Ubiquinone biosynthesis in Leishmania major promastigotes

GOURI Department

RANGANATHAN

of Biological

Sciences, University U.S.A.

(Received

biosydk&

pdmy

Key words: kkishmaniu

and ANTONY

1 March

major;

of Cincinnati,

1994: accepted

in L. nra$or tbns shares daractenlstics

J. MUKKADA* Cincinnati,

OH 45221,

28 June 1994)

with -alulbactdsptenrs.

promastigotes; ubiquinone biosynthesis.

INTRODUCTION

The ubiquinone (UQ) molecule is comprised of a benzoquinonering and an isopreneside chain. The number of isopreneunits varies between6 and 10 in different organisms.The homologsof UQ can be denoted by reference to the number of isoprene units, e.g. UQs,UQs,etc. UQ is synthesizedde now in both procaryotic and eucaryotic cells. The two parts of the moleculeare synthesizedindependently and assembledlater. The isoprenoidportion of the molecule is synthesizedthrough the acetate-mevalonate pathway shownin Fig. 1 (Faust, Goldstein, & Brown, 1979;Wiss& Brubacher, 1961).Mammalian cells are generally incapable of synthesizing the aromatic ring. However, it is derived from aromatic amino acidssuchasphenylalanineand tyrosine (Fig. * To whom ail correspondence should be addressed. 279

1) which are the breakdown products of proteins in the diet or the medium. Procaryotes, on the other hand synthesizethe aromatic ring by the shikimete pathway (Fig. 1). Shikimete formed in this pathway goes to form parahydroxybenzoic acid which gets incorporated into the UQ ring (Cox & Gibson, 1964). Bacterial cells can incorporate [‘?I acetate into UQ but mevalonicacid is not thought to be an intermediatein isoprenoidsynthesisdue to the lack of incorporation of [t4C] mevalonateinto UQ (Cox & Gibson, 1966;Ramasarma,1968).Although UQ9 was identified in Criihidia fascicuhta and C. oiscopelti(Kusel & Weber, 1965;Lemonias,Kidder & Dewey, 1963) very little is known about the biosynthesisof UQ in protozoa. The studieswhich have beendone indicate that the pathway resembIes that in animal cells. However, a part of the acetateshikimate pathway for the synthesisof p-hydroxy-

280

G. Ranganathan and A. J. Mukkada

Isoprene synthesis ACETATE

-

in mammaiian

ACETYLCoA

cells

-

HMG CoA

-

MEVALONATE

-

LONG

ISOPENTENYL.PYROPHOSPHATE

I I

PHOSPHATE

UBIQUINONE

RING

CHAIN

PRBNYL

CHOLESTEROL

UBIQUINONE

Ring

synthesis

in mammalian

eelis

AROMATIC AMINO ACIDS

Ring

.

sgnthesis

ACETATE

-

p-HYDROXY BENZOIC ACID

-

b &mnxrgotes

SHBHMATE

-

CHOBISMATE

-

@‘TBBOXY BBNZOATB

-

UBIQBIBOBB RIXG

Fig. 1. Pathways of ubiquinone side ring and chain synthesis.

