Isolation and identification of a new pteridine, neopterinyl-3′-β-d -glucuronic acid from Bacillus subtilis

Comp. Biochem. Physiol., 1970, Vol. 33, pp. 201 to 207. Pergamon Press. Printed in Great Britain

ISOLATION AND IDENTIFICATION OF A NEW PTERIDINE, NEOPTERINYL-Y-/3-D-GLUCURONIC ACID FROM BACILLUS SUBTILIS* K. KOBAYASHI and H. S. FORREST Department of Zoology, University of Texas, Austin, Texas (Received 1 M a y 1969)

A b s t r a c t - - 1 . Bacillus subtilis produces two pteridines in relatively high amounts during growth. 2. One of these is 2-amino-4-hydroxypteridine. 3. The second is a new compound whose structure has been derived from degradative experiments. It is neopterinyl-3"-fl-D-glucuronic acid. 4. Its biosynthesis in B . subtilis from guanosine triphosphate has been demonstrated.

INTRODUCTION DURING g r o w t h o f B a c i l l u s subtilis considerable a m o u n t s of blue fluorescent c o m p o u n d s are p r o d u c e d . T h e two m a j o r ones have b e e n isolated a n d characterized; one is identical with 2 - a m i n o - 4 - h y d r o x y p t e r i d i n e , the s e c o n d is a n e w c o m p o u n d . T h i s p a p e r provides evidence t h a t it is a derivative of n e o p t e r i n [ 2 - a m i n o - 4 - h y d r o x y - 6 - ( l ' 2 ' 3 ' - t r i h y d r o x y p r o p y l ) p t e r i d i n e ] in w h i c h a g l u c u r o n i c acid residue is attached to the terminal h y d r o x y l g r o u p of the p r o p y l side-chain of neopterin. MATERIALS, METHODS AND RESULTS Bacillus subtilis (Strain 168, ind-; obtained from Dr. Orville Wyss, Microbiology De-

partment, University of Texas) was grown in a medium modified from that of Spizizen (1958) as follows (quantities in g/1.): (NH4),SO4, 2"0; K2HPO4, 14"0; KH,PO4, 6"0; MgSO4.7H,0, 0"2; sodium citrate, 1"0; glucose 5"0; L-tryptophane, 0-0005; pH adjusted to 7-2-7"5. Stock cultures were maintained at 4°C on agar slants of the same medium; they were transferred weekly. Cultures were grown on the liquid medium for about 40 hr at 37°C with shaking. Isolation of the new compound

After approximately 40 hr growth, the whole culture (1 1.) was acidified to p H 3 and manganese dioxide (200 mg) was added. After 2 hr at room temperature, the suspension was filtered (diatomaceous earth), the filtrate was treated with charcoal (Darco G60; ca. 1 g) and the whole stirred for 30 min. The charcoal was collected, washed with water and 50% acetone and then with 50% ethanol-1% ammonium hydroxide (1 : 1) to remove absorbed materials. * This work was supported in part by Grant G M 12323 from the National Institutes of Health, Public Health Service, and by a grant from the Robert A. Welch Foundation, Houston, Texas. 201

