Vl. 46, N. 6, December ]998 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL Pages 1219-1225 ROLE OF REDUCING CO-FACTOR IN CERULENIN-INSENSIT1VITY OF 6-HYDROXryMELLEIN SYNTHASE IN CARROT CELL EXTRACT Fumiya Kursaki, Kusuke Tgashi and Munehisa Arisawa Faculty f Pharmaceutical Sciences, Tyama Medical and Pharmaceutical University, Sugitani, Tyama 930-0194, Japan Received September 24, 1998 Summary - The activity f 6-hydrxymellein synthase, a multifunctinal plyketide bisynthetic enzyme f carrt, was nt inhibited by cerulenin in the presence f NADPH. Hwever, cerulenin shwed a marked inhibitry activity t the synthase if the reducing c-factr was mitted frm the assay mixture. The synthase was als sensitive t the antibitic even in the presence f NADPH when the acyl cndensatin site and the reducing dmain at the reactin center f the enzyme were dissciated under the high inic strength cnditin, In additin, the synthase activity was appreciably inhibited when NADH was emplyed instead f NADPH. These bservatins strngly suggest that a phsphate grup attached t 2'-psitin f adensyl miety f NADPH mlecule plays an imprtant rle in the apparent insensitivity f 6-hydrxymellein synthase tward cerulenin. Key Wrds - Daucus carm (carrt); cerulenin; plyketide bisynthetic enzyme; insensitivity; reducing c-factrs; reactin center. Intrductil~ Cerulenin (2R,3S-2,3-epxy-4-x-7,10-trans, trans-ddecadienylamide, Fig. 1, a, 1) is an antibitic prduced by Cephalsprium caerulens, and exhibits ptent inhibitry activity tward fatty acid synthases (FASs) belnging t bth type I (multifunctinal enzyme) and type II (rganized by separable cmpnents) [1]. It is als well knwn [2-4] that cerulenin inhibits the catalytic activities f many plyketide bisynthetic enzymes (PKSs) f varius surces, and a similar inhibitry mechanism f this antibitic is assumed fr these tw related enzymes, FASs and PKSs. Cerulenin frms a lactam ring in the prtic slvents (Fig. 1, a), and the reactivity f the epxide grup is highly enhanced [5]. The epxide is cleaved t frm a cvalent bnd with a Cys-SH at the reactin center f the acyl-ca cndensatin enzyme f FASs and PKSs, and the transfer f acetyl miety frm its CA ester t the SH grup is blcked t cause the irreversible inhibitin f the activities f these synthases [6]. 6-Hydrxymellein (6HM, Fig. 1, b, 2) is a direct precursr f 6-methxymellein, an antimicrbial cmpund prduced by fungi-infected carrt cells as a defense substance [7], and the enzyme catalyzing the frmatin f the cmpund, 6HM synthase, was fund t be a multifunctinal PKS [7, 8]. This synthase catalyzes the cndensatin f acetyl- and malnyl-cas (Fig. 1, b), and an NADPH-dependent ketreductin f the carbnyl grup takes place at the triketide intermediate stage t frm the partially reduced ketmethylene chain [7]. Recently, we fund that 6HM synthase is catalytically active nly in the dimeric frm [9], and tw subunits with multifunctinal prperties are aligned in an antiparallel 1219 10394712/98/181219-07505.00/0 Cpyright 9 1998 by Academic Press Australia. All rights [ reprductin in any./~rm reserved.
