In vitro carotenogenesis and characterization of the phytoene desaturase reaction inAnacystis

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 916-921

Vo1.163, No. 2, 1989

September 15,1989

In vitro

Carotenogenesis Desaturase

and C h a r a c t e r i z a t i o n of the Phytoene Reaction in Anacystis

Gerhard Sandmann and Susanne Kowalczyk

Lehrstuhl fur Physiologie und Biochemie der Pflanzen, Universit~t Konstanz, D-7750 Konstanz, FRG Received July 21, 1989 An in vitro system for carotenogenesis has been developed from the cyanobaeterium Anacystis. Precursor conversion is highly effective and almost no colored lhtermediates before R-carotene accumulate. These cell-free reactions have been employed to characterize the phyto~ne desaturation reaction. Phytoene desaturation is dependent on NAb(P) and oxygen but insensitive to inhibitors of plant-type monooxy~enases. This result suggests a hydride/proton transfer as mechanism ~or insertion of a double bond into phytoene. Furthermore, feed-back regulation of phytoene desaturase could be demonstrated for most of the subsequent carotenes. © i~89 Academic Press, Inc.

Carotenoids are synthesized in bacteria, fungi and photosynthetic eukaryotes. In photosynthetic organisms, carotenoids are essential for the function of the photosynthetic apparatus (i). Although the biosynthetic pathway is well established for decades, only a few in vitro systems are available to study formation of the whole carotene series in photosynthetic membranes (2). So far, the most efficient B-carotene synthesis in chloroplasts was achieved with intact spinach chloroplasts from photosynthates

(3) rather than with

solubilisates and isolated membranes from specific precursors

(4,5).

The most complete cell-free carotenogenlc system is the one from a unicellular cyanobacterium.

In two steps it is possible to form prenyl pyrophosphates

(6)

from mevalonic acid (MVA) as well as to convert geranylgeranyl pyrophosphate (GGPP) or phytoene via different carotene intermediates to R-carotene and further on to xanthophylls

(7,8).

In this report we present an in vitro carotenogenlc system from the cyanobacterium Anacystis.

It is advantageous for the study of the steps

that convert phytoene into R-carotene because of the very high conversion rates and an efficiency that prevents accumulation of intermediates under optimum conditions. This in vitro system comes nearest to the in vivo conditions in which no carotene precursors of R-carotene are detectable in photosynthetic membranes. We have used it to study the requirements of the phytoene-desaturase reaction and to draw conclusions on the mechanism of desaturation.

0006-291 X/89 $1.50 Copyright © 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

