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Isolation of a new naturally occurring pteridine from bacteria, and its relation to folic acid biosynthesis
ARCHIVES
OF
BIOCHEMISTRY
Isolation
AND
of a New and
BIOPHYSICS
Naturally
its Relation
M. GOTO, H. S. FORREST, Department
A new
naturally
8-14
111,
Occurring to Folic
LOIS HEARD of
occurring
(1965)
Pteridine
Acid
DICKERMAN,
AND
University of Texas, Received February 24, 1965 has been
Bacteria,
Biosynthesis’
Zoology,
pteridine
from
isolated
Austin,
from
T. URUSHIBARA Texas
two
different
micro-
organisms. It has been shown to be identical with synthetic xanthopterin sulfate and is presumed to arise as a biproduct of excess pteridine ring production (for folic acid synthesis)
in these
organisms.
Axotomonas ins&la (1) is a gram-negative, non-spore-forming bacterium capable of limited nitrogen fixation. When grown on synthetic medium on agar plates, it excretes a blue-fluorescent compound, identified as 2-amino-4-hydroxypteridine, in considerable quantity into the agar. Because of the relatively high production of this pteridine, it was thought that this organism might be useful to study the biosynthesis of the pteridine ring and, possibly, of folic acid-like compounds. However, some of the characteristics of the organism make it unsuitable for this work. It is slow growing (although this can be alleviated to some extent by the addition of L-asparagine to the medium) ; it invariably grows in clumps in liquid medium (making the production of mutants, for example, a difficult, if not impossible, task) ; and its folic acid content is about t.he same as that of other microorganisms, thus negating any special advantage in using it for the study of folic acid biosynthesis. However, when grown in liquid culture, Azotomonas excretes, in addition to 2-aminoa number of other 4-hydroxypteridine, pteridines of which the principal one is a new naturally occurring compound. The elucidation of the structure of this compound, which has also been isolated from
Escherichia coli, forms the main subject of this paper; in addition, the other pteridines found in this organism are catalogued, and some of the attempts to use Azotomonas for studies of pteridine ring biosynthesis are described. EXPERIMENTAL Isolation
of
the Mew
Compound
From Azotomonas insolita. The medium used for the growth of this organism was modified from one described by Skerman (2) and was made up as follows (quantities in grams per liter): NaN03 , 2.0; MgS04.7H20, 0.2; FeS04.7Hz0 or iron versenate, 0.1; NasHPOd , 0.21; NaHzPOl , 0.09; KCI, 0.04; CaClz , 0.015; CuS04.5H20, 0.005; H3B03 , 0.010; MnS0,.5H20, 0.010; ZnS04,7H,0, 0.010; MoOI , 0.010; glucose, 5; asparagine, 1. As mentioned above, asparagine is not necessary for the growth of Azotomonas, but its use reduces the growth period considerably (alanine and glycine have similar but less marked effects). Cultures grown in this medium for about 72 hours at 25” with aeration were autoclaved, acidified (acetic acid) to pH 4, and filtered with the aid of Celite. The filtrate was treated with charcoal (Darco G60; about 1 gm per liter). After stirring for 15 minutes, the charcoal was collected and washed with water and finally with ethanol-l% ammonia (1:l) to remove the absorbed materials. The eluate was evaporated to small bulk and the concentrated solution was st,reaked on heavy chromatographic paper (Whatman No. 17). The chromatogram was developed with n-propanol1% ammonia (2:l). The broad fluorescent band, Rf , 0.2-0.3, was cut out, and the fluorescent ma-
1 This work was supported in part by grant GM 12323 from the National Institutes of Health, Public Health Service, and by a grant from the Robert A. Welch Foundation, Houston, Texas. 8
ISOLATION
OF
XANTHOPTERIN
9
SULFATE
0.0
3
5
WAVELENGTH
11
13
15
(~VIICRONS) FIG. 1. Infrared occurring pteridine
spectra of synthetic from Azotomonas
xanthopterin (light line).
terial was eluted from it with 1% aqueous ammonia. Ammonia was removed from the eluate by evaporation in WWUO, and sufficient Dowex 1 (OHform) was added to remove all fluorescence. The Dowex resin was washed thoroughly with water, poured into a small column, and washed with 98yo formic acid. This formic acid eluate was allowed to drop into a tenfold excess of ether, and the resulting precipit,ate was collected and recrystallized after neutralization with sodium hydroxide as described below. It was identical, in all respects, with the material isolated from E. co&i. From Escherichia coli B. Escherichia coli B (wild type) was cultivated for 16 hours in a fermentation tank (lOOO-liter capacity) at 32”-33” with efficient aeration. The medium used was as follows: n-glucose, 3 kg; monosodium glutamate, 2.25 kg; NazHPOd , 19.15 kg; KH2P04 , 450 gm; NH&l, 1.5 kg; MgS04, 150 gm; CaCls , 7.5 gm; FeS04, 0.38 gm; yeast extract, 3.75 gm; water, 750 liters. After filtration, the medium from the fermentation (750 liters) was adjusted to pH 4 (cont. HCl), and the fluorescent compounds were absorbed on charcoal (2 kg) and subsequently eluted with aqueous alcoholic ammonia (ethanol-3ye aqueous ammonia, 1:3). The eluate was evaporated to dryness below 50” in vacua and an acidified solution of the brown residue (27 gm) was treated two more times according to the same procedure. The final residue was redissolved in 3% ammonium chloride, and the solution was added to a column of cellulose powder (70 X 300 mm, Toyo Roshi B, 200-300 mesh). The column was developed with 3% ammonium chloride, and the main fluorescent band
sulfate
(heavy
line)
and the naturally
