Tetrahydrobiopterin is synthesized by separate pathways from dihydroneopterin triphosphate and from sepiapterin in adrenal medulla preparations

ARCHIVES

Vol.

OF BIOCHEMISTRY

AND

227, No. 1, November,

BIOPHYSICS

pp. 272-278,

1983

Tetrahydrobiopterin Is Synthesized by Separate Pathways from Dihydroneopterin Triphosphate and from Sepiapterin in Adrenal Medulla Preparations GARY Department

of Medicinal

K. SMITH’

CHARLES

AND

A. NICHOL

Biochemistry, The Wellcome Research Laboratm~es, Research Triangle Park, North Carolina 27709 Received

May

3030

Cornwallis

Road,

31, 1983

Using Escherichia coli guanosine triphosphate cyclohydrolase, dihydroneopterin triphosphate was synthesized from guanosine triphosphate and was compared with sepiapterin as a substrate for tetrahydrobiopterin formation in bovine adrenal medulla extracts. The dihydrofolate reductase inhibitor, methotrexate, blocks the formation of tetrahydrobiopterin from sepiapterin but not from dihydroneopterin triphosphate. Reduced nicotinamide adenine dinucleotide phosphate and a divalent metal ion are required in partially purified preparations (gel filtration of 4060% ammonium sulfate fraction on Ultrogel ACA-34) for the biosynthesis of tetrahydrobiopterin from dihydroneopterin triphosphate. Sepiapterin was converted only to dihydrobiopterin in the same fractions since dihydrofolate reductase was removed. The evidence indicates that both dihydroneopterin triphosphate and sepiapterin are good precursors of tetrahydrobiopterin but they are not on the same pathway, contrary to previous proposals.

Previous reports have indicated that sepiapterin is an intermediate in the de nOuo pathway for the biosynthesis of HAbiopterir? (l-7), and the last step of the pathway has been proposed to be the reduction of H,biopterin to HIbiopterin by DHFR (8-10). Recently, however, it was found that MTX, a potent DHFR inhibitor, has no effect on the endogenous biosynthesis of H4biopterin in a variety of mammalian cells in culture and in rat tissues in vivo, or on the in vitro conversion of GTP to H,biopterin by extracts of bovine adrenal medulla. In contrast, the biosynthesis of the cofactor from exogenous se-

$3.00

Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.

MATERIALS

AND

METHODS

GTP was obtained from P. L. Biochemicals. Biopterin, sepiapterin, and Hzbiopterin were purchased from Dr. B. Schirks, Wettswill, Switzerland. Pterin and dithiothreitol were obtained from Sigma Chemical Company. Bovine adrenals were purchased from PelFreeze and stored at -70°C until used. All other chemicals were of reagent grade and were used without prior purification.

i To whom correspondence should be addressed. ’ Abbreviations used: H,biopterin, tetrahydrobiopterin; Hzbiopterin, 7,8-dihydrobiopterin; Hzneopterin-PPP, 7,8-dihydroneopterin triphosphate; MTX, methotrexate; DTT, dithiothreitol; DHFR, dihydrofolate reductase; Hepes, 4-(2-hydroxyethyl)-l-pipevazineethanesulfonic acid. 0003-9861/X3

piapterin or Hzbiopterin is markedly inhibited by MTX (11-13). These results suggest that in contrast to previous proposals, the de nmo biosynthetic pathway for H4biopterin does not use DHFR and that sepiapterin is not on this pathway. Instead, sepiapterin seems to be converted to H*biopterin by an alternate route utilizing DHFR. The present study was undertaken to compare Haneopterin-PPP and sepiapterin as precursors of H,biopterin in partially purified preparations of bovine adrenal medulla.

