Protein farnesyltransferase inhibitors interfere with farnesyl diphosphate binding by rubber transferase Christopher J. D. Mau 1 , Sylvie Garneau 2 , Andrew A. Scholte 2 , Jennifer E. Van Fleet 1 , John C. Vederas 2 and Katrina Cornish 1 1 USDA, Agricultural Research Service, Western Regional Research Center, Albany, CA, USA; 2 Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada Rubber transferase, a cis-prenyltransferase, catalyzes the addition of thousands of isopentenyl diphosphate (IPP) molecules to an allylic diphosphate initiator, such as farnesyl diphosphate (FPP, 1), in the presence of a divalent metal cofactor. In an effort to characterize the catalytic site of rubber transferase, the effects of two types of protein farnesyltransferase inhibitors, several chaetomellic acid A analogs (2, 4–7)anda-hydroxyfarnesylphosphonic acid (3), on the ability of rubber transferase to add IPP to the allylic diphosphate initiator were determined. Both types of com- pounds inhibited the activity of rubber transferases from Hevea brasiliensis and Parthenium argentatum, but there were species–specific differences in the inhibition of rubber transferases by these compounds. Several shorter analogs of chaetomellic acid A did not inhibit rubber transferase activity, even though the analogs contained chemical features that are present in an elongating rubber molecule. These results indicate that the initiator-binding site in rubber transferase shares similar features to FPP binding sites in other enzymes. Keywords: Hevea brasiliensis; Parthenium argentatum; chaetomellic acid A; hydroxyfarnesylphosphonic acid. Rubber transferase catalyzes the biosynthesis of natural rubber [1]. To form this polymer of cis-polyisoprene, rubber transferase adds up to thousands of molecules of isopente- nyl diphosphate (IPP) to a single initiating allylic diphos- phate, usually considered to be farnesyl diphosphate (FPP, 1,Fig.1)asthein vivo substrate. However, rubber transferase can also use other allylic diphosphates as initiators; this substrate flexibility is probably a reflection on the manner in which the catalytic site deals with the elongating rubber polymer. In addition, a divalent metal cofactor, such as Mg 2+ , is required. In spite of the dependence of modern industrial society on natural rubber, the biochemical properties of rubber transferase are only partially understood [1–6]. Several compounds are known to bind to FPP sites in other enzymes that use FPP as a substrate. Most of these substances have been discovered as a result of oncogenesis studies involving protein farnesyltransferases. Chaetomel- lic acid A (2) (Fig. 1), made by Chaetomella acutiseta,isan inhibitor of protein farnesyltransferases (PFTs), such as those that modify Ras, and competes for the FPP binding site of PFTs with an IC 50 of 55 n M [7]. Derivatives of chaetomellic acid A have also been found to inhibit PFTs [8]. a-Hydroxyfarnesylphosphonic acid (HFPA, 3) (Fig. 1) is another compound shown to inhibit PFTs [9] with an IC 50 of 30 n M [7]. In an effort to characterize the FPP binding site of rubber transferase, we have tested the ability of chaetomellic acid A and several analogs, as well as HFPA, to inhibit rubber biosynthesis in vitro. We have used rubber transferases from Hevea brasiliensis and Parthenium argentatum to determine if there are similarities in enzymatic behavior that might be characteristic of rubber transferases in general, as well as species-specific differences. Materials and methods Chemicals Chemicals were purchased from Sigma Chemical Com- pany unless otherwise noted. Farnesyl diphosphate (FPP), dimethyl allyl diphosphate (DMAPP) and [1- 14 C]IPP (2.04 GBqÆmmol )1 ) were purchased from American Radiolabe- led Chemicals, Inc. (St. Louis, MO, USA). a-Hydroxy- farnesylphosphonic acid (HFPA) was purchased from Calbiochem-Novabiochem Corp. Washed rubber particles (WRP) from P.argentatum and H. brasiliensis were purified ([10,11], respectively) and stored in liquid nitrogen [12]. Synthesis of chaetomellic acid A analogs Several analogs of chaetomellic acid, purified as lithium salts, were made according to Ratemi et al.