was collected from the column and the solution was treated with charcoal (200 mg). The fluorescent material was eluted as before and submitted to powdered cellulose chromatography two more times (charcoal being used to recover the compound from the eluates) using consecutively npropanol-17, aqueous ammonia (2:l) and nbutanol-acetic acid-water (4: 1: 1). The purified material (12 mg) was dissolved in 0.1 N hydrochloric acid, the solution was filtered to remove a small amount of insoluble material, and the filtrate was neutralized (NaOH). The pale-yellow precipitate (8 mg) was recrystallized twice from water to give almost colorless needles (5 mg), m.p. > 300”. Sample dried at 120” in aacuo over P20j . Anal. Calcd. for CBH105N&Na: C, 25.6; H, 1.4; N, 25.0; S, 11.4. Found: C, 25.1; H, 2.3; N, 24.5; S, 11.3. The sample gave a negative test for phosphorus. Spectra. The ultraviolet spectra of the compound gave the following values: X,,, (10%) 262 (16.4), 370 (6.0) in 0.1 N sodium hydroxide; 235 (9.4), 246 (shoulder), 290 (3.4), 324 (5.7) in 0.1 N hydrochloric acid. The infrared spectrum is given in Fig. 1. Hydrolysis. The compound, in hydrochloric acid (4 N), was heated for 2 hours at 95” in a sealed tube. The contents of the tube were evaporated to dryness in vacua, and the residue, dissolved in water, was compared chromatographically and spectrophotometrically with known compounds (Table I). The hydrolysis product was identical with xanthopterin. Aluminum amalgam reduction. Amalgamated aluminum wire (30 X 0.5 mm) was placed in a solution (0.05 ml) of the unknown compound. After hydrogen evolution had ceased, the precip-
10
GOT0
ET
TABLE Rr
VALUES
OF THE
NEW
Substance
New compound Synthetic xanthopterin Hydrolysis product Xanthopterin Hydrogenation product 2-Amino-4-hydroxypteridine 2-Amino-4-hydroxypteridine-6carboxylic acid
sulfate
PTERIDINE
FROM
AL. I
Azolomonas
AND
ITS
DEQRADATION
PRODUCTS
1
2
3
4
5
sb
0.02 0.02 0.36 0.36 0.29 0.29 0.11
0.13 0.13 0.15 0.15 0.31 0.31 0.09
0.36 0.36 0.62 0.62 0.55 0.55 0.45
0.66 0.66 0.47 0.47 0.49 0.49 0.52
0.54 0.54 0.47 0.47 0.46 0.46 0.55
78 78 -1 -1 -2 -2 59
p Solvents: 1, n-butanol-acetic acid-water (4:l:l); 2, n-propanol-1% ammonia (2:l); formic acid-water (8:2:5); 4, 3% ammonium chloride; 5, 5% sodium citrate, isa-amyl b Distance (in mm) to anode after electrophoresis at pH 4.65 (sodium acetate buffer) at 20 V/cm. itated aluminum hydroxide was removed by centrifugation and the supernatant was examined ehromatographically. The major product was shown to be identical with 2-amino4hydroxypteridine (Table I). Microhydrogenation. The unknown compound (0.51 mg) in sodium hydroxide solution (2 ml; 0.001 N) was hydrogenated in a Warburg vessel over Adams’ catalyst (2 mg). Hydrogenation virtually ceased after 2.1 moles of hydrogen were adsorbed (2 hours). The catalyst was removed and the mixture was allowed to stand overnight in air. Chromatography of the resulting solution showed that it contained a mixture of the original compound and 2.amino-4-hydroxypteridine. Preparation of xanthopterin-sulfate. A solution (105.3 mg) in of 2-amino-4-hydroxypteridine sodium hydroxide (15 ml; 0.1 N) was hydrogenated in the presence of Adams’ catalyst (26.0 mg) for 20 hours; the solution was no longer fluorescent. The catalyst was removed carefully by decantation and SO? was bubbled into the solution for 15 minutes; the flask was stoppered and the solution was stirred for 15 hours. It was then heated on a water bath for 10 minutes to expel excess SOZ , cooled to room temperature, neutralized (NHdOH), and treated with a slight excess of a saturated solution of KMn04 in 0.1 N sodium hydroxide. The fluorescent compounds, except 2-amino-4-hydroxypteridine and xant,hopterin sulfate, were destroyed by this process. Excess permanganate was destroyed with a little alcohol, and MnOs was removed by centrifugation. The precipitate was washed well with water and the solution and washings were combined (200 ml). This solution was placed on a 38 X 130-mm column of Dowex 1 X 8 (Cl-, 200-400 mesh); the fluorescent compounds were adsorbed and the column
3, sec-butanolalcohol. for 90 minutes
was washed with 8 liters of 0.03 M ammonium chloride, bringing about the elution of a-amino4-hydroxypteridine. The column was further washed with 0.03 M ammonium chloride and 0.1 N hydrochloric acid, the proportion of the solvents being changed to give a continuous pH gradient (total volume of washings, 16 liters). Xanthopterin sulfate was eluted with an additional 1.8 liters of 0.1 N hydrochloric acid. The eluted solution was adjusted to pH 3.0 with ammonia and the pteridine was adsorbed on Norit (509 mg); the charcoal was washed with water and the compound was eluted with eight portions (600 ml) of 27’n ammonium hydroxide-ethanol (3:l). The eluate was earlcentrated to dryness in vacua and the residue was crystallized from water; yield 26.9 mg. Anal. Calcd. for CH6 60 5N 6S.H*O: C, 26.0; H, 2.5; N, 25.3; 8, 11.6. Found: C, 26.9; H, 3.4; N, 25.8; S, 10.5. Mass spectrum. The mass spectrum was determined with a Hitachi mass spectrometer (model RMU-GA); the sample in the inlet system was heated to 260” and the isatron temperature was also maintained at 200”. The ionizing voltage was kept at 70 eV and the ionizing current at 60 ma. The spectrum is given in Fig. 2. Comparison of xanthopterin sulfate and naturally occurring material. Table I shows Rf values and electrophoretic data for the natural and synthetic sulfates. In addition, their ultraviolet absorption spectra were identical as were their infrared spectra (Fig. 1). Other pteridines in Azotomonas. A number of other less conspicuous fluorescent bands were observed on the original chromatogram of the charcoal-recovered material from A. inaolita culture fluid. The fluorescent materials in these bands were eluted in the usual way, purified by further
ISOLATION
OF
XANTHOPTERIN
11
SULFATE
257 w2r
FIG.