202

K. KOBAYASHI AND H . S. FORREST

T h e c o m b i n e d eluates f r o m 250 such cultures were evaporated to small bulk in vacuo and the concentrated solution was streaked on filter paper (20 sheets; Whatrnan No. 17). After d e v e l o p m e n t with the solvent [ n - p r o p a n o l - 1 % a m m o n i u m hydroxide ( 2 : 1)], two broad fluorescent bands RI, 0"1-0"2 (band B1) and RI, 0"3-0"4 (band B~), were cut out and the fluorescent materials were eluted from t h e m with 1 ~o a m m o n i u m hydroxide. T h e separate ammoniacal eluates were then evaporated to small bulk in vacuo. Further purification of B1. T h e concentrated solution from B1, brought to p H 2 with conc. hydrochloric acid, was absorbed on a D o w e x 50W x 8 column (H + f o r m ; 50-100 m e s h ; 4"5 x 11 cm), and the column was washed w i t h water (500 ml) until the eluate was neutral. A large a m o u n t of colored, non-fluorescent material was eluted at this stage. T h e blue fluorescent materials were then eluted with 1 ~o a m m o n i u m hydroxide (1 "5 I.). T h e appropriate fractions were evaporated to dryness and the residue was dissolved in 1 ~o a m m o n i u m hydroxide (12 ml) and the solution streaked on filter paper ( W h a t m a n No. 17). After d e v e l o p m e n t w i t h n-butanol-acetic acid-water (3 : 1 : 1), the broad band, Rf 0"05-0.10, was cut out and the fluorescent material again eluted from the paper with 1~o a m m o n i u m hydroxide. T h i s chromatographic step was repeated twice more, and the final product (53 mg) in a small a m o u n t of water was placed on a D o w e x 1 x 8 column (C1- f o r m ; 100200 m e s h ; 4"5 x 11 cm). After washing with water, the blue fluorescent material was eluted w i t h hydrochloric acid (0-02 N). T h e appropriate fractions of the eluate were combined, neutralized with concentrated a m m o n i u m hydroxide and the solution evaporated to small bulk. Finally, this was placed on a D o w e x 50W x 8 column (H + f o r m ; 100200 m e s h ; 4"5 x 5 cm), and the fluorescent material eluted in the same way as for the first ion-exchange column. T h e residue (30 mg) obtained by evaporation of the ammoniacal eluate was recrystallized from 70~/o ethanol to give the p r o d u c t (15 rag) which did not m e l t below 250°C. It was h o m o g e n o u s paper chromatographically. (Tables 1 and 2 give Rf values and electrophoretic mobilities.) T h e c o m p o u n d is insoluble in most organic solvents, but freely TABLE I - - Rl VALUES OF PTERIDINES FROM B.

subtilis AND DEGRADATION PRODUCTS

Substance

2-Amino-4-hydroxypteridine6-carboxylic acid Permanganate oxidation product B1 A c i d - h y d r o l y z e d B1 Enzyme-hydrolyzed BI Neopterin U r o n i c acid f r o m Bx (and lactone) D-Glucuronic acid (and lactone) 2-Amino-4-hydroxypteridine B~

Solvents 1

2

3

4

5

6

0" 12

0" 14

0"43

0'04

0"45

--

0"12 0"06 0"37 0"37 0"37

0"14 0"01 0"12 0-12 0"12

0-43 0"25 0"41 -0"41

0"04 0"03 0"23 0.23 0"23

0'45 0"69 0"62 -0"62

------

0"15

.

.

0"15 0"43 0"43

. 0"35 0"35

.

. . 0"53 0"53

.

0"29 (0"48)

. 0.40 0"40

0"47 0"47

0"29 (0"48) ---

* Solvents: 1, n - p r o p a n o l - 1 % a m m o n i u m hydroxide ( 2 : 1); 2, n-butanol-acetic a c i d water (4 : 1 : 1); 3, s e c - b u t a n o l - f o r m i c acid-water (8 : 2 : 5); 4, iso-propanol-5% boric acid (4 : 1); 5, 4 % sodium citrate; 6, ethyl acetate-pyridine-acetic acid-water (5 : 5 : 1 : 3).

203

A NEW PTERIDINE FROM BACILLUS SUBTILIS

soluble in water. Ultra-violet spectra: Anmx (~xe%m), 255 (600), 360 (180) in 0"1 N sodium hydroxide; 250 (320), 320 (200) in 0'1 N hydrochloric acid. Sample dried at 100°C for 4 hr in vacuo over P=O~. Analysis: calculated for CxsH x.N6Ox0 - - N , 16.3; f o u n d - - N , 15-5. T A B L E 2 - - E L R C T R O P H O R E T I C MOBILITIES OF PTERIDINE$ FROM B .

subtilis

System * Substance

2-Amino-4-hydroxypteridine-6-carboxylic acid Permanganate oxidation product B1 Acid-hydrolyzed B 1 Enzyme-hydrolyzed B1 Neopterin B~ 2-Amino-4-hydroxypteridine