[8].The structures of (Z)-2-octyl-3-methylbutenedioic acid dilithium Correspondence to K. Cornish, Western Regional Research Center, USDA-ARS 800 Buchanan Street, Albany, CA 94710, USA. Fax: + 1 510 559 5663, Tel.: + 1 510 559 5950, E-mail: kcornish@pw.usda.gov Abbreviations: DMAPP, dimethyl allyl diphosphate; FPP, farnesyl diphosphate; IPP, isopentenyl diphosphate; HFPA, a-hydroxyfarne- sylphosphonic acid; PFT, protein farnesyltransferase; WRP, washed rubber particles; UDP, undecaprenyl diphosphate. (Received 5 June 2003, revised 8 July 2003, accepted 31 July 2003) Eur. J. Biochem. 270, 3939–3945 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03775.x salt (4)(Z)-2-nerolyl-3-methylbutenedioic acid dilithium salt (5)(Z)-2-farnesyl-3-methylbutenedioic acid dilithium salt (6), and (Z)-2-geranyl-3-methylbutenedioic acid dilithium salt (7) are shown in Fig. 1. Concentrated stock solutions of chaetomellic acid A analogs (4–7) were prepared in dimethylsulfoxide at 2 m M . Reaction conditions Rubber transferase assays were performed in multiwell plates as described in Mau et al. [13]. The typical reaction assay contained 1 m M [1- 14 C]IPP (7.03 MBqÆmmol )1 ), 1.25 m M MgSO 4 ,5m M dithiothreitol and 100 m M Tris pH 7.5 in a total volume of 50 lL. Various concentrations of initiators (FPP or DMAPP) were added, ranging from 10 p M to 1 m M , along with the stated concentrations of chaetomellic acid A analogs (4–7)orHFPA(3)(thatwas dissolved in ethanol). For rubber transferase assays (all performed in triplicate), 0.5 mg of H. brasiliensis WRP was used; 0.25 mg of WRP was used per assay involving P.argentatum rubber transferase. The reactions were started by the addition of WRPs to the other components and were incubated at 25 °CforH. brasiliensis WRP and 16 °CforP.argentatumWRP. The assays were incubated for 4 h and were stopped by the addition of 0.5 M EDTA pH 8 to a final concentration of 20 m M . The incorporated 14 C was measured by liquid scintillation counting of the newly synthesized rubber which had been trapped on filters and subsequently washed to remove unincorporated [ 14 C]IPP. Results Effects of various organic solvents and lithium salts on rubber transferase activity Prior to the first assays involving the analogs, several organic solvents that could be used to dissolve the chaeto- mellic acid A analogs were added to the standard rubber transferase assay [13] to determine what effects the presence of these solvents had on enzymatic activity. The range of concentrations tested were typical working dilutions. Dimethylsulfoxide and ethanol did not inhibit rubber transferase activity at the final concentration of 10% (v/v) in the rubber transferase assay, so these were the chosen conditions for conducting the inhibitor studies (data not shown). As the chaetomellic acid A analogs (4–7) were synthes- ized as lithium salts, the effect of lithium cations on rubber transferase activity was also evaluated. The presence of LiCl in the amounts of 1 l M to 1 m M did not affect rubber transferase activity (data not shown). As a result, all subsequent assays involving analogs 4–7 were compared to internal controls containing comparable amounts of LiCl and dimethylsulfoxide. Within any experiment in which the chaetomellic acid A analogs were diluted serially, the final dimethylsulfoxide concentration was kept constant at 10%. Assays involving HFPA were compared to control reactions supplemented with ethanol, and serial dilutions of HFPA were made to maintain a final ethanol concentration of 10%. Fig. 1. Structures of various chemicals tested for effects on rubber transferase activity in vitro. Farnesyl diphosphate 1 is the presumed initi- ator in vivo. Chaetomellic acid A (SG-2–29, 2) and a-hydroxyfarnesylphosphonic acid (HFPA, 3) are known inhibitors of protein farnesyltransferases, which covalently modify proteins with a FPP molecule. (Z)-2-octyl-3- methylbutenedioic acid dilithium salt (SG-2–96, 4)(Z)-2-nerolyl-3-methyl- butenedioic acid dilithium salt (SG-1–27, 5) (Z)-2-farnesyl-3-methylbutenedioic acid dilithium salt (SG-1–29, 6), and (Z)-2-geranyl- 3-methylbutenedioic acid dilithium salt (SG- 1–30, 7) are analogs of chaetomellic acid A. 3940 C. J. D. Mau et al.(Eur. J. Biochem. 