MATERIALS AND METHODS

Anacystls R2 (= Synechococcus PCC 7942) was cultivated as described for other unzcellular cyanobacterza ~7). The carotene-deficient Fusarium monlliforme SG4 was grown in a 2.4% potato-dextrose broth (w/v; Difco LaDoratorles, Detroit, USA) for 5 days and the carotene mutants of Phycomyces blakesleeanus C5 and C9 according to re(. (9). In vitro carotenogenesis was carried out according to a given procedure (6) with the following modifications: Anac~stis membranes were obtained by French-press treatment (500 bar) in 0.i M rrls-N~l Duffer, pH 8, containing 5 mM dithlothreltol. After centrifugation (12000 x g, 15 min) the pellet was resuspended in the same buffer and the membranes were ready for the assay. Alternatively, this suspension was brought to 1% by Tween 40 and stirred on ice for 30 min. The supernatant was used~f~r incubation. Fusarium SG4 was the source of I~C-GGPP synthesized from R - / 2 - z ~ C ~ m e v a l o n i c acid in most of the experiments. For experiments i ~ t a b l e I P ~ c o m y c e s C5 and C9 were used additionally for generation of ~ C - p h y t o e n e ana ~ ~-lycopene, respectively. After incubation with membranes equivalent to i00 - 120 ~g chlorophyll for 2 h in the l%ght at 30~C and hydrolysis of chlorophylls with 6% KOHofor 15 min at 60~C, the carotenes were partitioned into petrol (b.p. 40-60 Q) and separated by HPLC on a Spherosorb ODS 5 ~ column with acetonitrile/methanol~2-propanol 85:10:5 (v~v/v) as eluent ~I0). Radioactivity was continuously recorded by a radioactivity flow detector (Ramona LS). The radiochromatogram was recorded together with the absorbance of added tracer carotenes (phytoene, ~-carotene, lycopene a n d ~ - c a r o e n e ) by a programable Jasco UV/visible photospect~ometric detector, m o d e l ~ 2 0 1 . Comparison of both traces allows a clear identification of the radioactivity peaks of all intermediates in the pathway from phytoene to B-carotene. By recording of absorbance spectra at the slopes of the carotene peaks with a Phillps diode array detector PU 4021 we ensured that the HPLC system employed was able to separate all the carotenes mentioned. For further demonstration of radioactive purity of the separated carotenes we have occasionally collected the peak fractions and purified them further by TLC (AIoO 3 plates with 7% toluene in petrol (6)).Except for lycopene we found ~o increase of specific radioactivity which indicates that purity is sufficient. Only for lycopene subsequent purification from contaminants by TLC on silica gel G plates with 15% toluene in petrol was necessary. In the feed-back experiments 25 ~g of unlabeled carotenes (for source see re(. i0 ) were ~laced in I ml of acetone in the test tube, 100 ~i of a Tween-40 solution (2.5%, w/v) was added and the acetone evaporated in a stream of N O . Then the complete reaction mixture was transfered into this tube. Semi-~naerobic incubations were carried out in Thunberg tubes by evacuatin~ three times and Dur~ing each time with N~. Conversion rates for phytoene ~esaturase were calculated as the radioactivity ratio ~-carotene/phytoene + ~-carotene. Chlorophyll (II) and protein (12) was determined as described. The fungal mutants were from the Departamento de Genetica. Universidad de Sevilla, Spain. LAB 117682 (3-(l,2,4-triazolyl-l)-l-(4-chlorophenyl)4,4-dimethylpentan-l-one) was a gift from BASF Company, Limburgerhof, FRG.

RESULTS AND DISCUSSION

Attempts

have been made to characterize

formation and interconversion. co(actor

requirements

date (2). Experiments

the reactions

However,

and mechanisms to approach

of the phytoene

extract from the Fusarium mutant SG4 generates pyrophosphate.

This substrate

Alternatively,

14C-phytoene

subsequently

the Phycom~ces

or lycopene as substrates

Saponification hydrocarbons

of the products

to B-carotene

sequence

14C-geranylgeranyl

is converted

mutants

by the Anac~stis

C5 and C9 generated

for carotenogenesis,

and partitioning

(in this case carotenes

(6, 8) in which a soluble

respectively.

into petrol enriched only

and squalene)

and prenyl alcohols

including phytol in the organic phase. By HPLC the carotenes were completely separated and purified subsequently

to

this problem have been performed with a

coupled in vitro system as in previous investigations

membranes.

involved in carotene

little is known about the regulation,

except for lycopene which had to be chromatographed

by TLC to obtain this carotene

917

free from contaminants.

Vol. 163, No. 2, 1989

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

In Table I we have assayed the membrane-associated

steps in carotenogenesis

with a thylakoid preparation from Anacystis. From the substrates employed, GGPP was the first intermediate in the series which was converted to g-carotene. Also the other g-carotene precursors, phytoene and lycopene, were used as effective substrate for g-carotene biosynthesis by the membranes. When phytoene was the substrate, hardly any intermediates were detectable. This lack of intermediate accumulation may reflect the high intactness of the enzymes and their tight association which was not lost during the preparation of the membranes. A high structural order of the enzymes of the specific carotenogenic pathway has been proposed by several authors as a pre-requisite for their cooperation (I). In contrast to other intermediates phytoene was accumulated when GGPP was the substrate. Nevertheless, a conversion rate of more than 50% is comparably high for an in vitro system. Cell-free systems from other photosynthetic organisms exhibit much lower phytoene-desaturation activity or are even blocked behind phytoene (2,13).