2. Mass
spectrum
of xanthopterin
TABLE R,
VALUES
OF OTHER
FLUORESCENT
Band I (new compound) Band II Band III FMN Band IV Isoxanthopterin Band V 2-Amino-4-hydroxypteridine Band VI Biopterin Q Compounds are numbered b Solvents: as in Table I. c Distance (in mm) to anode at 10 V/cm. chromat,ography, authentic materials in liquid medium flavine-5-phosphate, 4-hydroxypteridine, is very similar to (Band II) which chloric acid for amino - 4 -. hydroxy
in order after
250
200
m/e
sulfate.
II
COMPOUNDS IN Azotomonas OF KNOWN COMPOUNDS
COMPARED
WITH
THOSE
1
2
3
4
5
0.02 0.02 0.02 0.02 0.15 0.15 0.29 0.29 0.30 0.30
0.13 0.12 0.07 0.07 0.17 0.17 0.31 0.31 0.31 0.31
0.36 0.43 0.46 0.46 0.42 0.42 0.55 0.55 0.60 0.60
0.66 0.79 0.49 0.48 0.33 0.33 0.48 0.49 0.69 0.66
0.54 0.32 0.51 0.51 0.34 0.34 0.46 0.46 0.72 0.69
of increasing
electrophoresis
and finally compared with (Table II). Azotomonas grown was thus shown to contain riboisoxanthopterin, a-aminoa compound (Band VI) which biopterin, and another compound on acid hydrolysis (2 N hydro30 minutes at 100”) yields a 2- 6 - trihydroxypropylpteridine
RI in propanol-lo/, at pH 4.65
(sodium
ammonia acetate
buffer)
52 20 17 17 -2 -2 -3 -3 -3 -3
(2: 1). for
130 minutes
(Table III). These latter two compounds have not been complet,ely identified. Attempts to produce mutants in Azotomonas. As stated above, Azotomonas tends to grow in clumps even on defined liquid medium. Attempts to prevent this clumping by incorporation of surfaceacting agents (e.g., Tween 80, disodium versenate, sodium dodecyl sulfate, pectinase, hyaluronidase, Alconox, or Vel) in the medium were unsuccessful.
12
GOT0
ET
TABLE FURTHER
AL. III
CHARACTERIZATION
OF BAND
II
Solvents
Substance Band II Hydrolysis product Neopterin KMn04 oxidation product 2-Amino-4-hydroxypteridine6-carboxylic acid
1
2
3
4
5
6
0.02 0.30 0.30 0.23 0.23
0.12 0.29 0.29 0.14 0.14
0.43 0.48 0.48 0.42 0.42
0.79 0.62 0.62 0.54 0.54
0.82 0.62 0.62 0.47 0.47
58 -3 -3 96 96
I
1
7Pn d d
C
&......“”
FIG.
I
I
I
4
6
12
3. Comparison
of growth
I
I
16 20 TIME (hours) rate
and pteridine
To remove the large clumps and obtain a relatively uniform suspension of cells for mutagenic studies the liquid culture was passed through a sterile filter of loosely packed glass wool. Attempts were made to produce a purine-requiring mutant by the use of heat on dried cultures (100”/2 hours) (4); ultraviolet light followed by penicillin treatment (5) ; nitrous acid (6) ; and X-ray irradiation. None of these methods was succesful, presumably because of the associations of cells still present in the filtered cultures. Attempts to incorporate radioactive purines into the pteridines of Azotomonas. Azotomonas cultures in the log phase were used as inocula for flasks of regular medium containing 10 pg per milliliter of one of the following compounds: uric acid-6-Cr4, hypoxanthine-2-P, 4-hydroxypteridine-2-C14, and guanylic acid-U-W. After reaching maximal growth, the cultures were boiled and filtered, and the filtrates were treated separately with excess
I
I
24
28
production
in Azotomonas.
