The cells were collected by centrifugation, washed twice with distilled water, then suspended in 80% ethanol and heated in a boiling water bath for I min. The precipitate was removed by centrifugation, washed with 807~ ethanol, and hydrolyzed in a sealed tube with 6 K HCl for 6 hr. at 105110°C. (In one experiment, one half of the alcohol precipitate was extracted thoroughly with hot SC/e trichloroacetic acid, and the precipitate was washed with ether.) The hydrolyzate was filtered and the HCl removed by evaporation of the warmed solution in a stream of air. The residue was dissolved in water and treated by ion-exchange chromatography and paper chromatography as described below.
CHECKS of the cultures or infusion (Difco) was
ISOTOPIC SUBSTRATES Aspartic acid-4-Cl4 was purchased from commercial sources. DL-AAA-6-V was synthesized by the method of Rothstein and Claus (20) and purified on a column of Dowex l-acetate with 0.5 A’ acetic acid as eluant. The product was sterilized by filtration (Millipore), since autoclaving caused nearly half of the AAA-B-Cl4 to cyclize to piperidonecarboxylic acid. The product used in the experiments reported in Table I was free of this contaminant. nn-Pipecolic acid-6.Cl4 was synthesized as reported for nn-pipecolic acid204 (21).
(B) CHLORELLA A %-day-old culture (100 ml.) was collected by centrifugation under sterile conditions and resuspended in sterile potassium phosphate buffer (0.2 fi!l pH 4.6). Sterile AAA-6-Cl4 (Table I) was added, and the cells were shaken in the light for 24 hr. After being harvested by centrifugation, the cells were washed twice with water and then suspended in cold 5’7, TCA. The suspension was centrifuged after one half hour, and the residue
EXPERIMENTAL
(A) EUGLENA In typical experiments, Euglena was grown for 3 days in 250.ml. cotton-plugged Erlenmeyer flasks containing 100 ml. R medium. The respecTABLE RADIOACTIVITY Expt.
lc
2 3” 4
Organism
Euglena Euglena Euglena Chlorella
OF PRODUCTS
SAFFRAN
I
RECOVERED
FROM ALQAL
Counts/min.”
Substrateb
DL-AAA-~-C’~ DL-Pip-6-Cl4
HYDROLYZATES
GIU
A&
0 Trace”
0 Tracee 20,500 32,800
DI,-As~-~-C’”
19,200
DI,-AAA-~-C~~
66,300
Lysine
27,100 29 ) OOOf 2,730 0
Soluble
12,8W 62, OOOe 2,250 67,000’
a Except as noted, values are based on aliquots of fractions from Dowex 50 columns. * The amounts of radioactivesubstrateare as follows: Expt. 1, 15,~c., 2pmoles; Expt. 2,5rc., 5pmoles; Expt.3,2~~.,25~moles;Expt.4,5rc.,5pmole.s. c Three experiments were performed with varying amounts of AAA-6-Ci4. Paper chromatograms of protein hydrolyzates showed detectable activity only in the lysine area in each case. d Alcohol extract; on paper chromatograms, only lysine appeared to be radioactive. e A total of 250 counts/min. was obtained in the combined glutamic acid, AAA, and aspartic acid. f Radioactivity in the protein hydrolyzate. Only the lysine area contained detectable radioactivity on paper chromatograms in two sets of solvents and on high-voltage electrophoretograms. Samples were removed for chromatography before counting so that the actual figure should be several thousand counts/min. larger. g All the activity appeared to be present as pipecolic acid. h A second experiment yielded similar results, but labeling was lower in all categories. i Trichloroacetic acid extract (5%); most of the activity was in AAA. None was detectable in the lysine area of paper chromatograms.
LYSINE was washed with ether material was hydrolyzed ISOL.~TIOS
BIOSYNTHESIS
IK
ALGAE
375
and dried. The resulting with 6 S HCl as above.
OF AMINO
ACIDS
The Eziglena hydrolysate from one of the AA.4C” incubations (Expt. 1, Table I) was chromatographed on Dowex l-acetate and Dowex 50 (H+) by the method of Hirs et al. (22). In one case, 5 Hmoles of carrier lysine was added before treatment on the columns. The Euglena hydrolyzate from the aspartic acid-J-C’” and the ChloreUa hydrolyzates were treated as above, but the Dowex 50 (H+) column was shortened to 18.5 cm. (“short column”). This was treated first with 1 S HCl (65 ml.) and then with 2.5 A’ HCl at a flow rate of approximately 0.2 ml./min. Fractions of -i ml. were collected. This procedure yielded lysine in a discrete peak at approximately 140-150 ml. of effluent (including the 1 A- HCI). IDEKTIFICATIOK
OF LYSINE
Portions of the fractions containing lysine were chromatographed on paper in butanol-acetic acid-water, -1: 1:l (BAW), ethanol-ammoniawater, 18: 1: 1 (EAW), and butanol-pyridinewater, 1: 1: 1 (BPW), and the chromatograms were scanned for radioactivit,y. After chromatography on Dowex 50, part of the lysine peak from the Euglena experiment with AAA-B-Cl4 (Table I, Expt. 1) was used to prepare the phenylthiohydantoin derivative after addition of 200 mg. of carrier nL-lysine. The product was recrystallized to constant specific activity. PREP~R~TIOS
OF LYSIKE
“ARTIFACT”
A 3-day-old culture of Euglena was collected and treated with ethanol as described above. To the washed precipitate was added 20,000 counts/ min. of nL-lysine-6-C14, which had been shown to yield one radioactive spot coinciding with the position of authentic lysine in BPW (as well as BAW). The mixt,ure was hydrolyzed in a sealed tube as described above. The hydrolyzate was dried, taken up in a little water, filtered, and chromatographed on paper in BPW. The remainder was treatted on a short column of Dowex 50 (H+) as described above. The lysine peak was collected, dried, and chromatographed on paper in BPW, BAW, and EAW. RESCLTS
Hydrolysis of Euglena protein yielded a radioactive “Iysine artifact” rather than lysine. This artifact could be distinguished from lysine on paper chromatograms de-
FIG. 1. Paper chromatography of radioactive lysine and “lysine artifact.” The chromatograms were scanned after development in butanolpyridine-water (1:l:l). d, B, Dr.-lysine-6-C11 and “lysine artifact” from Ezrglena protein hydrolyzate, respectively, run on the same chromatogram; C, “lysine” fraction after treatment of hydrolyzate on Dowex 50.