270) Ó FEBS 2003 Chaetomellic acid A analogs inhibit rubber transferase Hevea brasiliensis. Incubating H. brasiliensis WRP with 20 l M chaetomellic acid A (2) and varying amounts of DMAPP or FPP initiator in a rubber transferase enzymatic assay demonstrated that chaetomellic acid A (2) was inhi- bitory when present at 20-fold molar excess of DMAPP or 200-fold molar excess FPP (Fig. 2). The related compound 4 inhibited H. brasiliensis rubber transferase by 40% when included at 180 l M in the presence of 10 n M FPP (data not shown). Compounds 5, 6 and 7 (Fig. 1) were tested for inhibitory effects on H. brasiliensis rubber transferase in vitro.Com- pound 5 was made to resemble the first cis-elongation product formed when using DMAPP as an initiator, while compounds 6 and 7 were synthesized to resemble FPP and geranyl diphosphate, respectively. Compound 6 inhibited activity by 25% when present in the assay at 180 l M in 1800-fold molar excess of FPP. Compounds 5 and 7 were not inhibitory when included in the assay at 180 l M in over a million-fold molar excess of initiator. Parthenium argentatum. All five chaetomellic acid A ana- logs (2, 4, 5, 6 and 7) were tested using the rubber transferase from guayule, P.argentatum, a species phylogenetically distant from H. brasiliensis. Compounds 2 and 6 reduced rubber transferase activity by 27% (when added at 180 l M in a 18,000-fold molar excess) and by 48% (when present at 180 l M in a 180-fold molar excess), respectively. Compounds 4, 5 and 7 did not inhibit rubber transferase activity under the conditions tested (supplemented in the assay at 180 l M in over a million-fold molar excess). Because compounds 2 and 6 were more inhibitory than 4 and 7, respectively, the long hydrophobic tails on these molecules might have some important role in determining efficacy of inhibition. To examine this possibility, palmitic acid and stearic acid were tested to see if either had any effect on rubber transferase activity. When present at 180 l M , neither palmitate nor stearate inhibited IPP incor- poration into DMAPP- or FPP-initiated newly synthesized rubber by H. brasiliensis or P.argentatum (data not shown). a-Hydroxyfarnesylphosphonic acid also inhibits rubber transferases. As chaetomellic acid A had been described initially as an inhibitor of protein farnesyltransferase, another type of PFT inhibitor was also tested to determine if it also could inhibit rubber transferase activity. HFPA inhibited FPP utilization by H. brasiliensis and P.argent- atum WRP by 36–37% when present at 20 l M at 2000-fold molar excess. Determination of kinetic constants for chaetomellic acid A analogs and HFPA. Double reciprocal plots (1/v vs. 1/[FPP]) of kinetic experiments indicated that compounds 2 and 3 were competitive inhibitors of H. brasiliensis rubber transferase (Fig. 3A,B), whereas compound 6 was a noncompetitive inhibitor (Fig. 3C). In contrast, all three compounds behaved as competitive inhibitors of the P.argentatumrubber transferase (Fig. 4A–C). The apparent K i s for the chaetomellic acid A analogs and HFPA were determined from plots (not shown) of the slope of the each reciprocal plot vs. the concentration of the inhibitory compound [14] (Table 1). Discussion Rubber transferases exhibit a considerable degree of toler- ance and can bind to a variety of different sizes of allylic diphosphate initiator molecules, at least up to solanesyl (C 45 ) diphosphate (M. H. Chapman and K. Cornish, unpublished data). Furthermore, the affinity of rubber transferase for the initiator increases with the size of the initiator, up to FPP (C 15 )inP. argentatum and geranylger- anyl diphosphate (C 20 )inH. brasiliensis. As a result, a model was proposed for the rubber transferase active site, envisioning the presence of non–specific hydrophobic interactions, which increased the affinity for longer allylic diphosphate substrates [15]. Nevertheless, it was uncertain if the chaetomellic acid analogs with the diacid groups could occupy the initiator-binding site with enough affinity to inhibit rubber biosynthesis. As seen in Fig. 2, chaetomellic acid A (2) was able to interfere with IPP incorporation in the rubber transferase assay. Almost 10-times more DMAPP than FPP was needed to overcome the