KMn04 in alkali. After complete oxidation, excess KMn04 was destroyed by addition of ethanol, and the suspensions were filtered to remove MnO* . The latter filtrates were adjusted to pH 4 (HCI), and the pteridines in them were absorbed on charcoal and eluted from this with ethanol-lG7o am monium hydroxide (1:l). After removal of the solvent, the residues were submitted to paper chromatography using successively n-propanol1% ammonium hydroxide (2:1), 6% aqueous acid-water ammonia, and set-butanol-formic (8:2:5). The fluorescent materials (2.amino-4hydroxypteridine and 2-amino-4-hydroxypteridine-6-carboxylic acid) were eluted and counted. No significant radioactivity was found in these compounds with any of the radioactive materials used. Comparison of growth rate and pteridine production in Aeotomonas insolita. It was of interest to determine the relationship, if any, between the
ISOLATION
OF
XANTHOPTERIN
growth rate of the organism and the production of 2amino-4-hydroxypteridine and folk acid. The growth rate was determined by measurement of the optical density at 540 rnp of IO-ml samples removed from a growing culture (500 ml) at 4hour intervals. Pteridine ring synthesis was measured on the same samples, after centrifugation of the cells, by comparison of the fluorescence of the supernatant, with that of a standard solution of 2-amino-4-hydroxypteridine. The folic acid content of the culture was also determined by using Streptococcus juecalis R on an aliquot of the boiled centrifuged samples. The results of all three assays are roughly parallel (Fig. 3). However, the folic acid content of the organism is, at most, 306 times less than the pteridine content. Precursors for the enzymic synthesis of jolic acid. A series of simple pteridines and one purine derivative were tested as potential folic acid precursors on cell-free extracts (prepared by sonication of cells from a log-phase culture) of A. insolita. The presence of guanosine, guanylic acid, 2-amino-4hydroxypteridine (with ATP and Mg++)) (2. amino-4.hydroxy-6-pteridinyl)methyl phosphate, phos(2.amino-4.hydroxy-6.pteridinyl)glycerol phate, 2-amino-7,8-dihydro-4.hydroxy-G-propionylpteridine (isosepiapterin), 2.amino-4-hydroxyor 2.amino-4-hy5,6,7,&tetrahydropteridine droxy-5,6,7,8-tetrahydro-6.trihydroxypropylpteridine did not increase the folic acid content, measured as above. 2.Amino-4-hydroxy-6-hydroxymethyl-5,6,7,%tetrahydropteridine stimulated folic acid production slightly, and the corresponding phosphate brought about a tenfold increase in folic acid content of the incubated extract. DISCUSSION
The new naturally occurring compound described above has an ultraviolet absorption spectrum almost identical with that of 2-amino-4-hydroxypteridine-6-carboxylic acid. However, it could be separated chromatographically from the latter compound, and alkaline permanganate oxidation-which, in fact, destroyed the compound-demonstrated that it did not have a substituent linked by carbon to the ring at the 6-position. The presence of the parent pteridine ring system was demonstrated by hydrogenation and reoxidation of the reduction product. This yielded 2-amino-4-hydroxypteridine in addition to regenerated starting material. Production of xanthopterin by acid hydrolysis showed that there was a substituent on the 6-position, and its
13
SULFATE
nature was demonstrated by comparison of the compound isolated from Axotomonas with synthetic xanthopterin sulfate. The compounds were indistinguishable. The structure of the synthetic compound was confirmed by the fact that its mass spectrum (Fig. 3) showed a peak at 257 (M-2). This compound appears to be identical wit’h the “2-amino-4-hydroxypteridine-6sulfonic acid” (m.w. 243) synthesized, in essentially the same way, by Viscontini and Weilenmann (3). The mass spectrum leaves no doubt as to the correct formula for the compound.2 The question then arises as to the origin of this compound, the first naturally occurring pteridine sulfate to be described. The most likely explanation is that it is produced in the culture medium from reduced 2-amino-4-hydroxypteridine in exactly the same way as it can be synthesized chemically, i.e., by reaction of SOS’ with 2 - amino - 4 - hydroxy - 7,8 - dihydropteridine (3), followed by oxidation and aromatization. This implies that the dihydro- or tetrahydro-pteridine is excreted by the cells, which, in turn, is consistent with the fact that 2-amino-4-hydroxypteridine is found in cultures grown on agar plates. Bearing in mind the level of folic acid found in this organism, it appears that Axotommas produces a large excess of the pteridine moiety of the cofactor (or cofactors) with respect to the amount used for coenzyme synthesis. A small amount is converted by oxidation into isoxanthopterin, and the rest is excreted or, under the appropriate conditions, converted into the sulfate. The problem of the origin of reduced 2-amino-4-hydroxypteridine still remains. Current theory (7) supposes that the pteridine ring arises from guanosine or guanylic acid, the first actual pteridine being a reduced 2-aminol-hydroxy-B-trihydroxyalkylpteridine or the corresponding 3’-phosphate ester. Either of these compounds in the reduced state is known to be unstable, and they could readily give rise to a reduced 2 - amino - 4 - hydroxypteridine. However, guanylic acid-U-Cl4 (which was demon* A more extended study pteridines will be published
of the mass elsewhere.
spectra
of
14
GOT0
strated by paper chromatography to contain both guanine and guanosine as impurities) was not incorporated into pteridines in growing Axotomonas cultures (a failure which of course could simply be due to nonpermeability) ; in addition, guanosine and guanylic acid failed to stimulate the production of folic acid in cell-free extracts. Qualitatively, however, guanine plus glucose added to Axotomonas medium increased the production of isoxanthopterin, although other purines (along with glucose) or guanine or guanosine alone, did not. Thus, although no direct evidence could be obtained, these experiments suggest that the same pathway exists for the conversion of a purine to a pteridine in Azotomonas as in E. coli (7) or in Corynebacterium (8). Furthermore, the most active pteridine tested as a precursor for folic acid was (2-amino-4-hydroxy-5,6,7, %tetra-hydro-6-pteridinyl)methyl phosphate which presumably can be readily converted into (2 - amino - 4 - hydroxy - 7,8 - dihydro 6-pteridinyl)methyl pyrophosphate the compound postulated by Weisman and Brown (9) as the immediate precursor of folic acid-
ET
AL.
like compounds. The over-all pathway in Azotomonas thus appears to be the same as in other organisms, the one unique feature being the vast overproduction of the pteridine ring, leading to the excretion of 2-amino4-hydroxypteridine or xanthcpterin sulfate. REFERENCES 1. STAPP, C., Zentr. fur Bakt. II Abt. 102, 1 (1940) [see also: Abs. of Communications, 3rd. Int. Congr. of Microbial., 30F (1939)l. 2. SKERMAN, V. B. D., “A Guide to the Identification of the Genera of Bacteria,” p. 151. Williams di Wilkins, Baltimore, Maryland (1959). M., AND WEILENMANN, H. R., 3. VISCONTINI, Helv. Chem. Acta 42. 1954 (1959). 4. ZAMENHOF, S., Proc. Natl. Acad. Sci. U.S. 46, 101 (1960). 5. GORINI, H., AND KAUFMAN, H., Science 131. 604 (1960). 6. KAUDEWITZ, F., Nature 183, 1829 (1959). 7. cf. BROWN, G. M., AND REYNOLDS, J. J., Ann. Rev. Biochem. 32, 419 (1963). 8. VIEIRA, E., AND SHAW, E., J. Biol. Chem. 236, 2507 (1961). 9. WEISMAN, R.. A., AND BROWN, G. M., J. Biol. Chem. 239, 326 (1964).