Incubation in the dark resulted in a partial block behind ~-carotene. We regard this light effect as an in vitro artefact, especially as we observed complete disruption o f ~ - c a r o t e n e

desaturation

in membranes with conversion rates for

phytoene desaturase of only 40% or less (= moderate intact membranes) compared to a small degree of inhibition of d a r k , - c a r o t e n e membranes showed phytoene-converslon

Conversion of phytoene

desaturation when the

rates of 60 to 90%.

to g-carotene was very efficient provided that the

Anacystis membranes were resuspended in a buffer with comparably low ionic strength, e.g. 0.I M Tris (Table II). Increase in the Tris concentration to i M or treatment with the detergent Tween 40 for 30 min disrupted the biosynthetic chain b e h i n d g - c a r o t e n e

which was almost exclusively accumulated. Obviously,

the coupling of ~-carotene desaturase

to phytoene desaturase and/or the

Table I Carotenogenic activity of thylakoid membranes from Anacystis for various substrates

14C-labeled substrate MVA IPP GGPP Phytoene Lycopene

(i0~ (i0° (4 x (4 x (6 x

dpm) dp~) I0- dpm) I0~ dpm) 193 dpm)

Radioactivity in reaction products (dpm) Phytoene g-Carotene 0 0 6211 -

0 0 4155 6734 2881

MVA = mevalonic acid; IPP = isopentenyl pyrophosphate; GGPP = geranylgeranyl pyrophosphate.

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table II Optimization of the preparation for in vitro assay of the carotene biosynthetic chain with GGPP as substrate Radioactivity (dpm) incorporated into Phytoene ~-Carotene Lycopene g-Carotene

Preparation I. Soluble fraction 2. Membranes resuspended in 0.I M Tris buffer 3. Membranes resuspended in 1.0 M Tris buffer 4. Membrane solubilisate with I% Tween 40 5. as 2. but incubated in darkness

substrate

transfer

conditions

protein

of Table

phytoene-desaturation

repeated

6147

10655

164

308

6655

3508

199

766

step in the in vitro

phytoene

reactions

had no effect.

we can exclude

The stimulatory nucleotides

deplete

activity.

an oxidative

Under

and hydrogen mediator

as proposed

that we found

(table

whereas

to demonstrate

the membranes Phytoene

from

desaturation

decreased

this

the same experimental

by more

mechanism

fully supports

hydroxylation

than 70% (15). by NADP.

which includes

for the desaturation

reaction

on oxidized

In Comparing

a sequence

nicotine

an alternative

by other authors

(16,17).

mechanism

It should be

the same NAD(P) + and oxygen dependence

of

of phytoene.

reaction with NAD(P) + as an electron

919

was

117682 which are both inhibitors

on NADPH and inhibited

dehydrogenation

of a

An absolute

the monooxygenase-catalyzed

a possible

desaturation

desaturation

conditions

effect of oxygen and the dependence

of phytoene

with water elimination

is difficult

in Aphanocapsa

and water elimination

on the

for the insertion

phytoene

CO and LAB

this reaction was dependent

were formed.

mechanism

this reaction.

Semi-anaerobic

decreased

carotenes

to obtain information

to completely

to g-cryptoxanthin

these results, hydroxylation

nor colored

chain.

activity

a basic activity was observed

carotenogenic

Furthermore,

assay where unfavorable

of the biosynthetic

or hydroxylation

desaturase

3B).

0

showed no carotenogenic

did not affect

(table

both inhibitors

of ~-carotene

mentioned

248

are two alternatives

destroy

by one third.

of monooxygenase

involving

174

transferase)

washing-steps

also oxygen-dependent

addition,

5477

A dehydrogenation

of phytoene

nucleotides

conditions,

5012

III were performed

nucleotides

NAD(P) + requirement

reaction

3990

of either NAD or NADP supported

nicotine

nicotine

216

(14). With washed membranes

3A). Addition

because

104

Neither

reaction.

(dehydrogenase-electron

reduced

4519

from Anacystis

(dehydrogenase-monooxygenase) double bond

0

lead to a disconnection

fraction

with GGPP as the substrate.

The experiments

0

is the crucial

or manipulations

The soluble

0

for the

VoI. 163, No. 2, 1989

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table III Nucleotide dependence of phytoene desaturase (A) and interaction with monooxygenase inhibitors (B) Radioactivity (dpm) in Phytoene ~ -Carotene

Conditions A. no nicotine nucleotide + NAD + NADP + NADH + NADPH

% Conversion

1049 1212 1105 2219 1004

473 1304 1252 873 411

31 52 53 28 29

2307 5330 2249 1655

6055 4949 5329 4452

72 48 69 73

~o

Control Semi anaerobic Saturated with CO + 0.i mM LAB

Incubations were carried out wlth membranes resuspended in 1.0 M Tris buffer in the dark; in experiments A the membranes were washed once additionally before resuspension; nicotine-nueleotide concentrations were 1.5 mM.

subsequent

desaturation

step c a t a l y z e d

by~-carotene

desaturase

(data not

shown).