Organism and growth conditions. L. major LEM 513 promastigotes were grown in a monophasic liquid medium supplemented with sodium penicillin (1 x 10” units G’) and streptomycin sulfate (1.65 g t’) as previously described (Shaefer, Bell & Etges, 1970). L. donovani Sudan l.S, L. tarentolae TAR II and Crithidia jksciculata A.T.C.C.# 11745 were grown in the same liquid medium. Incorporation of radioactive precursors into ubiquinone. Cells were incubated with the radioisotopes for 24 h during 48 to 72 h growth. At the end of the incubation period, the cells were harvested by centrifugation (3300 g, for 10 mm). Cells were washed free of adhering culture medium with 0.85% NaCl. Extraction of Zip&. Lipids were isolated using chloroform-methanol extraction (Garbus, Daluca, Loomas & Strong, 1963). In brief, cells (approx. 1 g) were suspended in 10 ml saline and extracted in 40 ml chloroform: methanol (1:2). To improve the recovery, 50 nmol of UQs was added. Lipids were partitioned into the organic phase by the addition of 10 ml water and 10 ml chloroform. MATERIALS AND METHODS Separation of ubiquinone and cholesterol. The lower phase Chemicals. The following isotopes were purchased from was separated on thin layer silica gel sheets in a solvent New England Nuclear (Boston, MA): [DL -2-14C] Mevalonolactone (45.0 mCi mmoll’ and [L- U 14C] system of 12% acetone in petroleum-ether. Ubiquinone and cholesterol were visualized upon spraying with iodine Tyrosine (505 mCi mmof’. [U -14C Glucose (200 mCi (2-3 times) to constant mmoll’) and [U-‘4C] parahydroxy benzoate (33 mCi vapours, rechromatographed radioactivity, and the radioactivity determined by liquid mrnol-) were obtained form Amersham (Arlington Heights, IL). Sodium [l-l40 acetate (55.5 mCi mrnol-) was scintillation counting. UQ was quantitated by measuring the change in absorbance at 275 mn before and after reducpurchased from International Chemical and Nuclear Corp., (Irvine, CA). UQ9 and UQlO were purchased from Sigma tion with sodium borohydride. A solution of 1 pm01 in-’ shows an optical density change of 12.2 when reduced with Chemical Co, (St Louis, MO).

benzoic acid has been demonstratedin the protozoan Tetrahymenapyriformis (Miller, 1965). Our results indicate that in Leishmania major, the isoprene side chain is synthesizedby the acetatemevalonatepathway and that the ring is synthesized from acetateor aromatic amino acids. Earlier studiesfrom our laboratory demonstrated the presenceof an unbranched electron transport chain in L. major promastigotes (Martin & Mukkada, 1979 a, b). The participation of ubiquinone (UQ) in electron transport was inferred from the observationthat dicoumarol, a UQ specific inhibitor, inhibited oxidation of NADH, succinate and proline. A study was, therefore, initiated to follow the synthesisand role of this moleculein L. major.

Ubiquinone biosynthesis in Leishmania major sodium borohydride (Ramasarma, 1968). Ubiquinone (RF 0.9) separates ciearly from cholesterol (RF 0.4) but the homologues of UQ move together in this system and were separated by reverse phase thin-layer chromatography using para& coated silica gel plates and 90% a&on&water as the developing solvent (Wagner & DengIer, 1962). Protein determination. Protein determination was done using standard techniques (Lowry, Rosebrough, Farr & Randall, 1951). Radioactivity determination. Samples were added to 5 ml Aquasol and the radioactivity determined with a Packard Tri Carb liquid scintillation spectrometer. Results are expressed as d.p.m.mg“ proteinor d.p.m. g-’ wet weight. Enz:ynte assays. Hydroxymethylglutaryl coenzyme A (HMG CoA) reductase activity was assayed by determining the formation of [14C] mevalonate from [14C] HMG CoA (Imblum & Rodwell, 1974). Mevalonolactone formed after acidification was extracted in ether and isolated by thinlayer chromatography using benzene:acetone (1: 1) as the developing solvent. The band corresponding to standard [14C] mevalonates was scraped and counted. Mevalonate kinase was assayed by measuring the incorporation of [2‘“c] mevalonate into 5-phosphomevalonate (Tchen, 1958). 5Phosphomevalonate was separated from the reaction mixture by thin layer chromatography. Cell extract for

absorbancespectrumof this extract, beforeand after the addition of Na-borohydride (oxidized and reduced,respectively)is shownin Fig. 2. It showsan absorbancemaximum between 273 and 275 nm which coincideswith the spectrumof the ubiquinone standard.Table I compares the ubiquinone contents of three speciesof Leishmania,promastigotesand the non-parasitic protozoan Crithidia fasciculata. Reverse-phasethin-layer chromatography shows

tostrictive ultrasonic oscillator operated at 4A, and monitoring for complete disruption of the cells microscopically. Preparation of lJQ9 derivative. Cells were grown in the presence of f4C] acetate and [14C] parahydroxybenzoate (PHB). UQ was isolated and the labeled quinone was diluted with 50 mg of carrier UQ. The diacetate was