OF
BIOCHEMISTRY
Isolation
AND
of a New and
BIOPHYSICS
Naturally
its Relation
M. GOTO, H. S. FORREST, Department
A new
naturally
8-14
111,
Occurring to Folic
LOIS HEARD of
occurring
(1965)
Pteridine
Acid
DICKERMAN,
AND
University of Texas, Received February 24, 1965 has been
Bacteria,
Biosynthesis’
Zoology,
pteridine
from
isolated
Austin,
from
T. URUSHIBARA Texas
two
different
micro-
organisms. It has been shown to be identical with synthetic xanthopterin sulfate and is presumed to arise as a biproduct of excess pteridine ring production (for folic acid synthesis)
in these
organisms.
Axotomonas ins&la (1) is a gram-negative, non-spore-forming bacterium capable of limited nitrogen fixation. When grown on synthetic medium on agar plates, it excretes a blue-fluorescent compound, identified as 2-amino-4-hydroxypteridine, in considerable quantity into the agar. Because of the relatively high production of this pteridine, it was thought that this organism might be useful to study the biosynthesis of the pteridine ring and, possibly, of folic acid-like compounds. However, some of the characteristics of the organism make it unsuitable for this work. It is slow growing (although this can be alleviated to some extent by the addition of L-asparagine to the medium) ; it invariably grows in clumps in liquid medium (making the production of mutants, for example, a difficult, if not impossible, task) ; and its folic acid content is about t.he same as that of other microorganisms, thus negating any special advantage in using it for the study of folic acid biosynthesis. However, when grown in liquid culture, Azotomonas excretes, in addition to 2-aminoa number of other 4-hydroxypteridine, pteridines of which the principal one is a new naturally occurring compound. The elucidation of the structure of this compound, which has also been isolated from
Escherichia coli, forms the main subject of this paper; in addition, the other pteridines found in this organism are catalogued, and some of the attempts to use Azotomonas for studies of pteridine ring biosynthesis are described. EXPERIMENTAL Isolation
of
the Mew
Compound
From Azotomonas insolita. The medium used for the growth of this organism was modified from one described by Skerman (2) and was made up as follows (quantities in grams per liter): NaN03 , 2.0; MgS04.7H20, 0.2; FeS04.7Hz0 or iron versenate, 0.1; NasHPOd , 0.21; NaHzPOl , 0.09; KCI, 0.04; CaClz , 0.015; CuS04.5H20, 0.005; H3B03 , 0.010; MnS0,.5H20, 0.010; ZnS04,7H,0, 0.010; MoOI , 0.010; glucose, 5; asparagine, 1. As mentioned above, asparagine is not necessary for the growth of Azotomonas, but its use reduces the growth period considerably (alanine and glycine have similar but less marked effects). Cultures grown in this medium for about 72 hours at 25” with aeration were autoclaved, acidified (acetic acid) to pH 4, and filtered with the aid of Celite. The filtrate was treated with charcoal (Darco G60; about 1 gm per liter). After stirring for 15 minutes, the charcoal was collected and washed with water and finally with ethanol-l% ammonia (1:l) to remove the absorbed materials. The eluate was evaporated to small bulk and the concentrated solution was st,reaked on heavy chromatographic paper (Whatman No. 17). The chromatogram was developed with n-propanol1% ammonia (2:l). The broad fluorescent band, Rf , 0.2-0.3, was cut out, and the fluorescent ma-
1 This work was supported in part by grant GM 12323 from the National Institutes of Health, Public Health Service, and by a grant from the Robert A. Welch Foundation, Houston, Texas. 8
ISOLATION
OF
XANTHOPTERIN
9
SULFATE
0.0
3
5
WAVELENGTH
11
13
15
(~VIICRONS) FIG. 1. Infrared occurring pteridine
spectra of synthetic from Azotomonas
xanthopterin (light line).
terial was eluted from it with 1% aqueous ammonia. Ammonia was removed from the eluate by evaporation in WWUO, and sufficient Dowex 1 (OHform) was added to remove all fluorescence. The Dowex resin was washed thoroughly with water, poured into a small column, and washed with 98yo formic acid. This formic acid eluate was allowed to drop into a tenfold excess of ether, and the resulting precipit,ate was collected and recrystallized after neutralization with sodium hydroxide as described below. It was identical, in all respects, with the material isolated from E. co&i. From Escherichia coli B. Escherichia coli B (wild type) was cultivated for 16 hours in a fermentation tank (lOOO-liter capacity) at 32”-33” with efficient aeration. The medium used was as follows: n-glucose, 3 kg; monosodium glutamate, 2.25 kg; NazHPOd , 19.15 kg; KH2P04 , 450 gm; NH&l, 1.5 kg; MgS04, 150 gm; CaCls , 7.5 gm; FeS04, 0.38 gm; yeast extract, 3.75 gm; water, 750 liters. After filtration, the medium from the fermentation (750 liters) was adjusted to pH 4 (cont. HCl), and the fluorescent compounds were absorbed on charcoal (2 kg) and subsequently eluted with aqueous alcoholic ammonia (ethanol-3ye aqueous ammonia, 1:3). The eluate was evaporated to dryness below 50” in vacua and an acidified solution of the brown residue (27 gm) was treated two more times according to the same procedure. The final residue was redissolved in 3% ammonium chloride, and the solution was added to a column of cellulose powder (70 X 300 mm, Toyo Roshi B, 200-300 mesh). The column was developed with 3% ammonium chloride, and the main fluorescent band
sulfate
(heavy
line)
and the naturally
