Regulation of Rubber Biosynthetic Rate and Molecular Weight in

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Regulation of Rubber Biosynthetic Rate and Molecular Weight in Hevea brasiliensis by Metal Cofactor Bernardo M. T. da Costa,† Jay D. Keasling,† and Katrina Cornish*,‡,§ Department of Chemical Engineering, University of California, Berkeley, California 94720, and USDA-ARS, Western Regional Research Center, 800 Buchanan Street, Albany, California 94710 Received July 7, 2004; Revised Manuscript Received September 22, 2004

Metal ion cofactors are necessary for prenyltransferase enzymes. Magnesium and manganese can be used as metal ion cofactor by rubber transferase (a cis-prenyltransferase) associated with purified rubber particles. The rubber initiation rate, biosynthetic rate, and molecular weight produced in vitro from HeVea brasiliensis rubber transferase is regulated by metal ion concentration. In addition, KIPP‚Mg varies significantly with m [Mg2+]. KIPP‚Mg decreases from 8000 ( 600 µM at [Mg2+] ) 4 mM to 68 ( 10 µM at [Mg2+] ) 8 mM and m increases back to 970 ( 70 µM at [Mg2+] ) 30 mM. The highest affinity of rubber transferase for IPP‚Mg occurred when [Mg2+] ) Amax (metal concentration that gives highest IPP incorporation rate). A metal ion is required for rubber biosynthesis, but an excess of metal ions interacts with the rubber transferase inhibiting its activity. The results suggest that H. brasiliensis could use [Mg2+] as a regulatory mechanism for rubber biosynthesis and molecular weight in vivo. Introduction Natural rubber, a strategic raw material used in enormous amounts, is currently only obtained commercially from HeVea brasiliensis (Muell. Arg.), the Brazilian or para rubber tree. Although 2500 plant species make natural rubber,1,2 H. brasiliensis is one of the few that produces the high molecular weight rubber required for high quality and high product performance.3 Because molecular weight is related to quality, studies of the rubber molecular weight regulation in vitro can suggest ways of regulating the rubber molecular weight in vivo, which may allow the production of rubber with high molecular weight in other plant species. Natural rubber is synthesized by the rubber transferase enzyme (EC 2.5.1.20), a membrane bound cis-prenyltransferase.4-9 The rubber transferase requires an allylic pyrophosphate (APP) to initiate the rubber molecule, isopentenyl pyrophosphate (IPP) to elongate the rubber molecule, and a divalent cation, such as Mg2+ or Mn2+ as cofactor4,5,7,8,10-12 (Figure 1, parts A and B). In vitro, rubber transferases can accept a variety of APP’s as initiator.4,5,9 However, the low binding constant of farnesyl pyrophosphate (FPP) (Figure 1C) compared to other APPs13 and its synthesis in the cytosol of the laticifer, the same compartment as the rubber transferase, indicates that FPP is probably the main initiator in vivo in H. brasiliensis, a view directly supported by NMR data indicating that the rubber molecule has a transC15 tail.12 Although rubber transferase can use a number of metal ions as cofactors,4,5,7,8,12 Mg2+ is the main cofactor in * To whom correspondence should be addressed. E-mail: kcornish@ yulex.com. Phone: +1-760-476-0320. Fax: +1-760-476-0321. † UC Berkeley. ‡ USDA-ARS. § Current address: Yulex Corporation, 1945 Camino Vida Roble, Suite C, Carlsbad, CA 92008.

Figure 1. Structure of IPP (A); Structure of APP, where APP is dimethyl allylic pyrophosphate (DMAPP) if R ) H, APP is geranyl pyrophosphate (GPP) if R ) C5H9, APP is farnesyl pyrophosphate (FPP) if R ) C10H17 (B); Structure of FPP (C). In solution, the pyrophosphate groups will be ionized to varying degrees.

vivo as its concentration is three orders of magnitude higher than Mn2+ and is at a meaningful physiological concentration.11 In vitro, alteration of the rubber molecular weight of H. brasiliensis, Parthenium argentatum Gray, and Ficus elastica Roxb. can be achieved by changing the concentrations of IPP and APP.14,15 It was shown, in general, that for H. brasiliensis, F. elastica, and P. argentatum at limiting [FPP] an increase in the [IPP] leads to a higher molecular weight. When FPP is not limiting, increasing the FPP concentration leads to lower mean molecular weights at all IPP concentrations. Unlike other cis-prenyltransferases and trans-prenyltransferases that have products of predetermined size,16,17 rubber transferases are able to produce a wide range of molecular weight rubber in vitro.14,15 In vitro, under identical [IPP] and [FPP], F. elastica produces rubber with twice the molecular weight than that made by H. brasiliensis or P. argentatum.14 It has been suggested that this is likely a direct consequence of the 3-fold-higher affinity of the F. elastica rubber transferase for IPP in the presence of FPP, in this species.14 However,

