Katsuki, Hirohiko

Nishino, Tokuzo

Suzuki, Nobuhiro

Takatsuji, Hiroshi

English

Kyoto University Research Information Repository

RIGHT:URL:CITATION:AUTHOR(S):ISSUE DATE:TITLE:Formation of Dehydrosqualene inMicrosomal Fraction ofRhodotorula GlutinisNishino, Tokuzo; Suzuki, Nobuhiro; Takatsuji,Hiroshi; Katsuki, HirohikoNishino, Tokuzo ...[et al]. Formation of Dehydrosqualene in Microsomal Fraction ofRhodotorula Glutinis. Memoirs of the Faculty of Science, Kyoto University. Series ofphysics, astrophysics, geophysics and chemistry 1981, 36(1): 67-721981-12http://hdl.handle.net/2433/257577Meraolrs of the Faculty of Science, Kyoto University, Series oÅí Physics, Astrophysics,        Geophysics and Chemistry, Vol. XXXVI, No. 1, Article 3, l981FORMATION OF DEHYDROSQUALENE IN       MICROSOMAL FRACTION OF          RHODOTORULA GLUTII?VIS                         ByTekuze NISKINO, Nebuhiro SUZVKI, Hiroshi TAKATSUjl                        aBd                 Hirohiko XATSUKIDepartment of Chemistry, Factilty of Science, Kyoto University, Kyoto                 (Received March 6, I979)                           SUMMARY   When the microsomal fraction obtained from Rhodotoruta glutinis was incubatedwith [i`C]farnesyl pyrophosphate, i`C-incorporation iRto dehydrosqualene was dernon-strated. The incubation in the presence of NADPH gave squalene instead of dehydro-squalene. When the microsopaal fraction was incubated with ['`C]geranylgeranyipyrophosphate, in contrast, radioactivity was incorporated neither into phytoeRe normto carotene.   Dehydrosqualene, in the pathway for C3e-diapocarotene biosynthesis, is a compoundwhich is beJieved to occ"py a position corresponding to that of physoene in the pathwayÅíor carotene biQsynthesis. The fact that dehydrosqualene, not phytoene, was formed inthe reaction using microsomal fraction of this yeast suggested that dehydrosqualeneformeation was catalyzed by the enzyme for squalene synthesis similarly to the case ofSaeeharoniyces cereyisiae, bvt not by that for phytoene syntlaesis.                            INTRODUCTION    We reported that dehydrosqualene was formed when the microsomal fractionobtained frorn Saccharomyces cerevisi.ae was incubated with farnesyl pyrophosphate.When the reaction was carried out in the presence of NADPH, only squalene wasformed instead of dehydrosquaiene. These results suggested that dehydrosqualenewas formed being catalyzed by the enzyme for squalene synthesis (1).    Dehydrosqualene, in the pathway for C3e-diapocarotene synthesis, is a com-pound which occupies the position corresponding to that of phytoene in the pathwayfor carotene synthesis. Before the publication of our report (1), the compound hadbeen found only from three species of bacteria, Staphylococcus aureus (2), Halo-bacterium cutirubrum (3) and Streptoeoccus faecium (4) which all belong to pro•-caryote and had been reporte(l to contain caroteRes (3, 5, 6). With celi-free extractsof Staph. aureus, formation of dehydrosqualene from mevalonate was reported (7).After that, the presence of C3o-diapocarotene was reported from Strep. faecium (4, 8)and Staph. auretts (9). Although the possibility that dehydrosqualene fuRctions asa precursor of C3o-diapocarotenes were pointed out, no demonstration has been made.As aiiother possibility, if these bacteria do contain C4o-carotenes and if phytoenesynthetase in the bacteria has a low substrate specificity, dehydrosqualene may beformed from farnesyl pyrophosphate being catalyzed by the enzyme.68 T. NISKINO, N. SUZUKI, K. TAKATSUJI and H. KATSUKI    As for sterol synthesis of bacteria, in contrast, no sterol has been demonstratedfrom most of bacteria and squalene also had been believed not to be present untilabout ten years ago. However, the presence of squalene was net the case and thecompound was found in some bacteria including the abeve three bacteria (3, 4, le),Especially with Staph. aureus, the incorporation of radioactivity from [i4C]farnesylpyrophosphate into squaiene was reported (IO). This suggests the possibility thatdehydrosqualefie formation is concerned with squalene synthesis even in the bacteriawhich produce carotenes.    