was collected from the column and the solution was treated with charcoal (200 mg). The fluorescent material was eluted as before and submitted to powdered cellulose chromatography two more times (charcoal being used to recover the compound from the eluates) using consecutively npropanol-17, aqueous ammonia (2:l) and nbutanol-acetic acid-water (4: 1: 1). The purified material (12 mg) was dissolved in 0.1 N hydrochloric acid, the solution was filtered to remove a small amount of insoluble material, and the filtrate was neutralized (NaOH). The pale-yellow precipitate (8 mg) was recrystallized twice from water to give almost colorless needles (5 mg), m.p. > 300”. Sample dried at 120” in aacuo over P20j . Anal. Calcd. for CBH105N&Na: C, 25.6; H, 1.4; N, 25.0; S, 11.4. Found: C, 25.1; H, 2.3; N, 24.5; S, 11.3. The sample gave a negative test for phosphorus. Spectra. The ultraviolet spectra of the compound gave the following values: X,,, (10%) 262 (16.4), 370 (6.0) in 0.1 N sodium hydroxide; 235 (9.4), 246 (shoulder), 290 (3.4), 324 (5.7) in 0.1 N hydrochloric acid. The infrared spectrum is given in Fig. 1. Hydrolysis. The compound, in hydrochloric acid (4 N), was heated for 2 hours at 95” in a sealed tube. The contents of the tube were evaporated to dryness in vacua, and the residue, dissolved in water, was compared chromatographically and spectrophotometrically with known compounds (Table I). The hydrolysis product was identical with xanthopterin. Aluminum amalgam reduction. Amalgamated aluminum wire (30 X 0.5 mm) was placed in a solution (0.05 ml) of the unknown compound. After hydrogen evolution had ceased, the precip-
10
GOT0
ET
TABLE Rr
VALUES
OF THE
NEW
Substance
New compound Synthetic xanthopterin Hydrolysis product Xanthopterin Hydrogenation product 2-Amino-4-hydroxypteridine 2-Amino-4-hydroxypteridine-6carboxylic acid
sulfate
PTERIDINE
FROM
AL. I
Azolomonas
AND
ITS
DEQRADATION
PRODUCTS
1
2
3
4
5
sb
0.02 0.02 0.36 0.36 0.29 0.29 0.11
0.13 0.13 0.15 0.15 0.31 0.31 0.09
0.36 0.36 0.62 0.62 0.55 0.55 0.45
0.66 0.66 0.47 0.47 0.49 0.49 0.52
0.54 0.54 0.47 0.47 0.46 0.46 0.55
78 78 -1 -1 -2 -2 59
p Solvents: 1, n-butanol-acetic acid-water (4:l:l); 2, n-propanol-1% ammonia (2:l); formic acid-water (8:2:5); 4, 3% ammonium chloride; 5, 5% sodium citrate, isa-amyl b Distance (in mm) to anode after electrophoresis at pH 4.65 (sodium acetate buffer) at 20 V/cm. itated aluminum hydroxide was removed by centrifugation and the supernatant was examined ehromatographically. The major product was shown to be identical with 2-amino4hydroxypteridine (Table I). Microhydrogenation. The unknown compound (0.51 mg) in sodium hydroxide solution (2 ml; 0.001 N) was hydrogenated in a Warburg vessel over Adams’ catalyst (2 mg). Hydrogenation virtually ceased after 2.1 moles of hydrogen were adsorbed (2 hours). The catalyst was removed and the mixture was allowed to stand overnight in air. Chromatography of the resulting solution showed that it contained a mixture of the original compound and 2.amino-4-hydroxypteridine. Preparation of xanthopterin-sulfate. A solution (105.3 mg) in of 2-amino-4-hydroxypteridine sodium hydroxide (15 ml; 0.1 N) was hydrogenated in the presence of Adams’ catalyst (26.0 mg) for 20 hours; the solution was no longer fluorescent. The catalyst was removed carefully by decantation and SO? was bubbled into the solution for 15 minutes; the flask was stoppered and the solution was stirred for 15 hours. It was then heated on a water bath for 10 minutes to expel excess SOZ , cooled to room temperature, neutralized (NHdOH), and treated with a slight excess of a saturated solution of KMn04 in 0.1 N sodium hydroxide. The fluorescent compounds, except 2-amino-4-hydroxypteridine and xant,hopterin sulfate, were destroyed by this process. Excess permanganate was destroyed with a little alcohol, and MnOs was removed by centrifugation. The precipitate was washed well with water and the solution and washings were combined (200 ml). This solution was placed on a 38 X 130-mm column of Dowex 1 X 8 (Cl-, 200-400 mesh); the fluorescent compounds were adsorbed and the column
3, sec-butanolalcohol. for 90 minutes
was washed with 8 liters of 0.03 M ammonium chloride, bringing about the elution of a-amino4-hydroxypteridine. The column was further washed with 0.03 M ammonium chloride and 0.1 N hydrochloric acid, the proportion of the solvents being changed to give a continuous pH gradient (total volume of washings, 16 liters). Xanthopterin sulfate was eluted with an additional 1.8 liters of 0.1 N hydrochloric acid. The eluted solution was adjusted to pH 3.0 with ammonia and the pteridine was adsorbed on Norit (509 mg); the charcoal was washed with water and the compound was eluted with eight portions (600 ml) of 27’n ammonium hydroxide-ethanol (3:l). The eluate was earlcentrated to dryness in vacua and the residue was crystallized from water; yield 26.9 mg. Anal. Calcd. for CH6 60 5N 6S.H*O: C, 26.0; H, 2.5; N, 25.3; 8, 11.6. Found: C, 26.9; H, 3.4; N, 25.8; S, 10.5. Mass spectrum. The mass spectrum was determined with a Hitachi mass spectrometer (model RMU-GA); the sample in the inlet system was heated to 260” and the isatron temperature was also maintained at 200”. The ionizing voltage was kept at 70 eV and the ionizing current at 60 ma. The spectrum is given in Fig. 2. Comparison of xanthopterin sulfate and naturally occurring material. Table I shows Rf values and electrophoretic data for the natural and synthetic sulfates. In addition, their ultraviolet absorption spectra were identical as were their infrared spectra (Fig. 1). Other pteridines in Azotomonas. A number of other less conspicuous fluorescent bands were observed on the original chromatogram of the charcoal-recovered material from A. inaolita culture fluid. The fluorescent materials in these bands were eluted in the usual way, purified by further
ISOLATION
OF
XANTHOPTERIN
11
SULFATE
257 w2r
FIG.