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in vivo, F. elastica produces a lower molecular weight than the other two rubber producing species, and so plants must have some way other than [IPP] and [FPP] of regulating the molecular weight of the rubber molecules. Possible mechanisms could involve regulation of polymer termination reactions or availability of essential cofactors. It has been previously shown that the metal ion cofactor concentration affects the IPP incorporation rate by rubber transferases from F. benghalensis,18 F. carica,19 F. elastica,11,20 H.brasiliensis,11,18-20 and P. argentatum.11,20 The rubber transferase not only requires the metal ion as a cofactor but also as an activator as it is not tightly associated with the enzyme. At low concentrations, the metal ion deinhibits the rubber transferase activity, whereas at high concentrations, it inhibits the rubber transferase activity.11 Thus, IPP incorporation rate is maximal (Vmax) at a certain metal ion concentration (defined as [A]max11). Nevertheless, throughout the remainder of this paper, the metal ion is referred to as a cofactor. Magnesium and manganese similarly affect IPP incorporation rate in F. elastica, H. brasiliensis, and P. argentatum.11 Also, in these three species, the rubber transferase can bind FPP, FPP‚metal or metal ion alone, whereas it can bind IPP‚metal or metal ion alone, but not IPP alone.11 In the present study, we have determined the influence of magnesium and manganese concentrations on the initiation, polymerization and molecular weight of rubber produced in vitro by H. brasiliensis rubber transferase. A possible regulatory role for magnesium in rubber biosynthesis also is discussed. Experimental Section Materials. Living latex from H. brasiliensis line PB260 was donated by the Rubber Research Institute of India and was stored in buffer as previously described.21,22 Unlabeled IPP and FPP were obtained from Echelon Biosciences Incorporated (Salt Lake City, UT), and [14C]IPP (55 mCi/ mmol) and [3H]FPP (60 Ci/mmol) were obtained from American Radiolabeled Chemical Inc., St. Louis, MO. Multiwell plates used in this study were MultiScreen R1 plates (MultiScreen - R1; 1 mm hydrophilic PTFE membrane; glass-filled PP plate; non-sterile with lid, Millipore, Bedford, MA; catalog number MAR1N1010). A vacuum manifold (Millipore catalog number MAVM0960R) was used for H. brasilienesis rubber transferase assays. Siliconized 1.5 mL tubes were supplied by USA Scientific (Ocala, FL). ScintiVerse BD Cocktail was purchased from Fisher Scientific (Santa Clara, CA). Chemicals, unless otherwise noted, were purchased from Sigma (St. Louis, MO). In Vitro Assay of Rubber Biosynthesis. Enzymatically active washed rubber particles from H. brasiliensis were purified and stored as previously described.21,22 IPP incorporation rates and FPP incorporation rates were assayed in H. brasiliensis purified rubber particles (washed rubber particles, WRP) using a modification of the method by Mau et al.23 The reaction took place in individual wells of a 96well plate. The wells were siliconized with Sigmacote (Sigma-Aldrich, Corp., St Louis, MO, #SL-2) for 2 min,

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rinsed with deionized water and with 95% ethanol, and dried at room-temperature overnight. The reaction volume was 50 µL (7 mM EDTA; 100 mM Tris-HCl pH 7.5; 5 mM DTT; IPP; FPP; [14C]IPP; [3H]FPP and MgSO4 or MnSO4 as indicated). EDTA (7 mM) was used in all experiments, as this was previously determined to be the amount required to chelate the metal ions bound to the WRP.11 Purification of WRP without metal ions causes denaturation of the rubber transferase, probably due to the extended time periods involved, thus preventing studies of the rubber transferase activity. The reaction was begun by the addition of 0.25 mg WRP of H. brasiliensis into each well, and the filter plate was placed on a ceramic cooling plate (Amershan Biosciences, Piscataway, NJ) equipped with a circulating water bath to control the temperature. After 4 h at 25 °C, the reaction was stopped by the addition of 25 µL of 500 mM EDTA. For single label experiments, the filters were washed using the Millipore vacuum manifold with 100 µL of 95% ethanol, 3 × 150 µL of 95% ethanol, 2 × 150 µL of deionized water, and 2 × 150 µL of 95% ethanol to each well. For dual label experiments, the filters were washed using the Millipore vacuum manifold with 100 µL of 95% ethanol, 3 × 150 µL of 95% ethanol, 2 × 150 µL of 0.1% 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonatehydrate (CHAPS), 2 × 150 µL of 95% ethanol, 2 × 150 µL of 0.1% CHAPS, 2 × 150 µL of 95% ethanol, 2 × 150 µL of 0.1% CHAPS, and 2 × 150 µL of 95% ethanol to each well. Filter plates were oven-dried at 37 °C for 30 min, the filters were removed from the plate and placed individually into vials with 2.5 mL ScintiVerse BD Cocktail. The amount of [14C]IPP and [3H]FPP was determined by liquid scintillation spectroscopy using Beckman LS6500 (Beckman Coulter, Fullerton, CA). The mean molecular weights (MWrubber) were calculated based on the IPP incorporation rate (IPP inc) and the FPP incorporation rate (FPP inc), as shown by eq 1. Each value is the average of three replicates MWrubber )

IPP Inc + 3 × FPP Inc × MWIsopentenyl + MWPP FPP Inc (1)

where MWIsopentenyl is the molecular weight of the isoprene monomer (68) and MWPP is the molecular weight of the pyrophosphate group (176) Calculation of [IPP‚Mg] and [FPP‚Mg]. When magnesium, IPP, and FPP are present in solution the three species are in equilibrium according to reactions (A) and (B) Mg + IPP T IPP‚Mg