27 ‘2

TETRAHYDROBIOPTERIN Preparation and treatment of adrenal medulla he mogenates. Medullae were dissected from the cortices of thawed bovine adrenals. The medullary tissue was minced and homogenized with polytron homogenizer in 1 vol of cold 50 mM Tris-HCI buffer, pH 7.4, containing 1 rnM MgCIz and 20% glycerol. Cellular debris was removed by centrifugation at 35,000g for 30 min. Prior to assaying, the crude supernatant was desalted by passing 1 ml through a Sephadex G-25 (medium) column (10 ml bed volume) equilibrated with homogenization buffer (or the same buffer lacking glycerol). Ammonium sulfate fractionation was performed by adding solid ammonium sulfate to the supernatant of the previous step; the suspension was then stirred for 20 min at 4°C after total dissolution of the salt. Pellets were removed by centrifugation at 35,000g for 20 min and redissolved in 50 mM Tris-HCl, pH 7.4, containing 1 mM MgClz. All ammonium sulfate fractions were desalted by Sephadex G-25 chromatography before assay. Protein concentration was determined using the Bio-Rad protein assay kit on desalted protein fractions. Synthesis of H2neopterin-PPP. H*neopterin-PPP was synthesized using Esche-richia coli GTP cyclohydrolase and GTP. GTP was purified using a 1.6 X 25-cm DEAE-Sephacel column at 4°C with a linear gradient (250 ml of each) of 0.1 to 0.4 M NH,HCO$, pH 7.6, to remove any contaminating GDP and GMP which cannot be readily resolved from the product H,neopterin-PPP. The GTP was lyophilized, redissolved in 5 ml H,O, and stored at -20°C. The reaction mixture for the synthesis of H,neopterin-PPP consisted of 0.8 pmol of purified GTP, 0.17 pmol/h units of purified E. coli GTP cyclohydrolase (14), and 5 pmol of EDTA combined in a final volume of 2.25 ml of 0.05 M Tris-HCl, pH 7.8, with 10% glycerol. The reaction was carried out under N2 in a stoppered serum vial. After 24 h at 37°C in the dark, the reaction mixture was loaded onto a 1.6 X 25-cm DEAE-Sephacel column and eluted in the dark as described above for GTP. The product, identified by its uv spectrum (X,,, = 276, 330 nm), eluted just ahead of and well resolved from unreacted GTP. The H,neopterinPPP solutions were pooled, deoxygenated, Nz-saturated, lyophilized, redissolved in 5 ml HzO, again deoxygenated, Nz-saturated, and stored at -70°C. The yield from GTP was about 60%. The identity of the product as H,neopterin-PPP was verified by its uv spectrum (X,, 276 and 330 nm, pH 7.5, ratio l&l), elution position on DEAE-Sephacel compared to GTP, conversion to neopterin upon oxidation and dephosphorylation (identified by its elution position on HPLC, and uv spectrum), and phosphate assay (3.2 + 0.3 phosphates/reduced pterin moiety) (3, 15, 16). Hzneopterin-PPP concentration was determined from its uv spectrum, e330 = 6.4 mK’ (17). Biosynthesis of H,biopterin from H,neqptwin-PPP.

273

BIOSYNTHESIS

The assay typically consisted of 200 ~1 of one of the enzyme preparations (which contained 50 mM TrisHCl, pH 7.4,l mM MgClz), 50 ~1 of 40 pM HzneopterinPPP, 3 ~1 of 0.5 M DTT, 3 ~1 of 20 mM NADPH, and 25 ~1 of 50 mM sodium pyrophosphate, pH 7.5, to inhibit phosphatases. Total assay volume was 281 gl. The assays were run for 1 hr at 37°C in the dark. For assay of synthesized pterins, a 125-~1 aliquot of the reaction mixture was oxidized in acid as described below, and a second 125~~1 aliquot was oxidized in base as described below. (These oxidation procedures both stop the reaction and convert the products to the fully oxidized pterins which are then determined by HPLC as described below.) Biosynthesis of HJriopterin from GTP. This assay was performed as described for biosynthesis of H,biopterin from Hzneopterin-PPP; however, 30 ~1 of 20 mM GTP replaced the Hzneopterin-PPP, and sodium pyrophosphate was omitted since it inhibits GTP cyclohydrolase. Biosynthesis of H,biopterin from sepiapterin and H&iopterin Assays were performed as described for H,biopterin synthesis from Hzneopterin-PPP; however, 50 ~1 of 0.25 mM sepiapterin or H,biopterin replaced H*neopterin-PPP. GTP cyclohydrolase was assayed according to the method of Viveros et al (18). Dihydropteridine reductase was assayed by following the enzyme-catalyzed oxidation of NADH in 50 mM Tris-HCl, pH 7.4 (19). Assay of ptetim. Neopterin, biopterin, and pterin concentrations were determined by HPLC after acid or base oxidation of the assay products (16, 20-22). RESULTS