1

2

3

40t 40 28 - 5 - 5 - 5 -5 - 5

20 20 10 - 5 -- 5 ---

43 43 33 - 8 - 8 ---

* Buffer systems: 1, sodium acetate--acetic acid (1 : 1 ; each 0"05 M), pH 4"6; 2, ammon i u m acetate (0'05 M), pH 6"7; 3, sodium phosphate (0"05 M), pH 8"9. 1" Distance (in mm) to anode after electrophoresis for 60 min at 10 V/cm. Further purification of band B~. T h e concentrated solution from band B~ (R! in prop a n o l - l % ammonium hydroxide, 0"3-0"4) yielded about 1"2 g of solid material on complete evaporation. This was dissolved in hydrochloric acid (1 N ; 30 ml) and the solution placed on a Dowex AG50W x 8 column (H + form; 100-200 mesh; 4.5 x 15 cm). T h e column was washed with water to neutrality and then with 1% ammonium hydroxide causing elution of the blue fluorescent material. The appropriate fractions were collected, evaporated to dryness, and the residue, in 1% ammonium hydroxide, was applied to a Dowex 1 x 8 column (C1- form; 100-200 mesh; 4-5 x 15 cm). Again the column was washed with water and then with hydrochloric acid (0"02 N), and the acidic eluate containing fluorescent materials was evaporated in vacuo to about 10 ml. T h e yellow residue obtained by the addition of acetone was collected, washed with water and acetone and dried. T h e yield was 5 rag. This compound, when tested after treatment with sodium metaperiodate, gave no formaldehyde or acetaldehyde, and it was not affected by alkaline permanganate. Its identity with authentic 2-amino-4-hydroxypteridine was established by paper chromatographic and electrophoretic comparisons (Tables 1 and 2). Characterization of B 1 A stock solution of B1 containing 1"30 mg/ml was used for the following experiments. Permanganate oxidation. A n aliquot (0"1 ml) of the stock solution in sodium hydroxide (0"1 N ; 1"9 ml) was oxidized with potassium permanganate (10 rag) at 100°C for 40 min. Excess permanganate was destroyed and the precipitated manganese dioxide removed by centrifugation and washed with more sodium hydroxide (0"1 N ; 2 ml). The filtrate and washings were combined and made up to 10 ml. The amount of 2-amino-4-hydroxypteridine-6-carboxylic acid identified by chromatography (Tables 1 and 2) to be the sole pteridine in this solution was determined using the known extinction coefficient for this compound. The yield was 65/zg. On this basis the molecular weight of Bx was calculated to be 433, and of the oxidizable fragment to be ca. 270.