(A)

Mg + FPP T FPP‚Mg

(B)

) Equation 2 gives the IPP‚Mg dissociation constant (KIPP‚Mg d in terms of free IPP, free Mg, and IPP‚Mg concentration in solution ([IPPf], [Mgf], and [IPP‚Mg], respectively) ) KIPP‚Mg d

[IPPf][Mgf] [IPP‚Mg]

(2)

Equation 3 gives the FPP‚Mg dissociation constant (KFPP‚Mg ) d in terms of free FPP, free Mg, and FPP‚Mg concentration

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Figure 2. FPP and IPP concentration dependence of [14C]IPP incorporation by rubber transferase in Hevea brasiliensis purified rubber particles. Incorporation rate of [14C]IPP was measured in the presence of 1 mM Mg2+ and (A) varying [FPP] in 1 mM IPP or (B) varying [IPP] in 20 µM FPP.

in solution ([FPPf], [Mgf], and [FPP‚Mg], respectively) KFPP‚Mg ) d

[FPPf][Mgf] [FPP‚Mg]

(3)

Equations 4-6 are the mole balances for IPP, FPP, and magnesium, respectively [IPPt] ) [IPPf] + [IPP‚Mg]

(4)

[FPPt] ) [FPPf] + [FPP‚Mg]

(5)

[Mgt] ) [Mgf] + [IPP‚Mg] + [FPP‚Mg]

(6)

[IPP‚Mg] is expressed in terms of total IPP in solution, [IPPt], and [Mgf] by solving eq 2 for [IPPf] then substituting it in eq 4. Similarly, [FPP‚Mg] is expressed in terms of total FPP in solution, [FPPt], and [Mgf] by solving eq 3 for [FPPf] then substituting it in eq 5. Substitution of the expressions for [IPP‚Mg] and [FPP‚Mg] in eq 6 yields [Mgf]3 + R[Mgf]2 + β[Mgf] - δ ) 0

(7)

where + KFPP‚Mg + [IPPt] + [FPPt] - [Mgt] R ) KIPP‚Mg d d β ) KIPP‚Mg KFPP‚Mg + KFPP‚Mg [IPPt] + KIPP‚Mg [FPPt] d d d d [Mgt] - KFPP‚Mg [Mgt] KIPP‚Mg d d KFPP‚Mg [Mgt] δ ) KIPP‚Mg d d The three roots of eq 7 were determined using the function “roots” of Matlab [version 5.3.0.10183 (R11), MathWorks, Natick, MA]. The largest positive root corresponds to the concentration of free magnesium in solution. The other concentrations in solution were then determined using eqs 2-6. To calculate the concentration of IPP‚Mg and FPP‚Mg ) 520 µM24 and KFPP‚Mg ) 120 in solution, we used KIPP‚Mg d d µM.25 Results FPP and IPP Dependence of IPP Incorporation Rate. IPP incorporation rate was dependent on [IPP] and [FPP]

(Figure 2). IPP incorporation was highly dependent upon [FPP] when FPP was nonsaturating ([FPP] < 15 µM), but at saturating (nonlimiting) concentrations of FPP, the IPP incorporation rate was independent of increasing [FPP] (Figure 2A). Similarly, IPP incorporation was highly dependent upon [IPP] when IPP was at a limiting concentration ([IPP] < 1 mM), but at saturating concentrations of IPP, the IPP incorporation rate was independent of [IPP] (Figure 2B). Magnesium and Manganese Dependence of IPP Incorporation Rate. To determine the effects of [Mg2+] or [Mn2+] on the IPP incorporation rate, we selected four [IPP] such that they would represent a limiting level of IPP (0.1KIPP m ) 14), a saturating level of IPP 37.5 µM), near KIPP (375 µM m (1000 µM) and a supersaturating level of IPP (10KIPP m ) 3750 µM). A saturating level of FPP was selected for the experiment (10KFPP m ) 15 µM). IPP Incorporation Rate, Single Label Experiment. The IPP incorporation rate was dependent on both magnesium and manganese concentration, but the patterns of activation and inhibition differed with [IPP] and with the particular cofactor (Figure 3). In both cofactors, the maximum IPP incorporation rate was highest when [IPP] ) KIPP m (375 µM). At [IPP] g KIPP m the IPP incorporation rate dependence on the metal ion spread over a wider range when magnesium (5-50 mM) was used than when manganese was used (5-9 mM). When [IPP] was severely limiting (0.1 KIPP m , 37.5 µM) little difference was observed with cofactor Mg Mn (Figure 4). At [metal ion] e 1/2 Ametal max (Amax ) 8 mM; Amax ) 7 mM) the IPP incorporation rate increased with increasing [IPP]. However, when the [metal ion] approached its 1/2 Ametal max value, the IPP incorporation rate in 375 µM IPP rapidly increased, reaching a maximum at 8 mM Mg2+ and 7 mM Mn2+. Metal ion concentrations greater than Ametal max 2+ inhibited the IPP incorporation rate. At [IPP] g KIPP m , Mn 2+ was more inhibitory than Mg but the degree of inhibition greatly slowed beyond that achieved in 8 mM Mg2+. Mg2+ was a poor inhibitor of IPP incorporation at high [IPP]s but had a large effect in [IPP] e KIPP m . When severely limiting [IPP] was used (37.5 µM), the IPP incorporation rate was completely inhibited by [Mg2+] g 40 mM. Since there is a close overlap of IPP incorporation rate dependence of magnesium and manganese at this [IPP] (Figure 4), it is expected that Mn2+ would have the same effect.