In vitro

phytoene

carotenes showed

from

desaturation

an i n h i b i t o r y

desaturated

lycopene

desaturation

effect.

Under

certain

is also e v i d e n t

in~-carotene

desaturase

these

as e x p e c t e d

strains

feed-back fungus

control

Phycomyces

Interference

Additions

pathway

of one or even conditions

reaction

was

bonds.

made

regulation

in S c e n e d e s m u s

but also already

with

by available the fully

A lower degree

rings

only~-carotene

has

affected

the carotenes

was o b s e r v e d

feed-back

e.g.

Not

desaturase

All

two ionone

from the m u t a t i o n

of phytoene in vitro

(18).

double

this

cells

IV).

decrease

Ii c o n j u g a t e d

in intact

thylakoids

(Table

The s t r o n g e s t

cotaining

or the p r e s e n c e

less e f f e c t i v e . desaturase

with A n a c y s t i s

the b i o s y n t h e t i c

inhibition of p h y t o e n e

mutants

blocked

is a c c u m u l a t e d

phytoene.

for

(9).

Table IV of carotenes with in vitro phytoene desaturatlon by feed-back inhibition

No carotene P-Carotene Lycopene ?-Carotene ~-Carotene

52 18 6 8 13

Incubations were carried out with membranes resuspended in 1.0 M Tris buffer in the dark; 25 ~g of carotenes were added.

920

in

A similar

been d e m o n s t r a t e d

% Conversion of phytoene

of

the

Vol. 163, No. 2, 1989

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Since membrane-bound phytoene desaturase has not been purified successfully, we have used crude membranes to elucidate the properties of this enzyme. For a more detailed characterization, however, an enzyme purified to homogeneity is indlspensible. Progress on the isolation of phytoene desaturase from spinach chloroplasts may be reported soon.

This work was supported by the Deutsche Forschungsgemeinschaft. Due thanks are expressed to Mrs. S. Kuhn for excellent technical assistance.

REFERENCES

I. Britto~ G. (1982) Physiol. Veg. 20, 735-755. 2. Bramle~ P. (1985) Adv. Lipid Res. 21. 243-279. 3. Schulze-Siebert, D. and Schultz, G. (1987) Plant Physiol.84, 1233-1237. 4. Kushwaha, S.C., Subbarayan, C., Becker, D.A. and Porter, J.W. (1969) J. Biol. Chem. 244, 3635-3642. 5. LHtke-Brinkhaus, F., Liedvogel, B., Kreuz, K. and Kleinig, H. (1982) P!anta 156, 176-180. 6. Sandmann, G. (1988) Meth. Enzymology 167, 329-335. 7. Clarke, I.E., Sandmann, G., Bramley, P.M. and B~ger, P. (1982) FEBS Lett. 140, 203-206. 8. Sqndmann, G. and Bramley, P.M. (1985) Planta 164, 259-263. 9. Bramley, P.M. and Davies, B.H. (1976) Phytoche~istry 15, 1913-1916. i0. E r n s t S. and Sandmann, G. (1988) Arch. Microbiol. 150, 590-594. ii. McKinney, G. (1941) J. Biol. Chem. 140, 315-322. 12. Fanger, B.O. (1987) Analyt. Biochem. 162, 11-17. 13. Bugy, M.J., Britton, G. and Goodwin, T.W. (1974) Phytochemistry 13, 125-129. 14. Massey, V. and Ghisla, S. (1983) in: Biological Oxidations (Sund, H. and Ullrich, V.,eds) pp. 114-139, Springer, Berlin. 13. Sandmann, G. and Bramley, P.M. (1985) Biochim. Biophys. Acta 843, 73-77. 16. Britton, G. (1979) Z. Naturforsch. 34c, 979-985. 17. Goodwin, T.W. (1983) Biochem. Soc. Transactions ii, 473-483. 18. Britton, G. and Powls, R. (1977) Phy~ochemistry 16, 1253-1255.

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