From these coiisiderations, it is of importance to study the mechanism for de-hydrosqualene formatioR in the organism producing carotenes. For this purpose,the microsornal fraction was prepared from RJtodotorula glutinis and it was left toreact with [i4C]farnesyl pyrophosphate, [i`C]presqualene pyrophosphate or[i4C]gerafiylgeranyl pyrophosphate. As a result of experiments, dehydrosqualenewas fouRd to be formed fi'oma the former two compounds but phytoene was not formedfrom the latter compound. This strongly suggested that dehydrosqualene formationwas coficerned with squalene synthesis in R. glutinis as well as in S. cerevi.siae (1),                     MATERIALS AND METHODS    [i4C]Famesyl pyrophosphate and [i4C]presqualene pyrophosphate (specificradioactivities, 66 and 340 Cilmol, respectively) were prepared as reported previously(1). [i`C]Geranylgeranyl pyrophosphate (specific radioactivity, 114Cifmol) wasprepared with some modifications according to the method of Ogura et al. (11).Rhodotorttla glutinis IFe 0389 was grown iR a medium cofitaining 20/. glycerol asa sole carbon seurce for 2e h at 280C (12). The harvested cells were disrupted andthe microsomal fraction was prepared as described previously (1).   The mixture for Åíhe enzyme reaction contained, in a final volume of 1.e ml,2 pmnol of MnCl2 (or 5 ptmol of MgCl2), O.1 ptmol of dithiothreitol, 20 "mol of KF,30 "mol of potassium phosphate buffer (pH 6.8), the microsomal fraction, and theradioactive substrate described above and additions as indicated. The reaction wascarried out in the presence aBd absence of NADPH (2 "mol) for 1 h at 300C.   After the reaction, the mixture was heaÅíed with 2 N KOH for 1 h at 600Cand nonpolar Iipicls were extracted with petroIeum ether. The radioactive nonpolarlipids were separated by two kinds of TLC as described previously (1) and cotmtedfor their radioactivities. Normal-phase TLC was carried out on silica gel usingbenzene-ethyl acetate (4:1, V!V) as a solvent system. Reversed-phase TLC wascarried out on Kieselguhr G plate impregnated with liquid paraMn using 900/. aqueousacetone saturated with liquid paraMn,                              RESUL[IrSIdeRtification ef Deliydrosqualene Produeed from Farnesyl Pyrepkosphate    The microsomal fraction obtained from R. glutinis was incubated with [i4C]-farnesyl pyrophosphate (15S,OOO dpm) akd Mn2" for 2 h at 30eC. After the reaction,the mixture was treated with alkali and nonpolar lipids were extracted with petroleumether. Analysis of the lipids (28,OOO dpm) by normal-phase TLC showed that aboutone-fourth the radioactivities was located in the fraction corresponding to squalene.Further analysis of the radioactive lipid eluted from the fraction by reversed-phaseFORMATION OF DEHYDROSQUALENE IN MICROSOMAL FRACTION69TLC showed that radioactivity was in the fraction corresponding to dehydrosqualene,not to squalefie. The radioactive lipid was mixed with the authentic sample of eis-dehydrosqualene isolated from Staph. aureus (2) and the mixture was subjected tothe column chromatography on alumina reported previously (1). The elution patternof radioactivity accorded completely with that of absorbance at 285 nm where theabsorption maximum of cis-dehydrosqualene exists. For identification of the struc-ture of radioactive lipid, a part of the mixture, after irradiated under UV light inn-hexane contaiRing iodine for cis-trans isomerization (2, 13), was subjected to thealurnina chromatography sifr}ilarly as described above. Figure 1 shows coincidenceof elution pattern of radioactivity with that of absorbance at 285 nm. Two peaksappeared-one (correspoRding to ci.s-dehydrosqualene) eluted with O.70/. ether inn-hexane and the other (corresponding to trans-dehydrosqualene) eluted with l.50/.ether in n-hexane (13, 14). From these rest}lts, the radioactive lipid obtained byincubation of tke microsomal fraction with [i4C]farnesyl pyrophosphate aRd Mn2"was identified as cis-dehydrosqualene.Percent of Diethyl Ether in n--HexaneO.075      50      o.o    5    2     .o    oii'"--- O.6 ---e- -eq O.7 -- l.5 . --Nt------- l.6 -o           5 10 35 20 25 30                  Fraction NumberCoincidence of elution pattern of radioactivity with that of absorbance due totrans-dehydrosqualeRes in alumina chromatography. The microsomalof protein) was lncubatecl with Ii`C]farnesyl pyropkosphate (l.1 nmol,and 2mM MnCi2 (final volume, 1 ml) for 2h at 30eC. Nonpolar lipidsextracted with petroleum ether from the reaction mixture after alkaliseparated by normal-phase TLC 'iki.