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Figure 3. Metal ion concentration dependence of [14C]IPP incorporation by rubber transferase in Hevea brasiliensis purified rubber particles. (A) Varying [Mg2+]. (B) Varying [Mn2+]. Incorporation rates were measured in 15 µM FPP, 7 mM EDTA and different [IPP]: 37.5 µM, 0; 375 µM, ∆; 1000 µM, ]; 3750 µM, O. The error bars in the figure represent the standard deviation of triplicates.

Figure 4. Metal ion concentration dependence of [14C]IPP incorporation by rubber transferase in Hevea brasiliensis purified rubber particles. Incorporation rates were measured in 15 µM FPP, 7 mM EDTA, 37.5 µM IPP and either varying [Mg2+], 0, or varying [Mn2+], O. The error bars in the figure represent the standard deviation of triplicates.

When Mg2+ was used as the metal ion cofactor, an increase in [Mg2+] did not affect the rubber transferase activity at 2+ [Mg2+] e 1/2 AMg max (Figure 5A). Further increases in [Mg ] Mg Mg 2+ enhanced the enzyme activity (1/2 Amax e [Mg ] e Amax), whereas increasing [Mg2+] beyond AMg max led to inhibition of the enzyme activity (Figure 5A). Similarly, when Mn2+ was used as the metal ion cofactor, at low [Mn2+], it had no effect on the rubber transferase activity, increasing the [Mn2+] enhanced the enzyme activity (Figure 5B), whereas [Mn2+]

g AMn max inhibited the enzyme activity. The activity of the rubber transferase beyond Ametal max differed in the two metals, and was independent of the [IPP] when manganese was used, but not when magnesium was the metal ion cofactor (cf. Figure 5, parts A and B). Also, activation of the rubber transferase at severely limiting [IPP] occurred at a lower [Mg2+] than at other [IPP], whereas the activation occurred at a higher [Mn2+] at severely limiting [IPP] than at the other [IPP]. . We investigated Magnesium Dependence of KIPP‚Mg m the unusual maximum activity at [IPP] ) KIPP m (Figure 3), to determine if it resulted from effects on Km or Vmax. The IPP incorporation rate was measured at different [IPP]s and at three magnesium concentrations with saturating [FPP] (15 µM). The IPP incorporation rate was highly dependent on the [Mg2+] (Figure 6A). The affinity of rubber transferase for IPP‚Mg varied significantly with magnesium concentration (Figure 6B and Table 1). The highest affinity (lowest Km) was observed when [Mg2+] ) 8 mM (AmaxMg). Although varied by 2 orders of magnitude depending on KIPP‚Mg m [Mg2+], the Vmax for rubber transferase remained almost unchanged (Table 1). Magnesium and Manganese Dependence of Molecular Weight. To determine the effects of metal ion cofactor concentration on rubber molecular weight, we selected four concentrations for each metal, based on Figure 3, such that they would represent a strongly limiting level of metal ion 2+ (1 mM Mg2+ and 4 mM Mn2+), near 1/2 Ametal max (5 mM Mg

Figure 5. Hill plots of metal ion concentration dependence of [14C]IPP incorporation by rubber transferase in Hevea brasiliensis purified rubber particles. (A) for [Mg2+]. (B) for [Mn2+]. Incorporation rates were measured in 15 µM FPP, 7 mM EDTA and different [IPP]: 37.5 µM, 0; 375 µM, ∆; 1000 µM, ]; 3750 µM, O. The error bars in the figure represent the standard deviation of triplicates.



Figure 6. IPP concentration dependence of [14C]IPP incorporation by rubber transferase in Hevea brasiliensis purified rubber particles in different [Mg2+]. (A) Velocity versus [IPP]. (B) Hill plot. Incorporation rate of [14C]IPP was measured in the presence of 15 µM FPP, 7 mM EDTA, varying [IPP] and one of the following [Mg2+]: 4 mM Mg2+, 0; 8 mM Mg2+, ∆; and 30 mM Mg2+, ]. The error bars in Figure 6A represent the standard deviation of triplicates.

Figure 7. FPP and metal ion concentration dependence of [3H]FPP incorporation by rubber transferase in Hevea brasiliensis purified rubber particles. Incorporation rates were measured in 7 mM EDTA, one of the following [IPP]: 37.5 µM (A, D), 375 µM (B, E), or 3750 µM (C, F) and varying [Mg2+] (A-C) or [Mn2+] (D-F). Table 1. Variation of Kinetic Constants for Rubber Transferase in Hevea brasiliensis Purified Rubber Particles with Magnesium Concentration

Vmax (µmol/g dw/4 h) SEa

Mg (mM) 4 8 30 a

KIPP‚Mg (µM) m

14 8.7 8.7

1 0.3 0.3

SEa 8000 68 970

600 10 70

SE = standard error.