Vl. 46, N. 6, 1998 BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL directin t frm tw cmplete reactin centers (Fig. 1, c). Therefre, when the dimer enzyme is dissciated t mnmer subunits under the high inic strength cnditins [9], the ketreducing reactin f the enzyme-bund tfiketmethylene chain des nt take place and triacetic acid lactne (TAL, Fig. 1, b, 3) is liberated as the derailment prduct instead f 6HM. This abnrmal reactin was als bserved in the dimer enzyme when NADPH was mitted frm the assay mixture [7]. We reprted previusly [10] that the activity f 6HM synthase is nt inhibited in the presence f even millimlar levels f cerulenin, and, t ur knwledge, this was the first reprt which demnstrated the ccurrence f cerulenin-insensitive PKS. The mechanism f cemlenininsensitivity f 6HM synthase was extensively studied, and we fund [11, 12] that cemlenin is capable f frming a cvalent bnd with Cys-SH at the reactin center f 6HM synthase as well as FASs and ther PKSs. Unlike these enzymes, hwever, the Cys-cemlenin bnd f 6HM synthase is unstable and the synthase-bund cerulenin is readily replaced by the substrate f the enzyme, thus, the catalytic reactin apparently takes place withut inhibitin. In the present experiments, we shw the evidence that a phsphate grup attached t 2'-psitin f adensyl miety f NADPH mlecule (2'P) plays an imprtant rle in the apparent insensitivity f 6HM synthase tward cerulenin. Methds Chemicals. Cerulenin, 2-chlrethylphsphnic acid, acetyl-ca, malnyl-ca, NADPH and NADH were purchased frm Sigma. Dehydracetic acid was btained frm Nacalai Tesque while dithithreitl was frm Wak. 6HM was prepared by demethylating 6-methxymellein islated frm fungi-infected carrt rts with BBr 3 as described previusly in detail [7] TAL was synthesized frm dehydracetic acid accrding t the methd described previusly [7]. [2-14C]malnyl-CA (specific activity 2.2 GBq/mml) were btained frm New England Nuclear. All ther chemicals were reagent grade. Inductin and partial purificatin f 6HM synthase. 6HM synthase was induced in carrt rt tissues by the treatment with 2-chlrethylphsphnic acid. Carrt rts purchased frm a lcal market (200 g) were sliced in 2 mm thickness, and 2-chlrethylphsphnic acid slutin (ttal 5 ml, 10 mg/ml in 10 mm K-phsphate buffer, ph 7.5 ) was pured nt the surfaces f the slices. They were incubated at 25~ fr 3 days in rder t induce 6HM synthase activity. After the inductin, the synthase prtein was highly purified accrding t the methds described previusly in detail [9, 10], hwever, the chrmatfcusing step was mitted in the present experiments. Purity f the enzyme preparatin was assessed by sdium ddecyl sulfate plyacrylamide gel electrphresis, and the results were reprted previusly [9, 10]. Prtein cncentratins were determined by the methd f Bradfrd [16]. Assay f 6HM synthase activity. The standard assay mixture f 6HM synthase activity cnsisted f 10 mm K-phsphate (ph 7.5), apprximately 4 pkat f the synthase, 0.1 mm acetyl- CA, 0.1 mm [14C]malnyl-CA (3.7 kbq), 1 mm NADPH, 2 mm dithithreitl and varius cncentratins f cerulenin in a ttal vlume f 100 ~tl. Prir t the initiatin f the enzyme reactin, 6HM synthase prtein was incubated with either 0.1 mm f acetyl-ca r varius cncentratins f cemlenin at 37~ fr 5 min t frm the enzyme-substrate r the enzyme-inhibitr cmplex. The enzymatic reactin was run at 37~ fr 1 h, and the prducts were extracted with 200 ~tl f ethylacetate. The radiactivities incrprated int 6HM r TAL were determined after the separatin f the prducts by TLC n a silica gel plate [7, 9]. In sme experiment, the dimeric frm f 6HM synthase was dissciated t mnmer subunits by the treatment with 2 M 1220
O HO 0 ~ V V ~ ~ Fig. 1. a: Structure f cemlenin. Cemlenin (1) frms a lactam ring in the prtic slvents and the epxide is cleaved t frm a cvalent bnd with Cys-SH at the reactin center f cndensatin enzyme f FASs and PKSs. b: Bisynthetic sequence f 6I-IM (2) by catalytic reactins f 6HM synthase. 6HM synthase catalyzes the cndensatin f acetyl- and malnyl-cas, and an NADPH-dependent ketreductin takes place at the triketide stage. When NADPH is mitted r the dimer enzyme is dissciated t the mnmer subunits, this reductin des nt ccur, and TAL (3) is liberated instead f 6HM. c: Schematic presentatin f dimeric frm f 6HM synthase. Tw subunits f 6HM synthase with catalytic dmains f acyl cndensatin and ketreductin are aligned in an antiparalm directin t frm tw reactin centers. 2 M P ITI --4 -< C~ w" r- 1> -4 rl'l z r'- a C Cndensatin Ketreductln 1 140 H N O P ~ ~ Ketreductln Cndensatin b Acetyl-CA 4- MalnyI-CA 0 0 0 0 ~.- ~ S. E n z S-Enz NADPH S-Enz O HO H MalnyI-CA 3 O O O O OH O r MaI~176~S'Enz ~O