The desalted supernatant fraction of a bovine adrenal medulla homogenate contains the complete enzyme system for the conversion of GTP to H,biopterin. This synthetic activity and the proportion of H,biopterin to total biopterin are unaffected by the presence of MTX (Table I). The conversion of Hzneopterin-PPP to H,biopterin by the cell-free extract is also shown in Table I. As in the case of the synthesis from GTP, MTX has no effect on either the total biopterin synthesized or that found in the tetrahydro form. On the other hand in the same preparation, MTX very effectively inhibits H,biopterin synthesis from either H,biopterin or sepiapterin (Table I). Upon fractionation with ammonium sulfate, the complete enzyme system converting H2neopterin-PPP to H.,biopterin

274

SMITH

AND TABLE

NICHOL I

Pmol/h/mg

Substrate GTP GTP Hzneopterin-PPP Hzneopterin-PPP Sepiapterin Sepiapterin Hzbiopterin Hzbiopterin

protein

MTX (mM)

Total biopterinb

H4 biopterin’

Percentage biopterin

0 0.1 0 0.1 0 0.1 0 0.1

43 47 84 82 2150 2290 d d

41 42 75 72 395 3 809 14

95 88 89 88 18 0.1 18 0.3

Hq

’ Assay was carried out as described in the text with 2.9 mg of desalted crude enzyme. In the absence of substrate, no product was observed. *Since all forms of biopterin (fully oxidized, dihydro, and tetrahydro) are converted to the fully oxidized biopterin during acid oxidation, this number is calculated from biopterin found after acid oxidation. “Since H,biopterin is destroyed (converted to pterin) on base oxidation, this number is calculated by subtracting biopterin remaining on base oxidation from total biopterin. Pterin is produced quantitatively from the decomposition of Hlbiopterin in base oxidation. Thus, in the case of the assays with sepiapterin and Hzbiopterin, this number was calculated from pterin produced in base. This method affords higher sensitivity for the small percentages of H,biopterin produced by these substrates. dThe amount of biopterin produced in the acid oxidation of this sample is equal to the amount in the starting materials (0.05 mM, or 4500 pmol/mg protein) since both Habiopterin and H,biopterin are converted to biopterin on acid oxidation.

was found in the 40-60% ammonium sulfate saturation pellet. Specific activity was 270 pmol/h/mg protein, and H4biopterin was 87% of total biopterin in the presence or absence of MTX. Little or no activity was found in the O-40% pellet or the 60% supernatant. Recovery at this stage of purity was 48% of that in the crude homogenate. The fraction reduced Hzbiopterin to H,,biopterin at the rate of 1300 pmol/h/ mg protein, and the reduction was inhibited by MTX. Sepiapterin reductase activity was found to be 8000 pmol/h/mg protein in this fraction. A redissolved 40-60% ammonium sulfate pellet prepared from 30 g of adrenal medulla was dialyzed for 18 h against 50 mM Tris-HCl, pH 7.4, containing 1 mM MgClz, and then chromatographed on a 2.6 X lOOcm Ultro-Gel ACA-34 column equilibrated at 4°C with the same buffer. The elution profile shown in Fig. 1A indicates that the Hzneopterin-PPP - H,biopterin activity eluted in fractions 80 through 90 and peaked at fractions 82 to 85. Specific ac-

tivity at the peak was 2600 pmol Hqbiopterin/h/mg protein, indicating approximately lo-fold purification, and recovery at this step was 75%. The percentage of the total biopterin present as Hqbiopterin was approximately 85% across the peak. MTX was without effect on either total biopterin synthesized or the percentage present as H4biopterin. Thus, the entire pathway from Hzneopterin-PPP to H,biopterin was found in these fractions, and its MTX insensitivity was maintained. When a fraction from the leading edge of the peak was assayed in combination with a fraction from the trailing edge of the peak, enhancement of the activity was observed. Thus, fraction 82 in combination with 90 gave an activity of 3700 pmol/ml/ h; however, the expected activity, the average of the two fractions, was 2600. This may indicate partial resolutions of biosynthetic enzymes by the gel filtration. Figure 1B shows the elution profile for sepiapterin reductase and dihydropteridine reductase. Peak activities for both enzymes