2+ and 6 mM Mn2+), near Ametal and 7 mM max (8 mM Mg 2+ metal Mn ), and an inhibitory level (1/2 AmaxI 50 mM Mg2+ and 8 mM Mn2+). Initiation Rate. FPP incorporation rate increased with increasing [FPP] at all [IPP] and cofactor concentrations used (Figure 7). The FPP incorporation rate varied with [metal ion] x [IPP] x [FPP]. At [IPP] e KIPP m (37.5 and 375 µM) FPP incorporation rates were dependent on [Mg2+] at all

[FPP] tested (Figure 7, parts A and B). However, when [IPP] was nonlimiting (3750 µM), FPP incorporation rates were primarily regulated by [FPP] and became largely insensitive to [Mg2+], although 8 mM Mg2+ appeared to slightly inhibit the FPP incorporation rate. FPP incorporation rates followed a similar pattern in Mn2+ as was observed in Mg2+, except that FPP incorporation was still quite strongly [Mn2+] dependent at 3750 µM IPP (cf. Figure 7, parts C and F). Also, at severely limiting [IPP], no inhibition of the FPP incorporation rate was observed when [Mn2+] was increased from 7 mM (AMn max) to 8 mM (1/2 AmaxIMn), whereas inhibition was observed from 8 mM Mg (AMg max) to 50 mM (1/2 AmaxI ) (cf. Figure 7, parts A and D). Biosynthetic Rate. At all [Mg2+]s, when [FPP] g KFPP m (1.5-50 µM), the IPP incorporation rate was not much affected by increasing [FPP] but was very sensitive to [Mg2+] (Figure 8). A strong rate enhancement was observed at

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Figure 8. IPP and metal ion concentration dependence of [14C]IPP incorporation by rubber transferase in Hevea brasiliensis purified rubber particles. Incorporation rates were measured in 7 mM EDTA, one of the following [IPP]: 37.5 µM (A, D), 375 µM (B, E), or 3750 µM (C, F) and varying [Mg2+] (A-C) or [Mn2+] (D-F). IPP [Mg2+] ) 8 mM (AMg max) and 375 µM IPP (Km ), as also was seen in Figure 3. Similarly, IPP incorporation rates were highly dependent upon [Mn2+], but some distinct differences to rates in Mg2+ were apparent. [FPP] did not affect significantly the IPP incorporation rate at limiting levels of 2+ Mn2+ (4 mM). At [Mn2+] ) 6 mM (1/2 AMn max) or [Mn ] ) Mn IPP 7 mM (Amax) and [IPP] g Km , the IPP incorporation rate was dependent on [FPP], increasing as [FPP] was increased from 0.15 to 15 µM, while being slightly inhibited at [FPP] ) 50 µM. When [Mn2+] ) 8 mM and [IPP] g KIPP m , the IPP incorporation rate was inhibited at saturating levels of FPP. At severely limiting [IPP], IPP incorporation rate was independent of [FPP], but no inhibition of the FPP incorporation rate was observed when [Mn2+] was increased from Mn 7 mM (AMn max) to 8 mM (1/2 AmaxI ) (cf. Figures 7D and 8D). Molecular Weight. Although the divalent metal ion cofactor is essential for prenyltransferase activity and can affect the rate of reaction catalyzed by prenyltransferases, it is not considered to affect the actual mechanism of the prenyltransferase reaction. Thus, a rubber molecule synthesized in vitro under different reaction conditions will always have only one FPP initiator molecule incorporated along with a varying number of IPP molecules. Therefore, eq 1 allows the mean molecular weight produced in vitro to be calculated. In addition, dual-labeled gel permeation chromatography has demonstrated that, under saturating substrate conditions, the molecular weights calculated using eq 1 approximate the actual molecular profiles of the newly synthesized radiolabeled rubber (unpublished results). In general, in both cofactors, the higher the [FPP] the lower the rubber molecular weight, whereas increasing [IPP]

increased the molecular weight, especially at limiting [FPP], as has been reported previously14 (Figure 9). Molecular weight was highly dependent upon [Mg2+] and [Mn2+]. Maximum molecular weights were achieved at AMg max at all IPP and FPP combinations. When manganese was used, the highest molecular weight was at 8 mM Mn2+ rather than at 7 mM Mn2+. Also, at nonlimiting IPP, increasing Mn2+ from Mn 7 (AMn max) to 8 mM (1/2 AmaxI ) (Figure 3) did not cause a decrease in molecular weight (Figure 9D). At severely limiting [FPP] and [IPP] ) KIPP m the molecular weight was higher at AmaxIMn than at AMn max (Figure 9 E). Discussion Our previous results11 indicated that FPP can bind to the rubber transferases of H. brasiliensis, P. argentatum, and F. elastica in the presence and absence of metal ions, whereas IPP can only bind to the rubber transferase in the presence of a metal ion. Therefore, the rubber elongation step requires that an IPP‚Mg complex interact with the active site of the rubber transferase. There are three possible ways that the enzyme‚IPP‚Mg complex (E‚IPP‚Mg) may be formed Mg + IPP T IPP‚Mg + E T E‚IPP‚Mg

(I)

E + IPP T E‚IPP + Mg T E‚IPP‚Mg

(II)

E + Mg T E‚Mg + IPP T E‚IPP‚Mg

(III)

When magnesium and IPP are present in solution, some of the Mg and IPP exist as an IPP‚Mg complex (formed through the first step of reaction 1), which allows reaction I to proceed. Our previous work11 showed that IPP by itself does