Vl. 46, N. 6, 1998 BIOCHEMISTRYand MOLECULAR BIOLOGy INTERNATIONAL NaC1 (final cncentratin) fr 5 min at 37~ [9] prir t the frmatin f the cmplexes with acetyl-ca and cerulenin. In this series f experiments, the assay was carried ut in the presence f the same cncentratin f NaC1. Results In animal FAS, it has been shwn [13, 14] that cerulenin is nt able t bind t Cys-SH at the reactin center when the enzyme prtein was preincubated with acetyl-ca, a 'cmpetitr' f the antibitic. 9 Therefre, in the present study, inhibitry activity f cerulenin against 6HM synthase was systematically examined under varius reactin cnditins in rder t characterize the insensitivity f the synthase t the cmpund. 6HM synthase prtein was incubated with either cemlenin r acetyl-ca prir t the additin f the ther assay cmpnents, and the effect f the pretreatments n the enzyme activity was examined (Fig. 2). It appeared that the NADPHdependent reactin f 6HM synthase was nt inhibited in the presence f I mm f cerulenin when the enzyme was previusly treated with aeetyl-ca (Fig. 2, a) as is in FASs [13, 14]. Hwever, the synthase was als insensitive t cerulenin even if the cvalent attachment f the antibitic t the synthase was frmed by the preincubatin f the enzyme with the cmpund prir t the initiatin f the catalytic reactin [12]. These results, tgether with ur previus bservatins [12], suggest that the cvalent bnd frmed between cerulenin and Cys-SH f the cndensatin enzyme f the synthase is readily cleaved by the additin f acetyl-ca in the NADPH-m~.diated 6HM frmatin by the enzyme. In cntrast, a different mde f actin f cerulenin was bserved in the NADPH-depleted reactin f 6HM synthase in which TAL was liberated as the derailment prduct instead f 6HM (Fig. 2, b). As is in FASs and ther PKSs, 6HM synthase activity was appreciably inhibited in the presence f 10 btm f cerulenin upn the preincubatinn f the enzyme prtein with this antibitic, and the residual activity decreased with the increase in the cemlenin cncentratin. Hwever, the inhibitry effect f cemlenin was essentially nt bserved after the pretreatment f the enzyme with acetyl-ca. A similar set f results was btained in the reactin catalyzed by the mnmer subunits f the synthase in which it is expected that the acyl cndensatin site is nt able t interact with the NADPH-assciated ketreducing dmain. (Fig. 2, c). The catalytic activity f the mnmer synthase was strngly inhibited even in the presence f NADPH when cerulenin was previusly incubated with the synthase prtein t frm the cerulenin-enzyme cmplex. Hwever, the synthase preincubated with acetyl-ca did nt shw the sensitivity t the antibitic. It is very likely, therefre, that the assciatin f NADPH mlecule with the ketreducing dmain in the dimeric frm f 6HM synthase is essential fr the insensitivity f the enzyme tward cerulenin. We reprted previusly [15] that the ketreducing reactin f 6HM synthase-bund triketmethytene intermediate takes place even if NADPH is replaced by NADH thugh the synthetic rate f 6HM is significantly decreased. Recently, we have fund [15] that a phsphate 1222
1~ 8 9 J l 9 9 9 0k' ',,.lt '. I 0 0.01 0.1 1 0 0.01 0.1 1 Fig. 2. Effect f cemlenin n varius 6HM synthase-catalyzing reactins. 6HM synthase prtein was preincubated with 0.1 mm f acetyl-ca (O) r cemlenin (C)) at the cncentratins indicated n the hrizntal axis, and then, the assay was carried ut in the presence f the antibitic at the respective cncenl~atins. Results were expressed as percentages in which the cntrl values btained withut cerulenin were taken as 100%. a: The assay f 6HM synthase was carried ut in the presence f NADPH. b: NADPH was mitted frm the assay mixture, c: NADPHassciated 6HM synthase was dissciated t the mnmer subunits, d: NADH was emplyed as the reducing c-factr instead f NADPH. P O P CO ID m -tin 3= -,-t r'- rt'l e-" r-" r'- Q..< i m r-- 0 0 9 t,,,, ~ I L~ O ~ = I 9 9 9 I Jt 40 co S 20 a ~ @,,, II,,, 120r -- -- -- ~" -- -- 40 Cerulenin cncentratin (mm)