-Fig, l.o                                                         cis- and                                                   frac i n (1 mg                                                     155,OOO dpm)                                                  which were                                                    treatment were                         Tke radioactive }ipid eluted from the fraction corre-sponding to dehydrosqualene was mixed wlth the autheRtic sample of cis-dehydrosqttaleReisolated from Staph auretts aBd the mixture was dissolved in rt-hexane containing iodine(about 2 % of dehydrosqualene in weight) aftd was irradiated under UV light to aliow thecis-trans isomerization. The mixture, after treatmeRt with sodium thiosulfate to reducethe iodine, was subjected to the colurnn chromaatography on alumina. EIution wasmade usiRg n-hexaRe containing ether by jncreasing the concentration of ether stepwiselyand radioactivity and absorbance at 285 nm were monitored.70 T. NISHINO, N. SUZUKI, H. TAKATSUJI and H. KATSUKIEffects of IN({n2+ and NADPH on Dehydrosqua}ene Fermation from FarnesylPyrophosphate    Table I shows effects of Mn2", Mg2+ and NADPH on i4C-incorporation from[i4C]famesyl pyrophosphate (24,OOO dpm) into nonpolar lipids. Total radioactivi-ties incorporated into nonpolar lipids after reaction in the presence of MR2+ were4,05e dpm. Almost 300/, of the radioactivity was found in dehydrosqualeAe and theremainder in other lipids mostly made up of farnesol and the coinpound whicla seemedto be presqua]efie alcohol. Replacement of Mn2" with Mg2" markedly decreasedthe iiacorporation into dehydrosqualene, indicating the effectiveness of Mn2" fordehydrosqualene formation similarly to the case of S. cerevisiae. When the reactionwas canied out in the presence of NADPH and Mn2', radioactivity was incorporatedonly into squalene, not into dehydrosqualene. Separate experimeis sliowed that theformation of squalene was not dependent on the kind of divalent metal iens. It isevident that the formation of dehydrosqualene was an enzymic reaction since replace-ment of the eRzyme with the boiled enzyme in the reaction did not give radioactiveproducts.Table 1. i4C-Incorporation from [i'iC]farnesyl phyrophosphate into nonpolarIipids. The reaction mixture (final volume, 1 ml) contained li`C]-farnesyl pyrophosphate (O.17nmoi, 24,OOOdpm), O.l ptmel ofdithi-othreitol, 20 ptmol of KF, 30 ptmol ef potassium phosphate buffer(pH6.8) and indicated additions (21xmol of MnC12, 5pmol ofMgC12, 2 ptmol of NADPH, the enzyme (O.5 mg ef protein or heat-inactivatedenzyme). The reaction was carried out for 1}i at 300Cand, after alkali treatment, nonpolar lipids were extracted. They wereseparated by normat-phase and reversed-pftase TLC. The fractionscorresponding to dehydrosqualene, farnesol, presqualene alcoholand squalene were scraped from the plates and radioactivity in everyfraction was counted.Additions     Radioactivity of Products (dpm)Total ?8L'Y.I,r.O.- others Squalenel Enzyme, MR2+Enzyme, Mga+Enzyrae, Mna+, NADPHBoiled enzyfne, Mn2+4,0503,4007,200 300l,150 220  50  202,9003,200 200 250g  o  8  6i4C-Incorporatien from [i4C] Presqualene ?yrophesphate inte Nenpolar Lipids    When the reactions were carried out using [i4C]presqualene pyrophosphate(O.04nmol, 30,OOOdpm) instead of [i4C]farnesyl pyrophosphate as a substrate inthe presence and absence of NADPH, radioactivities were incorporated inte squaleneand del3ydrosqualene, respectively. Also in this case, Mn2" was much more effectivethan Mg2" for the dehydrosqualene formation. Differently from the experimentusing [i4C]farnesyl pyropliesphate, a compound which seemed to be presqualenealcohol was formed, instead of the formation of farnesol, besides dehydrosqualene inthe incubation without NADPHLFORMATION OF DEHYDROSQUALENE IN MICROSOMAL FRAenON71'`C-incorporation from [i`C]Geranylgeranyl Pyrophosphate inte Nonpelar Lipids   When similar reaction was carried out using [i4C]geranylgeranyl pyrophosphate(O.11 nmol, 28,OOO dpm) instead of ['`C]farnesyl pyrophosphate, very little radio-activity, almost equal to that in control experiment using boiled enzyme, was in-corporated into nonpolar lipids. Separation of the lipids by normal-phase TLCshowed that yery little activities were found in geranylgeraniol, geranyllinalool andunknown compound which were thougltt to be contaminants in the sttbstrate. Noradioactivity was incorporated into phytoene and lycoperseBe. Similar pattern wasobserved in the control experiment. No variation was found in the pattern obtainedby the incubatioR with NADPH. These