Figure 9. FPP and metal ion concentration dependence of molecular weight produced by rubber transferase in Hevea brasiliensis purified rubber particles. Mean molecular weights were estimated based on FPP and IPP incorporation rates measured in 7 mM EDTA, one of the following [IPP]: 37.5 µM (A, D), 375 µM (B, E), or 3750 µM (C, F) and varying [Mg2+] (A-C) or [Mn2+] (D-F).

not interact with the rubber transferase, so it is likely that reaction II is not relevant at physiological conditions. It also has been demonstrated that magnesium is required for IPP binding26 by undecaprenyl pyrophosphate synthase (UPPS), another cis-prenyltransferase enzyme. This work gives further evidence that reaction II should not be relevant at physiological conditions in H. brasiliensis. Reaction III involves a magnesium ion binding to the enzyme and a free molecule of IPP binding to the enzyme-magnesium complex. Biosynthetic Rate. Our results showing that IPP incorporation rate is dependent on [metal ion] (Figure 3) are consistent with the conclusions of Scott et al.11 that rubber transferase can interact with FPP, FPP‚metal, IPP‚metal or metal ion, but not with IPP alone. As [metal ion] increase (at [metal ion] e Ametal max ) [IPP‚metal] also increases in solution, which leads to higher IPP incorporation rates, perhaps because of faster interaction with the rubber transferase. When the [metal ion] is increased beyond Ametal max , the IPP incorporation rate is inhibited. When there is a large excess of metal ions over IPP molecules, the first step of reaction I implies that all IPP is present as the IPP‚metal complex, which is confirmed by the measured KIPP‚Mg d (KIPP‚Mg ) 520 µM).24 Without free IPP the first step of d reaction II and the second step of reaction III cannot proceed. The amount of free IPP present in solution does not support the observed IPP incorporation rates. Thus, reaction I is the most likely pathway for rubber elongation. Since [IPP‚Mg] is constant, the decrease in IPP incorporation rate with increasing [metal ion] must be due to the interaction of free metal ion with the enzyme. The interaction of free metal ion with the enzyme prevents the elongation of the rubber

Figure 10. IPP‚Mg concentration dependence of [14C]IPP incorporation by rubber transferase in Hevea brasiliensis purified rubber particles. [IPP‚Mg] was calculated using KdIPP‚Mg ) 520 µM24 and KdFPP‚Mg ) 120 µM.25 Incorporation rates were measured in 15 µM FPP, 7 mM EDTA and [IPPt] 37.5, 375, 1000 or 3750 µM, as well as different [Mgt]: 20 mM, ∆; 30 mM, ]; 40 mM, 0; 50 mM, O.

molecule, which may be caused by impeding the IPP‚metal complex from accessing the rubber transferase active site. At high [metal ion] an increase in [IPP] increases the [IPP.metal] and the IPP incorporation rate also increases (Figure 10), supporting the conclusion that free metal ion interacting with the rubber transferase inhibits its activity. The divalent metal ion is thought to bind with the allylic pyrophosphate substrate and accelerate the formation of the allylic cation produced by elimination of the pyrophosphate ion from the allylic substrate.16,27 The formation of the allylic cation is a rate-limiting step in the 1′-4 condensation.27,28 The rate of allylic cation formation depends on the species

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of metal ion, and the rate is faster for Mg2+ than Mn2+.27,29 Therefore, it is not surprising that at high [IPP] (375-3750 µM) the IPP incorporation rate [Mg2+] and [Mn2+] dependency are different, as the identity of the IPP‚metal complex affects the enzyme activity (Figure 3). However, at severely limiting [IPP], the IPP incorporation rate dependence on [Mg2+] and [Mn2+] are very similar (Figure 4), which might happen because the low [IPP‚metal] allows the complex to easily interact with the rubber transferase independent of the metal ion identity. Manganese caused a greater degree of inhibition of IPP incorporation than magnesium when [IPP] g KIPP m (Figure 3). According to earlier work,30 this inhibition might be an artifact caused by IPP depletion by precipitation of the IPP‚Mn complex. In such a case, inhibition should become progressively greater as [Mn2+] increases beyond AMn max. However, this is not the case here as the IPP incorporation rate was virtually constant at [Mn2+] > 9 mM (Figure 3). When [IPP] > KIPP m , it is possible that the interaction between free manganese ions and the rubber transferase complex is stronger than the interaction between free magnesium ions and the enzyme complex. For both metal ion cofactors tested at [metal ion] e 1/2 Ametal max and any [IPP], an increase in [metal ion] did not lead to an enhancement of the rubber transferase activity, as the slope of the Hill plot does not change, keeping a value close to one (Figure 5). When the [metal ion] was in the range metal 1/2 Ametal max to Amax , an increase in [metal ion] activated the enzyme, as the slope of the Hill plot is greater than one. The enhancement in the rubber transferase activity could be due to the increased affinity of the enzyme for IPP‚Mg (Figure 6 A and Table 1). When [Mg2+] is increased from 4 to 8 mM the KIPP‚Mg decreases from 8000 to 68 µM, m reflecting a large increase in affinity that we believe may be due to a conformational change caused by binding of free magnesium ions to a site in the enzyme that either is, or affects, an activator site. If the activator site has a relatively low affinity for magnesium ions, even when most of the IPP is present as IPP‚Mg, the enzyme might not bind enough magnesium ions to cause the conformational change at only 4 mM magnesium but does bind enough of them at 8 mM [Mg2+] (Ametal max ) to undergo the change. Increasing the [metal ion] beyond Ametal max then inhibited the enzyme activity, as shown by the negative slope in the Hill plots. A possible explanation of this inhibition is that excess metal ions inhibit activity by interacting with a site within the enzyme that has too low of an affinity for metal ions to be adversely affected by lower concentrations. Alternatively, at least in case of Mg2+, such an inhibitory site may only be accessible after the proposed conformational change has occurred. When magnesium was used, the level of inhibition was dependent on the [IPP], which was not observed with manganese (cf. Figure 5, parts A and B). These results again indicate that the interaction of the rubber transferase with free manganese is likely to be stronger than its interaction with free magnesium. This might be due to the differences in ionic radii, as magnesium has a smaller radius (86 pm) than manganese (97 pm).31 It seems possible that the larger ion may be more difficult to dislodge, once it is bound to