376
ROTHSTEIN
veloped in butanol-pyridine-water, 1: 1: 1 (A, B, Fig. l), but not in BAW or EAW. An identical product was obtained by subjecting a mixture of authentic DL-lysine-6-C14 and unlabeled Euglena protein to the hydrolysis procedure. From columns of Dowex 50, the material emerged as a peak in the lysine position. Paper chromatograms of this peak showed the presence of both lysine and “lysine artifact” (C, Fig. 1). The phenylthiohydantoin derivative prepared from this material after addition of carrier lysine, led to a product which, after four recrystallizations, showed a constant specific activity (23 counts/min./mg.) through three additional crystallizations. The “artifact” appears to be formed during the evaporation of the protein hydrolyzate, rather than during hydrolysis of the protein; preliminary experiments show that addition of nL-lysine-6-C’” to previously hydrolyzed Euglena protein, followed by evaporation of the mixture, leads to formation of the artifact. This may be analogous to the formation of artifacts from glutamic acid (23). The results of the isotopic experiments are given in Table I. DISCUSSION
It is apparent from the data in Table I that Euglena uses the AAA pathway of lysine biosynthesis exclusively, whereas Chlorella uses only the DAP pathway shown by Vogel (7). Experiment 2 shows that pipecolic acid is converted almost exclusively to lysine in Euglena. The fact that no glutamic acid or, for that matter, any other intermediates are formed to any significant degree eliminates the possibility that lysine is being synthesized from carbon-chain fragments derived from the breakdown of the pipecolic acid. This finding supports the idea that pipecolic acid lies on the pathway of lysine biosynthesis. As to the order of formation of intermediates, Aspen and Meister (14) have implicated pipecolic acid in lysine biosynthesis by showing that mutants of AspeTgillus niger accumulate pipecolic acid in the medium after exposure to AAA. Thus, if pipecolic acid lies directly on the pathway, the mechanism of lysine
AND
SAFFRAN
biosynthesis should involve the steps AAA-+ pipecolic acid ---f lysine (pathway a below). pipecolic acid
La
allb AAASAAA-semialdehyde
lysine
7b
The above results do not exclude the possibility that AAA-semialdehyde plays a central role in the pathway, with pipecolic acid representing a side reaction (route b). AAA-semialdehyde has been reported by other investigat,ors to be an intermediate in lysine biosynthesis in yeast (4, 13). However, if AAA-semialdehyde is formed from pipecolic acid, one would expect to find at least a small amount of labeling in the isolated AAA. No such labeling could be detected either in the nonprotein cell extracts or the incubation medium. Of course, the reaction between AAA and the semialdehyde conceivably could be irreversible under the conditions of Expt. 2. The further steps of lysine synthesis are completely unknown. Pipecolic acid could be dehydrogenated to form Al-piperideine2-carboxylic acid e e-amino-cr-ketocaproic acid, and by transamination yield lysine. However, Aspen and Meister have reported that the latter compound does not support growth in lysine-requiring mutants of Aspergillus niger (14), although, an e-acyl derivative could be involved. A route such as this is essentially the reverse of the proposed breakdown pathway of lysine in mammalian species (15, 16, 21). Alternatively, the pathway may involve synthesis of the compound “saccharopine” (E-N-(L-glutaryl-2)-L-lysine) (13). REFERENCES 1. WORK, E., AND DEWEY, D. L., J. Gen. Microbiol. 9, 394 (1953). 2. VOGEL, H. J., Proc. Natl. Acad. Sci. U. S. 46, 1717 (1959). E., J. Biol. Chem. 192, 607 (1951). 3. WINDSOR, 4. SAGISAKA, S., AND SHIMURA, K., J. Biochem. (Tokyo) 49, 392 (1961). 5. BROQUIST, H. P., STIFFEY, A. V., AND ALBRECHT, A. M., Appl. Microbial. 9, 1 (1961). 6. VOGEL, H. J., Biochim. Biophys. Acta 41, 172 (1960). 7. VOGEL, H. J., Biochim. Biophys. ilcta 34, 282 (1959).
LYSINE
BIOSYXTHESIS
8. VOGEL, H. J., fiatwe 189, 1026 (1961). 9. (;II,VARG, C., J. Biol. Chem. 236, 1429 (1961). 10. PETERKOFSKY, B., AND GILVARG, C., J. Viol. Chem. 236, 1432 (1961). 11. RDELMAN, J. C., AND GILVARG, C., J. Biol. Chew. 236, 3295 (1961). 12. STRASSM.4N, M., AND WEINHOUSE, S., J. :17x. (*he,n. Sot. 75, 1680 (1953). 13. LARSON, It. L., SANDINE, W. I)., AND BROQCIST, H. P., J. Biol. Chem. 238, 275 (1963). 14. AWES, A. J., AND MEISTER, 8., Biochemistry 1, 606 (1962). 15. ROTHSTEIN, M., AND MILLER, L. L., J. Biol. Chem. 211, 851 (1954).
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