204

K. KOBAYASHI AND H. S. FORREST

Formaldehyde determination after periodate oxidation (McFadyen et al., 1945). An aliquot (0'1 ml), after treatment with sodium metaperiodate (0"3 M ; 0"2 ml) in sodium bicarbonate (1 M ; 0"2 ml) for 24 hr at room temperature followed by addition of sulfuric acid (1 N; 1"5 ml) and sodium arsenite (1 N ; 0"5 ml), gave no color reaction with chromotropic acid. Acetaldehyde determination after periodate oxidation (Shinn & Nicolet, 1941). An aliquot (0"1 ml), similarly treated, yielded no acetaldehyde on testing with p-hydroxydiphenyl in sulfuric acid. Alkaline hydrolysis. An aliquot (0"1 ml), made 0"1 N with respect to sodium hydroxide, was heated at 100°C for 1 hr. No decomposition was observed spectrally or paper chromatographically. After 2 hr, only a trace of 2-amino-4-hydroxypteridine-6-carboxylic acid could be detected in the starting material. Acid hydrolysis. A solution of B1 (4 mg) in 2 N hydrochloric acid was heated in a steam bath for 3 hr. The course of hydrolysis was followed by withdrawing samples at intervals and chromatographing these in the standard propanol-1% ammonium hydroxide solvent. Over 90 per cent of the original compound had disappeared after 3 hr. The hydrolysate was concentrated in a desiccator over sodium hydroxide pellets and the residue, dissolved in water, was compared chromatographically and electrophoretically with neopterin (Tables 1 and 2). Further purification of the hydrolysis product was achieved in the following way. An ammoniacal solution was streaked on Whatman No. 3 M M filter paper (4 sheets) ; the chromatogram was developed with propanol-1% ammonium hydroxide (2 : 1); the band with R! 0"3~)'4 was cut out, the blue fluorescent material was washed from it with 1% ammonium hydroxide, and the ammoniacal solution (30 ml) was placed on a Dowex 1 x 8 column (C1- form; 100-200mesh; 1"5 x6-0 cm). The column was washed with water, and the blue fluorescent material eluted with hydrochloric acid (0"004 N). The acidic eluate was evaporated to small bulk and the concentrated material again placed on a Dowex 1 × 8 column (H + form; 200-400 mesh; 1"5 x 6"0 cm). The blue fluorescent eluate, obtained with 1 °/o ammonium hydroxide, was evaporated to dryness, the residue dissolved in water and some insoluble material was removed by centrifugation. The supernatant was again evaporated and the residue (0"72 rag) in water (10 ml) used for determination of the u.v. spectrum [•max (El~n), 255 m/z (800) and 365 m/~ (320) in 0"1 N sodium hydroxide; 248 (450) and 321 m/z (300) in 0"1 N hydrochloric acid]. After periodate oxidation, in the same way as before, formaldehyde was estimated using chromotropic acid. The stock solution (1 ml), containing 72/~g of the hydrolysis product, gave 5"7 #g formaldehyde (theoretical for neopterin, 5"1 k*g)Acetaldehyde could not be detected after periodate oxidation of the hydrolysis product. Thus, by these criteria, the hydrolysis product was identical with neopterin. Quantitative periodate oxidation of the hydrolysis product. The spectrophotometric method of Dixon & Lipkin (1954) was used. Using a tool. wt. of 253, the consumption of periodate after 15 min, was 1'8 moles. The only fluorescent product which could be isolated from the reaction mixture was 2-amino-4-hydroxypteridine-6-carboxylic acid which presumably arises from over-oxidation during the working up. Small-scale experiments showed that the -6-aldehyde was being produced, but even in these it is always contaminated with the corresponding acid. Quantitative periodate oxidation of the original compound (B1). Using the same method, compound B1 consumed 3"1 moles/mole of periodate in a 24-hr period. The pteridinecontaining oxidation product was 2-amino-4-hydroxypteridine-6-carboxylicacid. Characterization of the acidic residue from acid hydrolysis orB1. A solution of B1 (0.4 rag) in hydrochloric acid (2 N ; 0"4 ml) was heated on a steam-bath for 2 hr after which solvent was removed in a desiccator over sodium hydroxide. The residue, in water, was spotted on Whatman No. 1 filter paper, and chromatograms were developed in n-butanol-acetic acidwater (4 : 1 : 1) and ethyl acetate-pyridine-acetic acid-water (5 : 5 : 1 : 3). After drying the papers, they were sprayed with aniline phthalate or lead tetra-acetate. The glycosidic

A NEW PTERIDINE FROM BACILLUS S U B T I L I S

205

residue was thus shown to have the same Ry value as the lactone of authentic glucuronic acid, obtained by a similar acid treatment (Table 1). A quantitative estimation by the method of Ashwell (1957) of the glucuronic acid was then carried out b y hydrolyzing 130/~g of B1 (in 0"1 ml stock solution) with" cone. sulfuric acid (1"3 ml) for 30 rain on the steam-bath. After cooling, an alcoholic solution of carbazole (0"1%; 0"2 ml) was added causing immediate development of pink color. After 1 hr the optical density at 525 m/~ (corrected for a blank) was read and compared with a standard curve made using authentic D-glucuronic acid. T h e amount of glucuronic acid released on hydrolysis was 44/~g (theoretical, using a molecular weight of 433, 47/zg). Enzymatic hydrolysis of Bt. A solution of Bx (24.7/~g in 1"5 ml acetate buffer, p H 4"6) was treated with fl-glucuronidase (1"6 mg; Worthington). T h e extent of hydrolysis was followed by chromatographing small aliquots in n - p r o p a n o l - l % ammonium hydroxide and estimating the neopterin produced (Tables 1 and 2) fluorometrically. After 30 min at 37°C 1.58/~g B t yielded 1"00/~g neopterin and 0"04 unchanged starting material (theoretical yield of neopterin from 1"54/Lg B1, 0-90/~g). Comparison of the growth rate and pteridine production. T h e amounts of the two blue fluorescent materials described above were estimated during growth of a culture of B. subtilis. Growth was determined by removing 50-ml aliquots from a growing culture (1 1.) at about 5-hr intervals, measuring the optical density of the sample at 600 m/z and converting this to dry weight. Each aliquot was then centrifuged, the cells were collected and resuspended in 4% acetic acid (5 ml). T h e suspension was treated with manganese dioxide (10 mg) and the whole heated on a steam-bath for 3 min. After centrifugation, the supernatant was streaked on Whatman No. 3 M M filter paper which was then irrigated with the standard propano1-1% ammonium hydroxide solvent. The two major fluorescent bands were eluted from the paper with ammonium hydroxide, the eluates were evaporated to dryness and the residues were again taken up in 1% ammonium hydroxide (1-5 ml). After centrifugation, fluorescence was measured on these using neopterin as a standard for Bx and 2-amino-4-hydroxypteridine as a standard for B2. T h e results are shown in Fig. 1. I 90-