da Costa et al.

the enzyme. Thus, increasing the [IPP] does not affect the inhibition by excess metal ion when manganese is used as the cofactor, because IPP‚Mn cannot easily dislodge the bound manganese ions. In contrast, when magnesium is used, increasing [IPP] leads to a higher [IPP‚Mg], which can displace the bound magnesium ions. Initiation Rate. The FPP incorporation rate by H. brasiliensis rubber transferase increased with increasing [FPP] at all [IPP], agreeing with earlier reports.14 In addition, FPP incorporation rates varied with [metal ion] x [IPP] x [FPP]. At limiting levels of IPP ([IPP] e KIPP m ), FPP incorporation rates were dependent on [Mg2+] for every [FPP] tested and increased with increasing [Mg2+] e AMg max (Figure 7, parts A and B). However, the FPP incorporation rate was largely independent of [Mg2+] at saturating [IPP] (Figure 7C). It has been previously shown that FPP alone can bind to the rubber transferase.11 Except at high [IPP] (3750 µM) and very low [Mg2+] (1 mM), most of the FPP is present as FPP‚Mg. Thus, any effect of magnesium on the FPP incorporation rate is likely to be due to the interaction of magnesium with the rubber transferase rather than to the formation of the FPP‚Mg complex or to the FPP interaction with the rubber transferase. At high [IPP] and very low [Mg2+], approximately half the FPP is present as FPP‚Mg, but when [Mg2+] is raised, and most of the FPP is complexed to magnesium, the FPP incorporation rate is little affected (Figure 7 C), suggesting that both FPP and FPP‚Mg are effective initiators of new rubber molecules. Thus, rubber transferase seems similar to other prenyltransferases in that the allylic pyrophosphate substrate can bind in the presence and absence of magnesium but the condensation reaction with the nonallylic IPP is absolutely magnesium-requiring.16,24,26,32 As [Mg2+] increases up to AMg max, the FPP incorporation rate increases because the [FPP‚Mg] increases, and the enzyme has a higher affinity for the complex than for FPP alone.11 At [Mg2+] > AMg max, magnesium alone associates with the rubber transferase inhibiting the initiation of rubber biosynthesis. This suggests that when magnesium alone associates with the enzyme it blocks FPP or FPP‚Mg access to the active site, whereas when FPP binds alone to the enzyme, magnesium can still access the FPP molecule and form FPP‚Mg in situ. The dependence of FPP incorporation rate on [Mg2+] lessens at higher [FPP]. The initiation rate is than at less dependent upon [Mg2+] at [IPP] ) KIPP m severely limiting [IPP], especially at lower [FPP] (cf. Figure 7, parts A and B). Also, at the saturating levels of IPP used, 15 µM FPP saturates the rubber transferase (Figure 2 A), and so the increased FPP incorporation rate seen when FPP is increased from 15 to 50 µM FPP is probably due to an increase of FPP‚Mg in solution. Similarly, at severely limiting [IPP] and low [Mn2+], FPP binds alone to the rubber transferase and FPP incorporation rate depends on [FPP] only. As [Mn2+] is increased, there is more FPP‚Mn in solution, which then binds preferentially to the enzyme, hence an increase in FPP incorporation rate (Figure 7 D). At [IPP] ) KIPP and 10 KIPP m m , the FPP incorporation rate shows a stronger dependence on [Mn2+] as the [FPP] increases, likely because [FPP‚Mn] increases with increasing [FPP], such that at high [FPP] the enzyme