TETRAHYDROBIOPTERIN

BIOSYNTHESIS

80 FRACTION

100

120

(4ml each)

FIG. 1. Elution profile from Ultrogel ACA-34. A lo-ml solution containing 64,300 pmol/h of H*neopterin-PPP + H,biopterin activity in 50 mM Tris-HCl, 1 ItIM MgC&, pH 7.4, was loaded onto a 2.6 X lOO-cm column equilibrated with the same buffer. Fractions of 4 ml each were collected. (A) Profile for synthesis of biopterin from Hcneopterin-PPP in the absence (0) and presence (0) of MTX; percentage of total biopterin found as H,biopterin in the presence or absence of MTX (Cl). (B) Profile for sepiapterin reductase (0) and dihydropteridine reductase (A). For linearity, sepiapterin reductase was assayed with 50 ~1 of enzyme and 150 ~1 of buffer; however, percentage biopterin found as H,biopterin (Cl) was zero even when the larger amounts of enzyme used in (A) (200 ~1) were used for the sepiapterin reductase assay. (C) Protein elution profile as determined by Am (-) and the Bio-Rad assay (0).

were observed in fractions 87 to 90. [Thus, based on these markers (sepiapterin reductase and dihydropteridine reductase), the enzymes which convert HzneopterinPPP to H,biopterin peaked at -70,000 M, (19, 23).] Interestingly, the Hzbiopterin produced from sepiapterin in the sepiapterin reductase assay was not then reduced to H,biopterin by these fractions (percentage H,biopterin = 0) (Fig. 1B). Similarly, when Hzbiopterin reduction to H,biopterin was assayed directly in these fractions, no H,biopterin was detected.

(The sensitivity of this assay was such that, based on the production of pterin from H,biopterin during base oxidation, 200 pmol of H,biopterin/h/ml of enzyme should have been observable in this particular case.) This result indicates that the enzyme reducing Hzbiopterin to H,biopterin, presumably DHFR (13), was either destroyed or removed by this gelfiltration treatment. In some preparations, some Hzbiopterin - H,biopterin activity (DHFR) was recovered. In these cases the activity was

276

SMITH

AND

found to peak at fraction 100 (peak activity = 2000 pmol Hlbiopterin/h/ml). No activity was found in the Hzneopterin-PPP H,biopterin activity peak. Thus, ACA-34 gel filtration separates the DHFR activity from the H,neopterin-PPP - H4biopterin activity. Figure 1C shows protein elution from the column. At this stage, the biosynthetic system was stable and could be stored at -20°C for 1 month with little loss in activity. GTP cyclohydrolase was removed by this procedure since it precipitated in the 3040% ammonium sulfate fraction and eluted near the AcA-34 void volume. Peak fractions were pooled and assayed to determine the effect of NADPH, Hepes, and EDTA on the synthesis of H*biopterin from Hsneopterin-PPP. In the absence of NADPH or the presence of EDTA, no biopterin is produced (Table II). When NADPH is omitted, although H,biopterin is not produced, pterin (which is normally formed from H4biopterin decomposition in base) is formed upon acid or base oxidation of the reaction mixture. Further, this pterin production is approximately twice as great after base oxidation as after acid oxidation. No pterin is produced in acid in the presence of NADPH. The presence of 0.1 M Hepes in the presence or absence of MTX has no effect on either total biopterin produced or on the percent reduction to H4biopterin. DISCUSSION

The present results indicate that the pathway for H4biopterin synthesis from Hzneopterin-PPP, like that from GTP, does not require DHFR and is different from that used by sepiapterin and Hsbiopterin which do require an enzyme which behaves like DHFR. This conclusion is supported by the following: (i) MTX inhibits the biosynthesis of H4biopterin in the adrenal medulla preparations by 98-99% when either sepiapterin or Hzbiopterin is used as the substrate; however, MTX has no effect on the synthesis when either GTP or Hzneopterin-PPP is the substrate. (ii) In the partially purified AcA-34 gel-filtration fractions which contain no DHFR, no detectable amounts of H,biopterin are