Table

that they all contain Biosynthesis

UQ,.

of ubiquinone

Acetate is a precursorof the isoprenesidechain in both procaryotes and eucaryotes. Growing cells of L. major incorporate [‘“Cl acetate into ubiquinone

and cholesterol (Table 2) indicating that it is a precursor of both compounds.Acetate incorporation into both compounds

is higher at log phase of

growth (48-72 h), compared to the earlier stages. Incorporation into cholesterol is 10 times greater than that into UQ. Table 3 shows that mevalonate, the sidechain precursorin mammals,is incorporated

into ubiquinone and cholesterol.Resultsin Table 4 enzymeassays weremadeby disruptingcellson icein phos- confirm the presence of hydroxymethylglutaryl coenzyme-A reductase(WMG-CoA reductase)and phate buffered saline using a Branson 20 KHZ magnel-Quantitative estimation of ubiquinone Leishmania and Crithidia

Species

Nanomol g dry weight

Leishmania major LEM 513

donovani Sudan 1s preparedby reductiveacetylationusingzincdustandacetic L. L. tarentolae TAR II anhydrideandcrystallizedto constantradioactivity(Crane, Crithidiafascictdata (A.T.C.C. 11745)

Lester, Hatefi & Widmer. 1959). Ozonblysis. The diacetate of UQs hydroquinone was dissolved in ethyl acetate. Ozonolysis was done at -7O”C, with damp ozone at the rate of 0.01 standard cu-ft min- for 60 min using an ozonator. The solvent was removed by vacuum distillation and the residue fractionated into

in

139 t l? 81 t 14

101 i 9 108 2 16

Cells were harvested at 72 h of growth and the lipids extracted using chloroform : methanol (1 : 2 v/v). UQ was

separated by thin layerchromatography andquanntitated by

measuring the change in absorbance at 275 nm before and after reduction. The resufts are expressed as mean t S.D. aqueousand ether extracts(Bentley,Ramsey,Springer, from triplicate analysis. Dialemeh & Olsen, 1961). The aqueous extracts were Table 2-Incorporation of [l-14C] acetate into ubiquinone pooled and its radioactivity determined. This fraction and cholesterol at different stages of cell growth contains the isoprene side chain as levulinaldehyde. The etherextractwasevaporatedto drynessandits radioactivDPM/mg protein ity determined. This fraction contains the benzoquinone Stage of cell growth (h) Cholesterol moiety of ubiquinone (Bentley, Ramsey, Springer, UQ Dialemeh & Olsen, 1965). RESULTS

in Leishmaniaand Crithidia To confirm the presenceof ubiquinone, in L. major, the lipid extract was separatedby thin layer chromatography and the spot corresponding to ubiquinone standard was extracted in ethanol. The Ubiquinone

O-24 24-48 48-72

160 c 13 380 2 10 570 F 46

1500 Y!I .?a 3100 -‘- 68

5010 L 94

Four hundred ml of medium was inoculated with L. major. Sampies werewithdrawnat specified timesandincubated with 25 u Ci of Il-t4Cl acetate for 24 h. Litids were extracted and UQ a&d chdle.sterol were sepa&d and radioactivity determined. Results are expressed as mean t S.D. from triplicate analysis.

282

G. Ranganathan and A. J. Mukkada

precursorsof the aromatic ring are alsoincorporated

0.5

into ubiquinone 0.4

8 5 e 8 $

0.3

other metabolic

(Table

3). The

role.

To establish the derivation of the ring and isoprene moieties of ubiquinone from p-hydroxybenzoate and acetate, the labeled molecule was cleaved

0.2

0.1

00 = o-o.5 300

260

nm Fig. 2. The ultraviolet spectrum of ubiquinone L. major. OX, oxidized; RED, reduced.

from

Table 3-Incorporation of f4C] labeled mevalonate, tyrosine, and p-hydroxybenzoate into ubiquinone and cholesterol Ubiquinone DPM/mg protein

Tracer [‘“Cl Mevalonate (15 &i) [14C] _ Tyrosine _ (25 &i) [14C] Parahydroxy benzoate (2 Ki)

Cholesterol DPM/mg protein

140?