2. Mass
spectrum
of xanthopterin
TABLE R,
VALUES
OF OTHER
FLUORESCENT
Band I (new compound) Band II Band III FMN Band IV Isoxanthopterin Band V 2-Amino-4-hydroxypteridine Band VI Biopterin Q Compounds are numbered b Solvents: as in Table I. c Distance (in mm) to anode at 10 V/cm. chromat,ography, authentic materials in liquid medium flavine-5-phosphate, 4-hydroxypteridine, is very similar to (Band II) which chloric acid for amino - 4 -. hydroxy
in order after
250
200
m/e
sulfate.
II
COMPOUNDS IN Azotomonas OF KNOWN COMPOUNDS
COMPARED
WITH
THOSE
1
2
3
4
5
0.02 0.02 0.02 0.02 0.15 0.15 0.29 0.29 0.30 0.30
0.13 0.12 0.07 0.07 0.17 0.17 0.31 0.31 0.31 0.31
0.36 0.43 0.46 0.46 0.42 0.42 0.55 0.55 0.60 0.60
0.66 0.79 0.49 0.48 0.33 0.33 0.48 0.49 0.69 0.66
0.54 0.32 0.51 0.51 0.34 0.34 0.46 0.46 0.72 0.69
of increasing
electrophoresis
and finally compared with (Table II). Azotomonas grown was thus shown to contain riboisoxanthopterin, a-aminoa compound (Band VI) which biopterin, and another compound on acid hydrolysis (2 N hydro30 minutes at 100”) yields a 2- 6 - trihydroxypropylpteridine
RI in propanol-lo/, at pH 4.65
(sodium
ammonia acetate
buffer)
52 20 17 17 -2 -2 -3 -3 -3 -3
(2: 1). for
130 minutes
(Table III). These latter two compounds have not been complet,ely identified. Attempts to produce mutants in Azotomonas. As stated above, Azotomonas tends to grow in clumps even on defined liquid medium. Attempts to prevent this clumping by incorporation of surfaceacting agents (e.g., Tween 80, disodium versenate, sodium dodecyl sulfate, pectinase, hyaluronidase, Alconox, or Vel) in the medium were unsuccessful.
12
GOT0
ET
TABLE FURTHER
AL. III
CHARACTERIZATION
OF BAND
II
Solvents
Substance Band II Hydrolysis product Neopterin KMn04 oxidation product 2-Amino-4-hydroxypteridine6-carboxylic acid
1
2
3
4
5
6
0.02 0.30 0.30 0.23 0.23
0.12 0.29 0.29 0.14 0.14
0.43 0.48 0.48 0.42 0.42
0.79 0.62 0.62 0.54 0.54
0.82 0.62 0.62 0.47 0.47
58 -3 -3 96 96
I
1
7Pn d d
C
&......“”
FIG.
I
I
I
4
6
12
3. Comparison
of growth
I
I
16 20 TIME (hours) rate
and pteridine
To remove the large clumps and obtain a relatively uniform suspension of cells for mutagenic studies the liquid culture was passed through a sterile filter of loosely packed glass wool. Attempts were made to produce a purine-requiring mutant by the use of heat on dried cultures (100”/2 hours) (4); ultraviolet light followed by penicillin treatment (5) ; nitrous acid (6) ; and X-ray irradiation. None of these methods was succesful, presumably because of the associations of cells still present in the filtered cultures. Attempts to incorporate radioactive purines into the pteridines of Azotomonas. Azotomonas cultures in the log phase were used as inocula for flasks of regular medium containing 10 pg per milliliter of one of the following compounds: uric acid-6-Cr4, hypoxanthine-2-P, 4-hydroxypteridine-2-C14, and guanylic acid-U-W. After reaching maximal growth, the cultures were boiled and filtered, and the filtrates were treated separately with excess
I
I
24
28
production
in Azotomonas.