Vl. 46, N. 6, 1998 BIOCHEMISTRYnd MOLECULAR BIOLOGY INTERNATIONAL residue in NADPH structure, 2'P, alters the micrenvirunmental circumstance arund the reactin center f the synthase prtein, and this structural change is respnsible fr the different reactin rates f NADPH- and NADH-dependent 6HM frmatin. Therefre, in the next experiment, NADH was emplyed instead f NADPH, and pssible difference between these tw reducing cfactrs in the sensitivity f 6HM synthase t cerulenin was examined (Fig. 2, d). Unlike the NADPH-mediated 6HM frmatin, the catalytic reactin f NADH-assciated 6HM synthase was markedly inhibited in a dse-dependent manner when the synthase prtein was preincubated with cerulenin t frm the enzyme-inhibitr cmplex. Hwever, as was in the ther series f the experiments in the present study, the synthase did nt shw the sensitivity t the antibitic upn the pretreatment with acetyl-ca. These bservatins strngly suggest that a phsphate grup in NADPH structure, 2'P, is an imprtant cmpnent in the apparent cerulenin-insensitivity f 6HM synthase. Discussin The results btained in the present experiments clearly indicate that the reducing c-factr assciated with the ketreducing dmain in the dimeric frm f 6HM synthase plays an imprtant rle in the apparent insensitivity f the enzyme against cerulenin (Fig. 2, a, b, c). In additin, a marked difference in the inhibitry mde f cerulenin was bserved between the NADPH- and NADH-assciated enzymes (Fig. 2, a, d). The NADPH-assciated synthase preincubated with either cerulenin r acetyl-ca was insensitive t the antibitic while the activity f the NADHenzyme was appreciably inhibited by the pretreatment with cerulenin. Therefre, it is very likely that the structural difference between NADPH and NADH mlecules, 2'P, is a key cmpnent in the apparent insensitivity f 6HM synthase t the antibitic. The phsphate miety wuld interact with a certain structure at the reactin center f cerulenin-6hm synthase cmplex, and the structural perturbatin results in the decrease in the stability f Cys-cerulenin attachment. This 2'-P-induced alternatin wuld allw the rapid replacement f the enzymebund cerulenin with acetyl-ca, therefre, the catalytic reactin f 6HM synthase takes place withut apparent inhibitin when NADPH is emplyed as the reducing c-factr. We have recently reprted [15] that the assciatin f NADPH mlecule with the ketreducing dmain f 6HM synthase results in the increase in the affinity f the enzyme t ne f the c-substrates, acetyl-ca. This alternatin was specifically bserved fr NADPH and culd nt replaced by NADH, therefre, 2'P f NADPH was implicated as an essential structure fr this change in the affinity f the synthase prtein t acetyl-ca [15]. The results btained in the present experiments demnstrate anther functin f 2'P f NADPH assciated with the ketreducing dmain f 6HM synthase in the bichemical prperties f this multifunctinal enzyme. Further elucidatin f the c-factr-induced alternatin f the characteristics f 6H~I synthase is in prgress in ur labratry. 1224
Vl. 46, N. 6, 1998 BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL References 1. Omura, S. (1981) in Methds in Enzymlgy (Lwenstein, J. M.Ed.) Vl. 72, pp. 520-532, Academic Press, New Yrk. 2. Kreuzaler, F. and Hahlbrck, K. (1975) Eur. J. Bichem. 56, 205-213. 3. Omura, S. and Takashirna, H. (1974) J. Bichem. (Tky) 75, 193-195. 4. Ohn, H., Ohn, T., Awaya, J. and Omura, S. (1974) J. Bichem. (Tky) 78, 1149-1152. 5. Funabayashi, H., Iwasaki, S., Okuda, S. and Omura, S. (1983) Tetrahedrn Lett. 24, 2673-2676. 6. Funabayashi, H., Kawaguchi, A., Tmda, H., Omura, S., Okuda, S. and Iwasaki, S. (1989) J. Bichem. (Tky) 105,751-755. 7. Kursaki, F., Kizawa, Y. and Nishi, A. (1989) Eur. J. Bichem. 185, 85-89. 8. Kursaki, F., Ith, M., Yamada, M. and Nishi, A. (1991) FEBS Lett. 288, 219-221. 9. Kursaki, F. (1995) Arch. Bichem. Biphys. 321,239-244. 10. Kursaki, F., Ith, M., Kizawa, Y. and Nishi, A. (1993) Arch. Bichem. Biphys. 300, 157-163. 11. Kursaki, F., Ith, M. and Nishi, A. (1993) Anal. Bichem. 210, 418-419. 12. Kursaki, F., Ith, M. and Nishi, A. (1994) Phytchemistry 35,297-299. 13. Rberts, G. and Leadlay, P. F. (1983) FEBS Lett. 159, 13-16. 14. Kawaguchi, A., Tmda, H., Nze, S., Omura, S. and Okuda, S. (1982) J. Bichem. (Tky) 92, 7-12. 15. Kursaki, F. (1998) Bichim. Biphys. Acta 1363, 6-10. 16. Bradfrd, M. M. (1976) Anal. Bichem. 72, 248-254. 1225