The conversion of dihydroneopterin triphosphate to sepiapterin by an enzyme system from Drosophila melanogaster

Vol. 67, No. 3, 1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

THE CONVERSION OF DIHYDRONEOPTERIN TRIPHOSPHATE TO SEPIAPTERIN BY AN ENZYME SYSTEM FROM Drosophila Ching

Liang

Fan,

Gwen G. Krivi

melanogaster

and Gene M. Brown

Department of Biology Massachusetts Institute of Technology Cambridge, Mass. 02139

Received

Ott

ober IO,1975

Summary : The enzyme system for the synthesis of the pteridine pigment, sepiapterin, from 2-amino-4-hydroxy-6(D-erythro-1 ‘,2’,3’-trihydroxypropyl)7,8-dihydropteridine triphosphate (dihydroneo:teriniphosphate) has been found in extracts NADP+ or NADPH and Mg2+ are required for this enzyof Drosophila melanogaster. No sepiapterin is produced when dihydroneopterin is supmatic transfcrmation. plied as substrate in place of dihydroneopterin triphosphate. Introduction: produced

from

hydrolase known

Dihydroneopterin’ GTP in the presence

I to distinguish

to occur

this

and other compound

mediate

it

in Drosophila

of several

ranthopterin,

role

(9)

kinds

its

of H2-neopterin-PPP

enzyme,

(5)

of all

from

report

and the

in the

melanogaster. of pterins

present pterin,

paper

the

in

with trivial

the

name for Pterin

has that

Syrian

golden

large

quanti-

These

include

(the

support

finding

view

of GTP cyclo-

contains

further

(10)

(11)

and drosopterin

we provide

as a key intermediate is

--et al.

as eye pigments.

sepiapterin

In the p-resent

1 Dihydroneopterin or H2-neopterin 6-(lt,2’,3’-trihydroxypropyl)7,8-dihydropteridine. for 2-amino-4-hydroxypteridine.

animal

acid

that

that

a key inter-

This

of biopterin

(l)), (6).

is

(6) on the presence

mainly

II

expressed

H2-neopterin)

by Fukushima,

The latter

GTP cyclo-

of folic

pterins.

biosynthesis

of Fan and Brown

portion

view has been form,

observations

(or

to be

melanogaster

of the pterin

naturally-occurring

recent

known

GTP cyclohydrolase

and Drosophila

dephosphorylated

isoxanthopterin,

eye pigment).

plants

is

enzyme GTP cyclohydrolase

a similar

in bacteria,

an intermediate

and by the

hydrolase

pterin

is

from

(H2-neopterin-PPP)

to be a precursor

(or perhaps

some support

hamsters

ties

is known pterins

of the

(2-4),

in the biosynthesis

received suggest

it

in bacteria

H2-neopterin-PPP (7,8)

triphosphate

major for

the

one of

2-amino-4-hydroxyis the trivial

name

Vol. 67, No. 3, 1975

BIOCHEMICAL

these pigments, sepiapterin,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

can be synthesized from HZ-neopterin-PPP in the

presence of an enzyme system from Drosophila melanogaster. enzymatic transformation shown later

The nature of this

becomesapparent from the formulas of the compounds

in Fig. 3 of this paper.

Materials and Methods: [U-14C]GTP was purchased from Amersham-Searle Corp., phosphocellulose and ECTEOLA-cellulose from Gallard Schlesinger, and piperazineN,N'-bis(2-ethane-sulfonic acid) (PIPES) buffer from Calbiochem. Purified GTP cyclohydrolase I from g coli was supplied by John Yim of this laboratory. Sepiapterin was prepared (12) from sepia mutants of Drosophila melanogaster. Crude extracts of Drosophila melanogaster, strain Oregon-R (obtained from Dr. Linda Hall), were prepared as described earlier (6). Radioactive H2-neopterin PPPwas prepared enzymatically by incubation (overnight at 42' in a total volume of 1.0 ml) of 100 uM [U-14C]GTP (10' cpm), 100 mMPIPES buffer (pH 7.5), 100 mM KCl, 5 mMEDTA (pH 7.5), and 10 mM2-mercaptoethanol with enough purified GTP cyclohydrolase I to achieve at least 90"0 conversion of GTP to H2-neopterin-PPP (measured as described earlier (6)). The standard reaction mixture for the assay of sepiapterin synthesis contained per 0.2 ml total volume: 75 mMPIPES buffer (pH 7.5), 20 mMMgC12, 3.3 mMNADPH,0.1 ml of the incubated reaction mixture described above as a source of H2-neopterin-PPP, and Drosophila extract. After incubation for 1 hour at 42", 4 units of alkaline phosphatase (Worthington) and 0.1 pmole of sepiapterin were added and the incubation was continued for an additional 30 minutes. Each mixture was heated at 100" for 5 minutes and subjected to centrifugation. The resulting supernatant material was applied to a phosphocellulose column (1.1 x 50 cm). The column was developed at 4" with water. Fractions of 5.0 ml were collected at a rate of 15 ml per hour. A portion of each fraction was analyzed for radioactivity in a scintillation counter (Packard 3320). The fractions containing standard sepiapterin were yellow. Sepiapterin synthesis was estimated from the radioactivity in the region of elution from the column of standard sepiapterin. Thin layer chromatography was carried out (ascending technique) at room temperature with 13255 cellulose Eastman Chromagramsheets (S x 10 cm). Protein was determined by the method of Lowry, et -- al. (13) with bovine serum albumin as the standard. Results:

Preliminary

experiments indicated that a radioactive

the chromatographic properties by incubation of radioactive melanogaster.

of sepiapterin

substance with

could be produced enzymatically

H2-neopterin-PPP with a crude extract

of Drosophila

Similar experiments revealed that the addition of NADP+or NADPH

stimulated the production of the putative MgC12was also stimulatory.

The results

sepiapterin

by 2- to 3-fold and that

of an experiment devised to measure the

enzymatic conversion of H2-neopterin-PPP to this radioactive presence of a crude extract

are given in Fig. 1.

substance in the

In this experiment, both NADPH

and MgC12were included in the incubation mixture to promote maximal synthesis of the product.

The data presented in the figure

1048

represent the elution pattern

BIOCHEMICAL

Vol. 67, No. 3, 1975

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

3Ok 6

m ‘0- 20 ; 2 15 2 .z IO ::

8

0;

5 -I 0

0

100

200

300

400

Elution

500

Volume,

600

700

ml

pattern from phosphocellulose of an incubated reaction mixture. Fig. 1. Elution The standard reaction mixture was prepared to contain either a crude extract of Drosophila (2.8 mg of protein) or heated (at 100” for 5 minutes) extract. After incubation (1 hour at 42”) and treatment with phosphatase, sepiapterin (0.1 nmole) The mixtures were applied to individual was added to each mixture as a marker. columns and each was developed with water (650 ml) and then with 1% NH3. Portions (1.5 ml) of fractions were analyzed for radioactivity. The standard sepiapterin appeared as a yellow substance in the fractions collected in the 135-150 The elution pattern of the reaction mixture containing unheated exml region. tract is represented with closed circles (0) and that of the control mixture, containing heated extract, with open circles (0).

from

a phosphocellulose

tion

of

of the

14

C-labeled

elution

pterin.

phosphatase)

GTP .

also

contained

Peak 1 coincided radioactive small

quantities

extract.

Peak 3 sepia-

the positions

of elution

of guanosine

and

must have been remained (Peak

the

void

presumably were

H2-

during

the develop-

produced

enzymatical

of the

identities.

in the

from unreacted

identified

volume

radioactive

1049

(by treatment

as a contaminant

and oxidation

of unknown

of unknown

derived

6) was produced

substances

components

incuba-

of standard

xanthine,

with

and unheated

after

of elution

treatment

radioactive

present

the position

and neopterin

Peak 2 contained

of these

compounds heated

GTP that

by the phosphatase

All

several

with

from unreacted

column.

chromatography.

with

The guanosine

preparation,

ment of the

with

coincides

respectively.

H2-neopterin-PPP

tained

H2-neopterin-PPP

pattern

neopterin-PPP

from

of radioactive

Peaks 5 and 6 coincide

neopterin, with

column

by thin column

layer and con-

Peaks 4, 7 and 8

compounds.

Approximately

ly

Vol. 67, No. 3, 1975

BIOCHEMICAL

94% of the radioactivity

applied

radioactivity

with

Peaks

associated

1, 3, 4 and 7 (418,000

PPP consumed

during

the

the material fractions the

that

concentrated

(Table

I).

tic

to be presented that

supports

material

the

in Peak 3 of Fig. The presence

I.

material

in 0.075

ml water.

subjected

conclusion

that

remainder

1 will in crude

we obtained

this

extracts

Identification by Thin Product as Sepiapterin

paper

peak.

to dryness, (0.004

ml,

and the

enzyma-

experiment evidence

was sepiapterin.

radioactive

product

of sepiapterin

Layer

and pyridine

present

Chromatography

in solvent

of the

nucleotides

Enzymatic

system

Compound

1

2

3

4

5

6

Sepiapterin

0.40

0.39

0.24

0.48

0.49

0.40

0.40

0.39

0.24

0.49

0.50

0.40

product

cpm)

to as sepiapterin.