~

t

1

0

- 2O le

E

E to

~12 .~

L) 5o

N

4

d// I I

A

i

I0

20 TIME

l

i

30

40

(hour)

FIO. 1. Comparison of growth rate and accumulation of pteridines in B, subtilis.

206

K. KOBAYASHIAND H. S. FORREST

Incorporation of guanosine triphosphate-U-laC into BI ush~g crude extracts. A 1-1. culture was allowed to grow for 24 hr at 37°C. The cells (wet wt., 1"5 g) were harvested, washed twice with this buffer (0.05 M; pH 8), resuspended in the same buffer (5 ml) and the suspension was sonicated for 3 min using a Raytheon 10Kc sonic oscillator. After centrifugation, a portion (2"5 ml) of the clear yellow supernatant was treated with 1/zc of guanosine triphosphate-U-14C, and the whole incubated for 3 hr at 37°C. The reaction mixture was then acidified to pH 3 with acetic acid, treated with manganese dioxide (20 mg) and the whole heated to 100°C for 2 min. The resulting heavy precipitate was removed by centrifugation and the supernatant was streaked on filter paper (Whatman, No. 3 MM). After development of the chromatogram with the standard propanol-ammonium hydroxide solvent, the band containing B1 was eluted and the Bt in the eluate was either oxidized with alkaline permanganate or hydrolyzed with acid as described above. The products from these reactions were purified by successive chromatography in propanol-ammonium hydroxide, and n-butanol-acetic acid-water (standard solvents, see Table 1), followed by paper electrophoresis at pH 4"6 (acetate buffer) for the permanganate oxidation product (2-amino-4-hydroxypteridine-6-carboxylic acid) or by chromatography in 3 % ammonium chloride for the acid hydrolysis product (neopterin). Radioactivity of these purified samples was determined using an Ansitron Liquid Scintillation Counter, and amounts were assayed by fluorescence (after oxidation to 2amino-4-hydroxypteridine-6-carboxylic acid, in the case of neopterin) with authentic material as a standard. Compounds were chromatographed to constant specific activity. The results of a typical experiment were: neopterin, 5"8× 103; 2-amino-4-hydroxy-6carboxypteridine, 4"6 x 103 counts/rain per /zM. (Theoretical for loss of two of the nine carbon atoms arising from the labelled guanosine triphosphate, 4"4 x 103 counts/min per/xM.) DISCUSSION C o m p o u n d B 1, isolated f r o m B. subtilis, has an absorption spectrum typical of a 6-substituted-2-amino-4-hydroxypteridine. Its oxidation b y alkaline p e r m a n ganate to 2-amino-4-hydroxypteridine-6-carboxylic acid confirms the presence of this pteridine residue. Acid hydrolysis of B 1 yields neopterin--identical with a synthetic s p e c i m e n - - a n d a second moiety with a molecular weight of about 180. Since B 1 is acidic in nature, the presence of a glucuronic acid residue was suspected, and this was confirmed by p a p e r chromatographic identification of the lactone of this acid f r o m acid hydrolysates of B 1. A quantitative uronic acid determination gave a 1 : 1 ratio of pteridine residue to uronic acid residue. T h e nature of this r e s i d u e - - a n d some evidence concerning its binding to the pteridine m o i e t y - - was further confirmed b y enzymatic hydrolysis of B 1 using the enzyme,/3-glucuronidase, the pteridine product of this hydrolysis, being neopterin. Therefore, the glucuronic acid is linked through the 1-position and this link has the fl-orientation. Finally, the position of attachment to the side-chain of the pteridine residue was investigated by the method of periodate oxidation. Since B1 consumed 3 moles of periodate (reasonably, 1 mole/neopterin residue and 2 moles/glucuronic acid residue) the linkage m u s t be t h r o u g h the 3'-position of neopterin. I f the glucuronic acid residue was attached at the 2'-position, periodate consumed would be 2 moles/mole, if at the l'-position periodate consumed would be 3 moles/mole but with production of formaldehyde. N o formaldehyde was detected after periodate oxidation of B 1.