KMn04 in alkali. After complete oxidation, excess KMn04 was destroyed by addition of ethanol, and the suspensions were filtered to remove MnO* . The latter filtrates were adjusted to pH 4 (HCI), and the pteridines in them were absorbed on charcoal and eluted from this with ethanol-lG7o am monium hydroxide (1:l). After removal of the solvent, the residues were submitted to paper chromatography using successively n-propanol1% ammonium hydroxide (2:1), 6% aqueous acid-water ammonia, and set-butanol-formic (8:2:5). The fluorescent materials (2.amino-4hydroxypteridine and 2-amino-4-hydroxypteridine-6-carboxylic acid) were eluted and counted. No significant radioactivity was found in these compounds with any of the radioactive materials used. Comparison of growth rate and pteridine production in Aeotomonas insolita. It was of interest to determine the relationship, if any, between the
ISOLATION
OF
XANTHOPTERIN
growth rate of the organism and the production of 2amino-4-hydroxypteridine and folk acid. The growth rate was determined by measurement of the optical density at 540 rnp of IO-ml samples removed from a growing culture (500 ml) at 4hour intervals. Pteridine ring synthesis was measured on the same samples, after centrifugation of the cells, by comparison of the fluorescence of the supernatant, with that of a standard solution of 2-amino-4-hydroxypteridine. The folic acid content of the culture was also determined by using Streptococcus juecalis R on an aliquot of the boiled centrifuged samples. The results of all three assays are roughly parallel (Fig. 3). However, the folic acid content of the organism is, at most, 306 times less than the pteridine content. Precursors for the enzymic synthesis of jolic acid. A series of simple pteridines and one purine derivative were tested as potential folic acid precursors on cell-free extracts (prepared by sonication of cells from a log-phase culture) of A. insolita. The presence of guanosine, guanylic acid, 2-amino-4hydroxypteridine (with ATP and Mg++)) (2. amino-4.hydroxy-6-pteridinyl)methyl phosphate, phos(2.amino-4.hydroxy-6.pteridinyl)glycerol phate, 2-amino-7,8-dihydro-4.hydroxy-G-propionylpteridine (isosepiapterin), 2.amino-4-hydroxyor 2.amino-4-hy5,6,7,&tetrahydropteridine droxy-5,6,7,8-tetrahydro-6.trihydroxypropylpteridine did not increase the folic acid content, measured as above. 2.Amino-4-hydroxy-6-hydroxymethyl-5,6,7,%tetrahydropteridine stimulated folic acid production slightly, and the corresponding phosphate brought about a tenfold increase in folic acid content of the incubated extract. DISCUSSION
The new naturally occurring compound described above has an ultraviolet absorption spectrum almost identical with that of 2-amino-4-hydroxypteridine-6-carboxylic acid. However, it could be separated chromatographically from the latter compound, and alkaline permanganate oxidation-which, in fact, destroyed the compound-demonstrated that it did not have a substituent linked by carbon to the ring at the 6-position. The presence of the parent pteridine ring system was demonstrated by hydrogenation and reoxidation of the reduction product. This yielded 2-amino-4-hydroxypteridine in addition to regenerated starting material. Production of xanthopterin by acid hydrolysis showed that there was a substituent on the 6-position, and its
13
SULFATE
nature was demonstrated by comparison of the compound isolated from Axotomonas with synthetic xanthopterin sulfate. The compounds were indistinguishable. The structure of the synthetic compound was confirmed by the fact that its mass spectrum (Fig. 3) showed a peak at 257 (M-2). This compound appears to be identical wit’h the “2-amino-4-hydroxypteridine-6sulfonic acid” (m.w. 243) synthesized, in essentially the same way, by Viscontini and Weilenmann (3). The mass spectrum leaves no doubt as to the correct formula for the compound.2 The question then arises as to the origin of this compound, the first naturally occurring pteridine sulfate to be described. The most likely explanation is that it is produced in the culture medium from reduced 2-amino-4-hydroxypteridine in exactly the same way as it can be synthesized chemically, i.e., by reaction of SOS’ with 2 - amino - 4 - hydroxy - 7,8 - dihydropteridine (3), followed by oxidation and aromatization. This implies that the dihydro- or tetrahydro-pteridine is excreted by the cells, which, in turn, is consistent with the fact that 2-amino-4-hydroxypteridine is found in cultures grown on agar plates. Bearing in mind the level of folic acid found in this organism, it appears that Axotommas produces a large excess of the pteridine moiety of the cofactor (or cofactors) with respect to the amount used for coenzyme synthesis. A small amount is converted by oxidation into isoxanthopterin, and the rest is excreted or, under the appropriate conditions, converted into the sulfate. The problem of the origin of reduced 2-amino-4-hydroxypteridine still remains. Current theory (7) supposes that the pteridine ring arises from guanosine or guanylic acid, the first actual pteridine being a reduced 2-aminol-hydroxy-B-trihydroxyalkylpteridine or the corresponding 3’-phosphate ester. Either of these compounds in the reduced state is known to be unstable, and they could readily give rise to a reduced 2 - amino - 4 - hydroxypteridine. However, guanylic acid-U-Cl4 (which was demon* A more extended study pteridines will be published
of the mass elsewhere.
spectra
of
14
GOT0
strated by paper chromatography to contain both guanine and guanosine as impurities) was not incorporated into pteridines in growing Axotomonas cultures (a failure which of course could simply be due to nonpermeability) ; in addition, guanosine and guanylic acid failed to stimulate the production of folic acid in cell-free extracts. Qualitatively, however, guanine plus glucose added to Axotomonas medium increased the production of isoxanthopterin, although other purines (along with glucose) or guanine or guanosine alone, did not. Thus, although no direct evidence could be obtained, these experiments suggest that the same pathway exists for the conversion of a purine to a pteridine in Azotomonas as in E. coli (7) or in Corynebacterium (8). Furthermore, the most active pteridine tested as a precursor for folic acid was (2-amino-4-hydroxy-5,6,7, %tetra-hydro-6-pteridinyl)methyl phosphate which presumably can be readily converted into (2 - amino - 4 - hydroxy - 7,8 - dihydro 6-pteridinyl)methyl pyrophosphate the compound postulated by Weisman and Brown (9) as the immediate precursor of folic acid-
ET
AL.
like compounds. The over-all pathway in Azotomonas thus appears to be the same as in other organisms, the one unique feature being the vast overproduction of the pteridine ring, leading to the excretion of 2-amino4-hydroxypteridine or xanthcpterin sulfate. REFERENCES 1. STAPP, C., Zentr. fur Bakt. II Abt. 102, 1 (1940) [see also: Abs. of Communications, 3rd. Int. Congr. of Microbial., 30F (1939)l. 2. SKERMAN, V. B. D., “A Guide to the Identification of the Genera of Bacteria,” p. 151. Williams di Wilkins, Baltimore, Maryland (1959). M., AND WEILENMANN, H. R., 3. VISCONTINI, Helv. Chem. Acta 42. 1954 (1959). 4. ZAMENHOF, S., Proc. Natl. Acad. Sci. U.S. 46, 101 (1960). 5. GORINI, H., AND KAUFMAN, H., Science 131. 604 (1960). 6. KAUDEWITZ, F., Nature 183, 1829 (1959). 7. cf. BROWN, G. M., AND REYNOLDS, J. J., Ann. Rev. Biochem. 32, 419 (1963). 8. VIEIRA, E., AND SHAW, E., J. Biol. Chem. 236, 2507 (1961). 9. WEISMAN, R.. A., AND BROWN, G. M., J. Biol. Chem. 239, 326 (1964).