RF values

Enzymatic

and

11,000

spectrophotometric product

the

chromatography

In another

the

Peaks 1,

purpose,

sepiapterin

radioactive

of this

be referred

layer

was sepiapterin.

paper,

in

For this

Portions

of standard

in

of H2-neopterin

sepiapterin

concentrated

to thin

The

in Peak 3 was sepiapterin,

analyses.

combined,

the product

the amount

putative

1-8.

present

present

present

were

were

in this

in the

Peak 3, the

of RF values

that

later

For convenience,

Table

the peak

indicate

in

in Peaks

materials

radioactivity

to chromatographic

The coincidence

product

was recovered

equalled

Of the

for

was redissolved

of the

column

enzymatically-produced

radioactive

was subjected

residue

the

incubation.

the

constituting

to the

cpm) approximately

3, 4 and 7, 54% was accounted To confirm

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

NH3 (2:l); 2, l-propanol-ethylacetate-H20Solvent systems: 1, 1-propanol-1% (7:1:2); 3, 3% NH4Cl; 4, l-butanol-acetic acid-H20 (4:1:2); 5, l-propanol1% ammonium acetate (1:l); 6, ethanol-ammonium borate (pH 8.0, 5% as boric acid)-3% NH4Cl (2:l:l). Standard sepiapterin was observed as yellow fluorescent zones under ultraviolet light. To locate the radioactive enzymatic product, appropriate areas were scraped from the plate and transferred to vials, each containing 1.7 ml of H20 and 5.0 ml of ScintiVerse (Fisher and radioactivity was measured in a scintillation counter. Scientific Co.),

1050

Vol. 67, No. 3, 1975

interferred pterin

with and (b)

quirement with

BIOCHEMICAL

.

(a)

the

thesis

To circumvent

tion.

of the

these

was recovered

40 and 60% saturation This

degree

that

fraction

for

and kind

the production

of pyridine

we fractionated

most

of the

enzymatic

in the protein

fraction

and that

was used

RESEARCH COMMUNICATIONS

analysis

difficulties,

and found

of sepiapterin

between

spectrophotometric

an assessment

ammonium sulfate

AND BIOPHYSICAL

in the

no sepiapterin remainder

of the

of sepia

nucleotide

re-

the

crude

extract

activity

for

that

precipitated

the syn-

was present

in this

frac-

experiments

to be reported

below. The synthesis concentration replace

HZ-neopterin-PPP

produced

requirement, is

added

nucleotide

from HZ-neopterin-PPP

up to 14 mg/ml.

ammonium sulfate pterin

of sepiapterin

is

enzymatically

from

is not

mixtures.

are:

(a)

(b) no sepiapterin

omitted

(e) no sepiapterin

to reaction

observations

as substrate,

fraction

and

Other

was linear

the

reaction

phosphorylated, is produced

Neither

protein

H2-neopterin

cannot

is produced mixture, (d)

unless

with

(c)

Mg2+ is either

if the

the sepia-

an absolute

NADP+ or NADPH

NAD+ nor NADH can satisfy

this

pyridine

requirement.

250

300

350 Wave

400 Length,

450 nm

Fig. 2. Absorption spectrum of the enzymatic product. Six standard reaction mixtures were prepared (see text). After incubation, the mixtures were heated and subjected to centrifugation to remove insoluble material. The combined supernatant fractions were applied to a standard phosphocellulose column and the column was developed with H20. The fractions constituting the region of elution of sepiapterin were combined (40 ml) and concentrated to 3.0 ml. The resulting yellow solution was applied to an ECTEOLA-cellulose column (1.1 x 55 cm) . The column was developed with H20. The yellow fractions were combined (20 ml), concentrated (1 ml) and subjected to chromatography on a second phosphocellulose column. The resulting yellow fractions were combined (40 ml) and concentrated to 1 ml. The spectrum of this material in H20 was determined with a Perkin-Elmer spectrophotometer, Model 202.

1051

Vol. 67, No. 3, 1975

In order analysis, added

is

of the

an amount

The absorption

along that

the

the

drolase

(6),

and the

results

compound pterins. tial tally

is

quantities

and Akino

(14)

is

in this

in Drosophila. is

H

for

legend

in Fig.

strongly

to

to Fig.

sepiapterin. I,

melanogaster

the

paper

conversion

on the

support

than

sepiapterin or not

presently

under is not

contains

2, This,

indicates

formation

contention of all

to occur

pterins

investigation converted

the

latter

naturally-occurring

are known these

of sepiapterin

that

in substan-

are made enzymatiin our

to sepiapterin

I H

H

GTP cyclohy-

of GTP to H2-neopterin-PPP,

enzymatic

to the

Whether

HZ-neopterin

P

mixtures

given

in Table

in the biosynthesis

other

that

Drosophila

further

a key intermediate

Our observation

presented

in the

No

sepiapterin.