A NEW PTERIDINE FROM B A C I L L U S

SUBT1LIS

207

Thus from degradative evidence the structure of compound B 1 is defined as neopterinyl-3'-fl-D-glucuronic acid. Attempts to synthezise this compound by direct condensation of neopterin and glucuronic acid, or by condensation of neopterin and glucose followed by selective oxidation have not been successful. The second pteridine occurring in major amounts is 2-amino-4-hydroxypteridine. In a preliminary experiment, it has been shown that crude cell-free extracts of B. subtilis are able to convert guanosine triphosphate into both of the compounds. Furthermore, radioactive neopterin, isolated from B 1 derived from such experiments, is degraded to 2-amino-4-hydroxypteridine-6-carboxylic acid with loss of radioactivity expected for the loss of two carbon atoms, out of nine contributed by the randomly labelled guanosine triphosphate. This strongly suggests that in this organism all five carbons of the ribose residue are incorporated into the neopterin residue of compound B 1, and that, therefore, the biosynthesis of the pteridine ring follows a pathway similar to that proposed for Escherichia coli as discussed by Kaufman (1967). The biological function of either of these compounds remains obscure. In one experiment, it was demonstrated that they are both synthesized at a rate parallel to the growth rate, although there is a drop in amount of B 2 near the stationary part of the growth curve. Thus the appearance of these compounds may simply reflect an overproduction of the pteridine ring system, in order to ensure a plentiful supply of folic acid coenzymes, as has been suggested for another organism (Goto et al., 1965), or B 1 at least may have a unique but unknown function. Since this work was completed, a report by Suzuki & Goto (1968) has appeared describing the isolation of what appears to be the same compound from A z o t o bacter agilis.

REFERENCES ASHWELr.G. (1957) The colorometric analysis of sugar. In Methods in Enzymology, Vol. III (Edited by COLOWICNS. P. & KAFLANN. O.), pp. 73-104. Academic Press, New York. DIxoN J. S. & LIPKIN D. (1954) Spectrophotometric determination of vicinal glycols. Analyt. Chem. 26, 1092-1093. GOTO M., FORRESTH. S., DICKERMANL. H. & URUSHIBARAT. (1965) Isolation of a new naturally occurring pteridine from bacteria, and its relation to folic acid biosynthesis. Arehs Biochem. Biophys. 111, 8-14. KAUFM~'~ S. (1967) Pteridine cofactors. Ann. Rev. Bioehem. 36, 171-184. McF~owN D. A., WATKINSH. D. & ANDERSONP. R. (1945) Estimation of formaldehyde in biological mixtures. ]. biol. Chem. 158, 107-133. SmNN L. A. & NICOLETB. H. (1941) The determination of threonine by the use of periodate. .7. biol. Chem. 138, 91-96. SFIZIZ~-NJ. (1958) Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonucleate. Proc. natn. Acad. SCI. U.S.A. 44, 1072-1078. SuzuKI A. & GOTO M. (1968) Neopterin-3"-fl-glucuronide: isolation from Azotobacter agilis. J. Biochem. 63, 798-801. Key Word Index--Bacillus subtilis, pteridines in; 2-amino-4-hydroxypteridine; new pteridine; neopterinyl-3"-fl-D-glucuronic acid; guanosine triphosphate, biosynthesis from.