8

1680 ? 21

152

4

0

1800 + 61

0

HMG-CoA reductase Mevalonate kinase

incorporated

into UQ. The incorporation

decreased

30% in the presenceof p-hydroxybenzoic acid and shikimic

acid. The lack of a complete

swamping

DISCUSSION

“Activities of HMG-CoA reductase mevalonate kinase in L. major

Enzyme

by ozonolysisand fractionated into the ring and side chain. Table 5 showsthat 65-80% of the PHB is incorporated into the ring moiety and about 15% is associatedwith the side chain. When UQ was labeledwith [14C]-acetate,60-70% of the radioactivity was associatedwith the side chain and 20-30% with the ring. In order to determine the possiblerole of the shikimate pathway in L. major for aromatic ring synthesis,incorporation of label from [14C]-glucose into UQ in the presenceand absenceof shikimicacid (5 mM) or p-hydroxybenzoic acid (5 mM) was studied.As shown in Table 6, label from glucoseis

effect of shikimicacid and p-hydroxybenzoic acid on [14C]glucoseincorporation may be becauseglucose is alsoincorporated into the sidechain of UQ.

50 ml of medium was inoculated with L. major. Tracers were added at 48 h of growth and incubated for 24 h. Lipids were extracted and UQ and cholesterol separated and radioactivity determined. Results are expressed as mean ? SD. from triplicate analysis. Table

but not cholesterol

low incorporation of tyrosine into ubiquinone may be becausemost of it is usedin protein synthesis,but p-hydroxybenzoic acid is almostexclusively incorporated into the UQ ring and is not known to have any

and

Activity (nmol min? rng-’ protein) 0.66 k 0.1 0.19 2 0.03

The cells were harvested at 48 h of growth, washed and suspended in 0.85% NaCl. The suspension was sonicated for 45 s and centrifuged at 800 g for 10 min. The supernatant was used for enzyme assays. The results are expressed as mean lr S.D. from triplicate analysis. mevalonate kinase. These are key enzymes of the acetate-mevalonate pathway and further support the involvement of the acetate-mevalonate pathway in UQ synthesis. Tyrosine and p-hydroxybenzoate,

Promastigotesof Leishmania major are free-living, aerobic microorganismswith ubiquinone content comparableto that in other protozoa (Lester et al., 1959).UQs has been reported in the ciliated protozoan Tetrahymena pyreformis and UQs in the flagellates Crithidia fasciculata and Strigomonas oncopelti (seeCrane, 1957).Our resultsshowthat UQs is characteristic of C. fasciculata and the three L&&mania specieslisted in Table 1. It appears that ciliates contain UQs while flagellatespossessUQs as was suggestedearlier. The isoprenoidportion of UQ and cholesterolin eucaryotes is commonly derived by the acetate-mevalonate pathway. In bacterial cellls, acetateis a precursor but mevalonatehas not been identified as an intermediate in isoprenoid biosynthesis. In the present study, using radiolabelled tracers,both acetateand mevalonatehave beenidentified as intermediates in ubiquinone biosynthesis in L. major promastigotesindicating that the acetate

mevalonatepathway is functional. Incorporation of radioactivity into cholesterol is lo-fold higher compared

to that into UQ, indicating

a higher rate

Ubiquinone biosynthesis in Leishmania Table

28.1

major

5--Incorporation of [1-‘4c] acetate and [U-14C] p-hydroxybenzoic benzoquinone ring and isoprene side chain of ubiquinone Expt