same degree of inhibition by chaetomellic acid A (2) (Fig. 2). The ability of DMAPP or FPP to displace chaetomellic acid A from the initiator- binding site in H. brasiliensis rubber transferase paralleled the ninefold lower affinity of the enzyme for DMAPP vs. FPP (K m,DMAPP of 13.2 and K m,FPP of 1.5 l M [1]). The length of the hydrophobic carbon tail also affected the inhibitory activity of the chaetomellic acid A analogs. Compound 6, which has a farnesyl tail, was weakly inhibitory at 180 l M , in both species, whereas 7,which has a shorter geranyl tail, was not inhibitory at all. Fig. 2. Chaetomellic acid A inhibits H. brasiliensis rubber transferase. Assays were performed using H. brasiliensis WRP, 1 m M [ 14 C]IPP, an allylic diphosphate initiator, and either chaetomellic acid A (SG-2–29) or its analog SG-2–96. DMAPP was varied in the presence of 20 l M SG-2–29 (d)or20l M SG-2–96 (s), while various concentrations of FPP were tested in combination with 20 l M SG-2–29 (.). The amount of [ 14 C]IPP incorporated in these reactions was compared to control reactions containing dimethylsulfoxide and LiCl instead of the chaetomellic acid A analogs. Ó FEBS 2003 Inhibition of rubber transferase (Eur. J. Biochem. 270) 3941 Chaetomellic acid A (2) with a 14-carbon aliphatic chain strongly inhibited H. brasiliensis rubber transferase at 20 l M while 4, with a similar structure but having a shorter 8-carbon tail, required 180 l M before it became inhibitory. On the other hand, P.argentatum rubber transferase activity was only weakly inhibited by 180 l M chaetomellic acid A (2) and not at all by 180 l M compound 4. In both cases, lengthening the hydrophobic surface of the analog added additional interactions, which increased the binding affinity of the inhibitor for the initiator-binding site. The difference in the inhibition of H. brasiliensis and P.argent- atum rubber transferases by chaetomellic acid A indicated that there may be differences in the catalytic site geometry between the two species. However, a hydrophobic aliphatic tail simply attached to anegativechargeisnotthesolecauseoftheobserved inhibitory activity, because neither palmitic nor stearic acid was inhibitory in either species (data not shown). High affinity binding of substrates in the catalytic site appears to require two negatively charged oxygen atoms at one end of the molecule. These criteria are met by a-hydroxyfarnesylphosphonic acid (3), which did inhibit rubber transferase activity in both species. That two types of protein farnesyltransferase inhibitors could also interfere with rubber transferase, a cis-prenyltransferase, indicates that the initiator binding site of rubber transferase shares similarities with the FPP binding site of the protein farnesyltransferases. For P.argentatumrubber transferase, compounds 2, 3 and 6 all appear to inhibit FPP binding (and subsequent IPP incorporation) competitively (Figs 4A–C). In contrast, although 2 and 3 both competitively inhibited the H. bra- siliensis rubber transferase (Figs 3A,B), 6 actedina noncompetitive manner (Fig. 3C). The calculated K i s show that the active sites of H. brasiliensis and P. argentatum rubber transferases have a higher affinity for 2 than for 6 (Table 1). While the allylic compound 6 more closely resembles the FPP initiator, compound 2 with its more flexible aliphatic backbone probably makes more extensive contact with the nonspecific hydrophobic surface lining the catalytic cavity [15]. Alternatively, the difference in length of the inhibitors allows 2 to interact with the polyisoprene molecules within the rubber particle. In both cases, the additional interactions result in tighter binding of 2 when compared to that of 6. The interpretation of the double reciprocal plots must be qualified because the rubber transferase active site is localized on the surface of a rubber particle [15]. This proximity to the membrane monolayer covering the rubber particle would preclude access to the active site from certain directions during the in vitro reactions. In addition, experi- mental manipulations of washed rubber particles can be challenging. The binding of competitive inhibitors and substrates are mutually exclusive, which is