NICHOL

formed from either sepiapterin or Hzbiopterin, while H4biopterin is formed at a high rate from Hzneopterin-PPP. (iii) In a cell-culture line which lacks DHFR (DIJKX subline of CHO cells), H4biopterin biosynthesis from GTP proceeds well while no H,biopterin is made from added sepiapterin (13). Thus, it is very unlikely that either sepiapterin or H,biopterin are intermediates in the de nova biosynthesis of Hlbiopterin from GTP. From the data presented, we propose that the de novo biosynthesis of Hqbiopterin proceeds as shown in pathway 1 below, and the synthesis from sepiapterin proceeds as in pathway 2. Pathway

1: GTP 1 Hzneopterin-PPP B -

--+

H4biopterin,

and Pathway

2: Sepiapterin Hzbiopterin

2 z H4biopterin,

where A = GTP cyclohydrolase, B = presently unidentified enzyme(s) and intermediate(s), C = sepiapterin reductase, D = DHFR. [D cannot be dihydropteridine reductase since it is known that 7,8Hzbiopterin is not a substrate for this enzyme (19).] The number and identity of the enzyme(s) and intermediate(s) at segment B of pathway 1 have not been determined; however, the partial resolution of activities during AcA-34 chromatography implies that at least two enzymes are involved. The characteristics of the enzymes reported in the present study are very similar to those identified in the pterin biosynthetic pathway in chicken kidney preparation (5), Drosophila (4), and human liver and kidney (7). Thus, with all of these preparations it was found that (i) the biosynthetic enzymes fractionate in the 40 60 or 65% ammonium sulfate fractions, (ii) the MW of the enzymes purified by gel filtration is in the range of 60,000 to 90,000, (iii) the enzyme system requires a divalent metal ion and NADPH, and (iv) in the absence of NADPH a labile compound accumulates.

TETRAHYDROBIOPTERIN TABLE EFFECT

OF NADPH,

EDTA,

277

BIOSYNTHESIS II

AND HEPES

ON ~~~~~~~~~~~ PRODUCTION BY AcA-34-PURIFIED ENZYME’

Pmol/h/mg

FROM

H~NEOPTERIN-PPP

Pterin (pmol/h/ mg protein)

protein H,Biopterin

Assay

Total

Complete Minus NADPH Plus

Plus

HIbiopterin was performed

0 2850 on

the were

and

(%I

Acid

0

87 -

910

0

-

0

86

0

2474 0

were performed activity. Assays

total biopterin, b This assay

H*Biopterin

2850

EDTA 0.1 M Hepesb

a Assays Hlbiopterin

biopterin

2460 AcA-34 carried

percentage in the presence

0

gel-filtration pool of the peak fractions out as described under Materials and H,biopterin were performed and absence of 0.1 mM

These similarities between our observations and earlier studies suggest that the enzymes in the bovine adrenal medulla are analogous to those reported earlier for various other organisms. However, the interpretation in previous studies that sepiapterin and Hzbiopterin are intermediates in the biosynthesis of H4biopterin (57) is not supported by the evidence herein for the reasons discussed above. It has also been proposed that the intermediates in H,biopterin biosynthesis are quinonoid Hapterins (quinonoid Hzneopterin-PPP and quinonoid H,biopterin) (24). This type of intermediate might also lead to a lack of MTX inhibition, even at the MTX concentration used here which is sufficient to inhibit dihydropteridine reductase (19), since DTT or NADPH could reduce quinonoid Hzbiopterin to Hlbiopterin (25). However, our observation (Table II) that addition of a catalyst for the rearrangement of quinonoid HBpterins to ‘7,8-Hzpterins, 0.1 M Hepes (26), does not decrease either total or percentage H4biopterin in our reactions, argues against this type of intermediate unless the intermediates are protected from solvent all along the pathway. In conclusion, in contrast to several previous reports that Haneopterin-PPP, sepiapterin, and Hzbiopterin are all intermediates on the de nova biosynthetic pathway for H,biopterin from GTP, the present results indicate that among these three compounds, only Hzneopterin-PPP is a true

MTX

Base

2500 1690

0 2500

for Heneopterin-PPP Methods. Calculations

as in Table with the

I. same

+ of

result.

intermediate. Studies are currently derway to determine the structures other intermediates on the pathway.

unof

REFERENCES 1. FAN,

C. L., KRIVI,

Biochem. 2. ETO,

I., FUKUSHIMA,

Biol.