Tracer

Total d.p.m. in UQ

% Incorporated in the ring

% Incorporated in side chain

60,800 58,700

65.0 80.0 28.6 17.8

14.8 14.0 68.6 57.0

[U-14C] p-Hydroxybenzoic acid (5 ,uCi) 1 2

[ 1-14C] acetate (10 &i)

acid into

1

1500 1400

2

The cells (50 ml) were grown for 48 h. The tracers were added and the incubation was continued for 24 h. The cells were harvested and ubiquinone was isolated. The ubiquinone molecule was derivatized and cleaved as described in Methods. Table

h--Effect

of p-hydroxybenzoic acid and shikimate incorporation into UQ

Addition f4C] Glucose

Expt

d.p.m. in UQ

1

1050

[14C]Glucose + p-hydroxybenzoic acid 1 2

[14C] Glucose + shikimic acid

1 2

998 700 706 749 695

on (U-14C)

d.p.m./g wet weight 32500 30464 24898 21780 21648 22800

glucose

% dilution

28.5 29.5

Cells (50 ml) were grown for 48 h and 7,~ Ci of [U-14C) glucose was added to the growth medium. Cells were harvested 24 h later, lipids were extracted and UQ was separated by thin layer chromatouraohv. Where indicated, 5 mM shikimate or 5 mM p-hydroxybenzoic acid were added aloigw;h [U-‘4C] glucose:

of synthesisof cholesterol.Two important enzymes of the acetate-mevalonate pathway, namely HMG-CoA reductaseand mevalonatekinase, are also active in cell free extracts, which further substantiatesthe participation of the acetate-mevalonate pathway for isoprenoid biosynthesis. HMG-CoA reductaseactivity is higher in the earlier stagesof growth in L. major. Enzyme activity is highest between 24 and 48 h but UQ levels are highestaround 60 h of cell growth. It is possiblethat HMG-CoA reductaseis a rate limiting enzyme in UQ biosynthesis and that side chain precursor synthesisis high just before UQ levels increase. Variation in UQ levelsin different stagesof growth were observed in Tetrahymena pyreformis (Crane, 1962). Aromatic amino acids, tyrosine and phenylalanine, are incorporated into UQ in higher animals which have lost the capacity to synthesize the aromatic ring. Theseamino acids act as precursors for the benzoquinone moiety (Olson, 19651.The present study showsthat [i4C]-tyrosine and [ 4C] phydroxybenzoic acid are incorporated into the UQ moleculein L. major. Incorporation of tyrosine into the UQ moleculecould be through p-hydroxybenzoic acid. There might alsobe an alternate pathway

available for aromatic:ring synthesisthrough shikimate. The shikimatepathway is functional in bacterial cells (Cox & Gibson, 1964, 1966).The dilution of [‘4C]-glucoseincorporation into UQ in the presence of 5 m&l shikimateand 5 mM p-hydroxybenzoic acid indicates that both pathways may participate in ring synthesisin L. major. High levels of incorporation of phydroxybenzoic acid into UQ of which 80% is distributed in the beazoquinone moiety indicate that p-hydroxybenzoic acid is an immediate precursor of the UQ ring in L. majvr. p-Hydroxybenzoic acid has been shown to be an important intermediatein UQ ring synthesisin all systemsthat have beenstudied (Raman, Rudney 6t Buzzeli, 1969).The distribution of radioactivity from [14C]-acetatein the UQ moleculeis about 65% in the isoprenoidpart of the molecule.About 20% of the radioactivity is found associatedwith the ring. Similar observationshave been made in bacterial systems (Threfall &: Glover, 1962) L. major promastigotesseemto be capable of synthesizing UQ through pathways which overlap between procaryotic and eucaryotic systems;isoprene side chain biosynthesisfollows the acetate-mevalonate pathway typical of mammaliancells while the ring may be synthesized from aromatic amino acids

284

G. Ranganathan and A. J. Mukkada

(mammaliancells)or through the acetate-shikimate pathway (procaryotes). Ubiquinone is an essentialredox componentof the respiratory chain in eucaryotes.It is believedto play a major role as a mobile carrier of electonsfrom NADH and succinate.This study further corroborates its place within the electron transport chain in Leishmania which we reported earlier (Martin 8~ Mukkada, 1979a,b). REFERENCES

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