not the case for noncompetitive inhibitors. Non-competitive inhibition results when binding of an inhibitor at a second site prevents catalysis at the normal active site, without causing any changes in the binding kinetics at the active site. The P. argentatum rubber transferase can only bind 6 in a manner which affects the apparent binding at the initiator substrate site. On the other Fig. 3. Chaetomellic acid A and SG-1–29 are inhibitors of H. brasil- iensis rubber transferase. Double reciprocal plots of 1/v vs. 1/[FPP] were created using kinetic data from H. brasiliensis rubber transferase assays. Rubber transferase assays were conducted in the presence of H. brasiliensis WRP, 1 m M [ 14 C]IPP, and the indicated amounts of FPP. ÔvÕ is measured in units of lmol [ 14 C]IPP incorporated per g dry weight rubber per 4 h. (A) Chaetomellic acid A (SG-2–29) was inclu- ded at concentrations of 10 (.), 20 (s)or50(d) l M during the assay while FPP was varied from 10 n M to 100 n M .(B)HFPAwaspresentat 10 (.), 20 (s), or 40 (d) l M while FPP was varied from 10 n M to 50 n M . (c). SG-1–29 was added at final concentrations of 50 (.), 100 (s), or 200 (d) l M while FPP was varied from 10 n M to 100 n M . 3942 C. J. D. Mau et al.(Eur. J. Biochem. 270) Ó FEBS 2003 hand, the noncompetitive inhibition by 6 of H. brasiliensis rubber transferase may be caused by interference resulting from the additional binding of compound 6 at the IPP binding site, which is in the proximity of the allylic diphosphate binding site. Competition between IPP and allylic diphosphate for the IPP binding sites has been observed at high substrate concentrations [1,16], and 6 was present at a range of 2500 to 10 000-fold molar excess to FPPintheassay(30-to130-timestheK m for FPP [17]). These results suggest that the spatial orientation between the IPP and initiator binding sites differs between the two rubber transferases, as well as the ability to bind 6 at other surfaces within the active site. The different behavior between the two rubber trans- ferases towards 6 should also be considered in light of the cooperative effects between IPP and FPP if one assumes that 6 is perceived as FPP by both enzymes. In the range of concentrations of 6 incubated in the assays, the two rubber transferases exhibit different degrees of negative coopera- tivity, with the P. argentatum enzyme showing the strongest effect [17]. Under these conditions, binding of the first initiator molecule decreases the ability of a free FPP to displace the bound FPP (or elongating polymer). The ability of the FPP-like compound 6 to bind noncompetitively at an additional location within the H. brasiliensis active site may explain some of differences in the degree of negative cooperativity between the two enzymes. Furthermore, the development of the P.argentatumrubber transferase with a low K m for FPP (about 150-fold lower in concentration than the corresponding constant for H. brasiliensis [17]) may also explain the difference in degree of negative cooperativity, because compound 6 can bind competitively into the initiator site of the P.argentatum enzyme, unlike the noncompetitive binding in the vicinity of the weaker affinity initiator binding site of the H. brasiliensis protein. More experimentation to elucidate the mechanism of the negative cooperativity can be performed, but the basis for this behavior will probably only become apparent after the crystal structures of the rubber transferases have been determined. The differing behavior of the various compounds tested, both in the amount of compound needed to create an observable effect and the different types of inhibition effected, supports earlier kinetic data on cosubstrate effects among the species [16,17]. Differences in binding constants, competitive effects, and in substrate activation exist between species [1,15–17]. Thus, although the rubber transferases Fig. 4. SG-1–29 and HFPA are competitive inhibitors of P. argentatum rubber transferase. Double reciprocal plots of 1/v vs. 1/[FPP] were created using kinetics data from P. argentatum rubber transferase assays. Rubber transferase assays were conducted in the presence of P.argentatumWRP, 1 m M [ 14 C]IPP, and the indicated amounts of FPP. ÔvÕ is measured in vitro of lmol [ 14 C]IPP incorporated per g dry weight rubber per 4 h. (A) Chaetomellic acid A (SG-2–29) was added at 50 (.), 100 (s), or 200 (d) l M while FPP was varied from 1.3 n M to 50 n M . (B) Assays contained HFPA at concentrations of 100 (.), 200 (s), or 500 (d) l M while FPP was varied from 5 n M to 100 n M .