results iRdicate that no formation of C4o-compound corresponding to squalene in C3o-series was found in the microsomalfraction of R. glutinis, though squalene or dehydrosqualene was formed dependingon the reaction condition. In this regard, Qureshi et al. reported that lycopersenewas formed by the catalysis of squalene synthetase in S. cerevisiae (15). This possi-bility seemed to be excluded by our present study. Our present studies stronglysuggest that dehydrosqualene is not formed by the catalysis of phytoene synthetaseowing to its low substrate specincity but the formation is closely related to the squalenesynthetase reaction.                            DISCUSSION    Some reports appeared oia the demofistration of C4o-carotenes in R. glutinis,such as the finding of 6-carotene, 7-carotene, torulene and torularhodin (12, 16, 17),and the demonstration of t4C-incorporation from [i4C]mevalonate into torularhodin(18). As for the formation of lycopersene, a coinpound related to carotene bio-synthesis, Qureshi et al. reported that squalene synthetase catalyzed a forfnation oflycopersene from geranylgeranyl pyrophosphate in S. cerevisiae (15). However,Ogura et al. studied on the substrate specificity of squalene synthetase using themicrosomal fraction of pig liver and reported that the enzyme had a high substratespecificity: the product with the longest carbon chain was bis-homo-squalene, andtetra-homo-squalene was not formed (l9). Our present studies also showed that themicrosomal fraction of R. glt{tinis catalyzed the formation of squalene and dehydro-squalene dependiRg on the reaction condition but llot the formation of phytoene orC4o-compound frorn geranylgeranyl pyrophosphate. This suggests tliat the dehydro-squalene formation was not catalyzed by phytoene synthetase owing to its low sub-strate specificity and that the C4o-compound synthesis was not catalyzed by squaleiaesynthetase. Studies on the intracellular distribution of phytoene synthetase are nowm progress.    Frofn the three species of bacteria which produce dehydrosqualene-Staph.aureus (2), H. cutirubrum (3), aRd Strep. faecium (4) -, squalene was demonstrated(3, 4, le). These facts suggest that dehydrosqualene is formed iR these bacteria in asimilar mechanism to that in S. cerevisiae afid R, glutinis. However, the possibilitythat a distinct enzyme from squalene synthetase is involved in the dehydrosqualeneformation and in the succeeding synthesis of C3o-diapocarotene can not be excluded,since many kinds of C3o-diapocarotenes were found in Strep. faeeium (4, 8, 20, 2D,From consideration of the squalene distributioB in some bacteria, its low content andof its unrevealed role in cellular metabolism, it can not be ruled out the possibility72 T. NISHINO, N. SUZUKI, H. TAKATSUjl and H. KATSUKIthat squalene is formed secondarily being catalyzed by "dehydrosqualene synthetase"in the above described bacteria. The mechanism for dehydrosqualene formation inthese bacteria are necessary to be elucidated.   The authors are grateful to Prof. K. Soda of Institttte for Chemical Research, Kyoto University,for klnd gift of R. gttttinis. This work was supported in part by research grants from the Ministryof Education, Science and Culture of Japan.il,i}gi}il)6l\l                         REFERENCESNishino, T., Takatsuji, H., Hata, S., & Katsuki, H,, (1978) Biochem. Biophys. Res. Cotnmun.85, 867-874.Suzue, G,, Tsukada, K., & Tanaka, S., (i968) Biochim. Biophys. Aeta 164, 88-93.Kushwaha, S. C., Pugh, E. L,, Kraraer, J. K. G., & Kates, M., (1972) Biochim. Biophys. Acta26e, 492-506.Tayior, R. F. & Davies, B. H., (l974) Biochem. J. 139, 751-760.Suzue, G. (1959) J. Biochetn. (Tgkyo) 46, 1497-1504.Cosenza, B. J. & Girard, A. E., (l970) Baeteriol. Proc. p. 45.Suzue, G., (1962) J. Biochem. (Tokyo) 51, 246-252.Taylor, R. F. & Davies, B. K., (1974) Biochem, J. 139, 761-769.Shinoi, K., Katayama, K., Kayami, M. & Kanemasa, Y., (1977) Proc. Jptt, Coqf. BiochentLipids (Japan) 19, 11-13.Suzue, G., Tsukada, K., Nakai, C. & Tanaka, S., (i968) Ai"ch. Biochem. Biophys. 123, 644.Ogura, K., Shinka, T. & Seto, S., (1972) f. Biochem. 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Formation of Dehydrosqualene in Microsomal Fraction of Rhodotorula Glutinis

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Formation of Dehydrosqualene in Microsomal Fraction of Rhodotorula Glutinis

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