G. G., AND BROWN,

Biwphys.

Chem

251,

Res. Commun K.,

AND

G. M. (1975)

67,1047-1054.

SHIOTA,

T. (1976)

J.

6505-6512.

K., RICHTER, W. E., JR., AND SHIOTA, J. Biol. Chem. 252, 5750-5755. 4. KRIVI, G. G., AND BROWN, G. M. (1979) B&hem. Genet. 17, 371-390. 3. FUKUSHIMA,

T. (1977)

5. TANAKA, K., AKINO, M., HAGI, Y., DOI, M., AND SHIOTA, T. (1981) J. Biol Chem. 256,2963-2972. 6. KAPATOS, G., KATOH, S., AND KAUFMAN, S. (1982) J. Newochem. 39, 1152-1162. 7. CURTIUS, H. C., HAUSERMANN, A., AND GHISLA, Clinical Aspects 50, de Gruyter, 8. KAUFMAN, 9. POLLOCK, rochem.

M., NIEDERWIESER,

S. (1982) in Biochemical and of Pteridines, Vol. 1, pp. 27New York.

S. (1967) J. Biol Chem. 242,3934-3943. R. J., AND KAUFMAN, S. (1978) J. Neu30,253-256.

10. SPECTOR, R., FOSBURG, M., LEVY, P., AND ABELSON, H. T. (1978) J. Neurochem. 30, 899-901. 11. DUCH, D. S., LEE, C. L., EDELSTEIN, M. P., AND NICHOL, C. A. (1982) Fed. Proc. 41, 1062. NICHOL, C. A., LEE, C. L., EDELSTEIN, M. P., CHAO, J., BOWERS, S. W., ANDDUCH, D. S. (1982) Fed. Proc. 41, 648. 13. NICHOL, C. A., LEE, C. L., EDELSTEIN, M. P., CHAO, J. Y., AND DUCH, D. S. (1983) Proc. Natl Acad.

12.

Sci. USA

80, 1546-1550.

R. L. (1979)

Anal

Biochem

14.

THEN,

15.

CHEN, P. S., JR., TORIBARA, T. Y., AND (1956) Anal. Chem 28, 1756-1758.

100,

122-128. HUBER,

W.

278 16.

SMITH

FUKUSHIMA,

T.,

Biochem 17.

T.,

19.

0.

H.,

NICHOL,

CRAINE,

AND

Biophys.

Biochem.

18. VIVEROS,

NIXON,

J.

NICHOL

AnaL

C. (1980)

and land,

102, 1’76-188.

FUKUSHIMA,

AND

AND

AND

LEE,

Chem

Arch

(1968)

Stience

M. M.,

T.,

21.

SHIOTA, FUKUSHIMA,

T. (1978) T., AND

KOBAYASHI,

istry

Biology

And. NIXON,

K.,

WOOLF,

I.,

(Kisliuk,

24.

in ChemR. L.,

G. M., eds.), York/Amsterdam.

J. H.,

NICHOL,

C. A.,

J. Chromatogr. T., AND

Acta

Elsevier/North-Hol-

KATOH,

AND

DUCH,

S.

Bit+

717,265-271.

25.

BUBLITZ,

C. (1977)

26.

ARCHER,

M.

Canad

D.

274, 398-402. S. (1982) B&him.

GAL, E. M., BYBEE, J. A., AND SHERMAN, (1979) J. Neurochem. 32, 179-186.

AND

89, ‘71-79.

J. C. (1979)

SUEOKA,

phys.

S. (1972)

ETO,

Biochem.

of Pteridines

23.

213,349-350.

E. S., AND KAUFMAN,

FUKUSHIMA,

22.

(1983)

247, 6082-6091.

20.

and

M.

C. L., ABOU-DONIA,

C. A. (1981)

J. E., HALL,

J. Biol.

AKINO,

128, l-5.

Brown, New

Biochem.

C., AND

J. Biochem.

Med.

SCRIMGEOUR,

48,278-287.

A.

17, 13-19. K.

G.

(1970)

D.