(C) SG-1–29 was present at final concentrations of 50 (.), 100 (s), or 200 (d) l M while FPP was varied from 5 n M to 100 n M . Table 1. Kinetic constants determined for the interaction between cha- etomellic acid A analogs or a-hydroxyfarnesylphosphonic acid with H. brasiliensis and P. argentatum rubber transferases. Compound H. brasiliensis P. argentatum K i (l M ) Type of competitor K i (l M ) Type of competitor SG-2–29 (2) 42 competitive 8.8 competitive HFPA (3) 64 competitive 420 competitive SG-1–29 (6) 140 noncompetitive 25 competitive Ó FEBS 2003 Inhibition of rubber transferase (Eur. J. Biochem. 270) 3943 from different species share many commonalities, they are not identical. These differences may have resulted from evolutionary divergence alone or in combination with the development of a different cellular environment for the rubber transferase in each species; H. brasiliensis rubber transferase is found on rubber particles in a free-flowing latex in laticifers, while P.argentatumrubber transferase is located on intracellular rubber particles in the bark parenchyma. The discovery that compounds known to bind to other FPP binding sites can interact with the initiator binding site of rubber transferases opens a new approach to modeling the catalytic site of this enzyme in the absence of crystal structures. We have already used other biochemical and physical studies to elucidate some features of the catalytic site [1,18]. Recently, two crystal structures of undecaprenyl diphosphate (UDP) synthase, a cis-prenyltransferase that catalyzes the formation of a 55-carbon carrier for glycosyl residues in peptidoglycan synthesis in bacteria, have been published [19,20]. Information from these structures may be helpful in our efforts to model the rubber transferase active site because both enzymes are cis-prenyltransferases with proposed catalytic sites near the cytosolic surface of the membrane. The active site contains a cleft flanked by hydrophobic amino acids that surrounds the aliphatic backbone of the substrate [20]. The chain length is apparently regulated by the size of the active site, and site- directed mutants, in which bulky, hydrophobic residues at the distal end of the catalytic site have been converted to alanine, can produce longer polyprenyl molecules. Similar mutagenesis of the avian FPP synthase catalytic site also extends the size of the product formed [21]. These structures suggest that the rubber transferase has a hydrophobic channel to direct the elongating biopolymer, in this case to the rubber particle interior [1,15,18]. Unlike the UDP synthase and the FPP synthase, rubber transferase appears to lack the bulky hydrophobic residues at the distal end of the active site. The normal mechanism for releasing the rubber molecule from the enzyme has not been determined. Thus, the results presented here further support the proposed model for the rubber transferase active site in which the presence of non–specific hydrophobic interactions increase the affinity for longer allylic diphosphate substrates [1,15]. These results also indicate that structural differences do exist between the rubber transferases from evolutionarily divergent species. Acknowledgements We thank Dr R. Krishnakumar at the Rubber Research Institute of India for supplying the H. brasiliensis latexasasourceforWRPand Dr Francis Nakayama at the US Water Conservation Laboratory in Phoenix, AZ for maintaining and harvesting P.argentatumplants for isolation of the P. argentatum WRP used in experiments described here. We also acknowledge the help of Ms. Mary H. Chapman and Dr Javier Castillo ´ n for isolating the H. brasiliensis and P. argentatum WRP used in our experiments. Ms. Saima Kint and Dr Thomas McKeon kindly provided palmitic and stearic acids for control experiments. 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