catalyzes

from H2-neopterin-PPP

reaction

product,

the enzyme that

pterins

of the

yellow

by Tsusue

and to each was

to 24 mg of protein.

of the purified

that

Several

equal

a spectrophotometric

prepared,

was as described

evidence

lend

were

fraction

The observation

from H2-neopterin-PPP

mixtures

for

product

chromatographic product

product

The processing

reported

reported

enzymatic

reaction

sepiapterin

that

enzymatic

Discussion:

of the

was included.

spectrum

with

with

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of ammonium sulfate

the putative

identical

enough standard

sepiapterin

purify 2.

to obtain

six

standard

BIOCHEMICAL

laboratory. by the

H+ In

reaction mechanism for the enzymatic conversion of a phosFig. 3. Possible phorylated form of H2-neopterin (I) to sepiapterin (V). @represents either one, two, or three phosphate residues. The carbon in compound I marked with an asterisk (*) is carbon 1’.

1052

Vol. 67, No. 3, 1975

enzyme system necessary occurs

of Drosophila

and suggests coincidently

how many of the neopterin

tionated

nor the

the

to produce

compound

sepiapterin

by the prior

a reduction

could (V).

might

should occurs

the pyridine

in the

of the

neopterin-PPP

for

the

and thus

provides

initial

By rearrangement,

NADP+, but

later

to regenerate proposed

through

might

this

a pyridine

involved

include

nucleotide,

group.

one might

expect

no oxidation

that or

knowledge

of the reaction

in the

and

to be facili-

More precise

and the mechanism

III

1’ to a keto

occurs

reaction.

include,

putative

does not

the NADP+ since

enzyme or enzymes

be involved. would

be expected

this

frac-

intermediates

for

on carbon if

H,I

or triphosphate)

mechanism

no role

of the OH group

H2-

neither

might

3.

know

the

of sepiapterin

reaction

require

since

pyrophosphate

step

yet

to allow

(or

be converted this

requirement

we do not

is

groups

and we have not yet

formation

Although

overall

of H2-neopterin

how many enzymes

in Fig.

then

in the

occur

nucleotide

the purification

to decide

shown

oxidation

Such an oxidation

reduction

II,

of phosphate

available,

of a phosphate

or a reduction

removal

to sepiapterin

has been

removal

but

need to be retained

directly

pathway

intermediate

an oxidation

tated

reaction

of sepiapterin,

groups

enough

form

of one or more of the phosphate

formation

phosphate

enzyme system

enzyme-bound

the

the

RESEARCH COMMUNICATIONS

a phosphorylated

removal

H2-neopterin-PP

step,

IV to give

the

to be converted

A plausible as a first

that

three

AND BIOPHYSICAL

shows that

with

moiety

neopterin-P

group

BIOCHEMICAL

must

conversion

about await

of H2-

to sepiapterin.

Acknowledgment: This work was supported by research grants from the National Institutes of Health (AM03442) and the National Science Foundation (GB33939). REFERENCES 1. Foor,

F.,

and Brown,

2. Burg,

A.W.,

G.M.

and Brown,

G.M.

3. Shiota,

T.,

and Palumbo,

4. Guroff,

G.,

and Strenkoski,

5. Mitsuda, H., Suzuki, l2-, 192-204. 6. Fan, 7. Burg,

C.L., A.W.,

and Brown, and Brown,

(1975)

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J.

(1966)

M.P.

G.M.

J.

(1966)

Tadera,

K.,

(1975)

Chem.,

Biochim.

(1965)

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G.M.

Biol.

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and Iwai,

Biochem.

(1968)

Biophys.

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in press. 245,

275-278.

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Vol. 67, No. 3, 1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

8. Shiota, T., Jackson, R., and Baugh, C.M. (1970) in Chemistry and Biology of Pteridines (K. Iwai, M., Akino, M. Goto, and Y. Iwanami, eds.), Int'l. Academic Printing Co., Tokyo, pp. 265-279. 9. Heine, M.C., and Brown, G.M. (1975) Biochim. Biophys. Acta, in press. 10. Brown, G.M. (1971) Adv. in Enzymology, 35, 35-77. 11. Fukushima, K., Eto, I., Saliba, D., and Shiota, T. (1975) Biochem. Biophys. Res. Commun.,65, 644-651. 12. Fukishima, T. (1970) Arch. Biochem. Biophys., 139, 361-369. 13. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (1951) J. Chem., 193, 265-275. 14. Tsusue, M., and Akino, M. (1965) Zool. Mag., 74, 91-94.

1054

Biol.