is binding to FPP‚Mn rather than to the FPP alone as in low [FPP]. Hence FPP binds alone to rubber transferase, and little variation is observed with [Mn2+] at low [FPP] (Figure 7, parts E and F). The rubber transferase initiation rate is more affected by changes in magnesium concentration than manganese (cf. Figure 7, parts A-C and D-F), which suggests that the enzyme has a higher affinity for FPP‚Mg than FPP‚Mn. This conclusion is supported by the higher FPP incorporation rates obtained when magnesium was used. Elongation. In agreement with ref 11, when [FPP] g FPP KFPP m (Km ) 1.5 µM), the IPP incorporation rate was not significantly affected by [FPP] but was by [Mg2+] (Figure 8A-C). At limiting [Mg2+], there is a limited amount of [IPP‚Mg] available to be incorporated by the rubber transferase. As the [Mg2+] increases, more IPP‚Mg is formed in solution and incorporation rates by the enzyme increase. However, at inhibitory [Mg2+] the excess metal ion present in solution can associate with the enzyme by itself hindering IPP‚Mg incorporation, which explains the observed decrease in IPP incorporation rate. When IPP is nonlimiting, the concentration of free Mg2+ is lower because of the greater amount of IPP‚Mg complex in solution, and less inhibition is observed. The biosynthetic rate of rubber was affected to a greater extent by [Mg2+] when [IPP] ) KIPP m , which suggests that varying cytosolic [Mg2+] may play a pivotal role in the regulation of rubber biosynthesis. At severely limiting [IPP], the IPP incorporation rate was [Mn2+] dependent but largely independent of [FPP] at all [Mn2+], because the levels of IPP‚Mn in solution were limited by the low [IPP] and the levels of FPP‚Mn in solution did not affect IPP incorporation rate (Figure 8D). At higher [IPP], increasing the [Mn2+] to AmaxMn caused an increase in IPP incorporation rate. When [IPP] g KIPP m , an increase of [Mn2+] to 1/2 AmaxIMn (8 mM, Figure 3B) caused an inhibition of IPP incorporation rate at saturating FPP, but not at limiting FPP (0.15 and 1.5 µM) (Figure 8, parts E and F). At limiting FPP, the number of rubber transferase sites producing rubber is smaller; thus, the excess manganese can interact with the empty sites without directly affecting the IPP incorporation rate. However, when saturating levels of FPP are used, the excess manganese is likely to block some of the active rubber sites from the complex FPP‚Mn, leading to a decrease in the IPP incorporation rate. Molecular Weight. In rubber biosynthesis, one APP molecule is always used to initiate the rubber molecule but only IPP is used for elongation; therefore, eq 1 can be used to estimate the mean molecular weight of rubber synthesized in vitro. As reported earlier,14 the more FPP molecules present in solution, the more likely that the elongating rubber molecule will be terminated by a chain transfer reaction, which is the displacement of the elongating rubber molecule from the rubber transferase active site by an FPP initiating a new rubber molecule. This leads to the observed reduction in molecular weight (Figure 9A-C) as [FPP] increase. A higher [FPP] leads to lower molecular weights, independent of the magnesium concentration used (Figure 9A-C), because FPP alone can associate with rubber transferase.11 For any fixed combination of [FPP] and [Mg2+], an increase in [IPP] leads to an increase in molecular weight, but this


effect was much greater at limiting [FPP]. As IPP alone does not associate with rubber transferase, increasing [Mg2+] up to AMg max led to an increase in IPP incorporation rate and consequently to an increase in rubber molecular weight (Figure 9A-C). At high [FPP], IPP incorporation rates were inhibited by high [Mg2+] (Figure 8A-C), whereas initiation rates were not significantly affected (Figure 7A-C), resulting in a decrease in the mean molecular weight (Figure 9A-C). Thus, at high [FPP], the dependence of rubber molecular weight on [Mg2+] is very similar to the dependence of IPP incorporation rate on [Mg2+] (cf. Figure 8A-C and Figure 9A-C). When manganese was used as the cofactor, any increase in [FPP] led to a decrease in molecular weight, similar to when magnesium was used (cf. Figure 9, parts A-C and D-F). When [Mn2+] e AMn max, and for all [FPP], an increase in [IPP] caused an increase in the rubber mean molecular weight, as the larger amount of IPP molecules in solution increased the [IPP‚Mn] in solution, allowing easier and more frequent interactions with the rubber transferase. However, when [Mn2+] ) AmaxIMn (8 mM) and at limiting [FPP] (0.15 µM) the highest observed rubber mean molecular weight was at KIPP m rather than at nonlimiting conditions (cf. Figure 9, parts E and F), due to the higher IPP incorporation rate observed at KIPP m than at nonlimiting IPP (3750 µM). In general, rubber molecular weight was higher when manganese was used as cofactor (Figure 9) than when magnesium was the cofactor. Hence, the metal ion identity is an important regulator of the rubber molecular weight in vitro. As in FPP synthase, the identity of the metal ion does not affect significantly the affinity of the enzyme for the IPP‚metal complex,24 and it is unlikely that the differences in rubber molecular weight can be attributed to the differences in enzyme affinity for the IPP‚metal complex. Nonetheless, the products of the octaprenyl, solanesyl and decaprenyl pyrophosphate synthases are longer in the presence of manganese than magnesium,27 which agrees with our results. Since the rate of allylic cation formation is higher for Mg2+ than Mn2+,29 the FPP incorporation rates are lower in the presence of Mn2+ (Figure 7), which causes the increase in the rubber molecular weight. In Vivo Implications. Km’s reflect the substrate concentration around which enzymes are most sensitive to substrate changes and they often reflect the endogenous substrate concentration in vivo.14,33 Thus, KIPP (375 µM) likely m approximates the endogenous level of IPP in H. brasiliensis latex when rubber is being produced. It is noteworthy that when [Mg2+] is in the range 6-10 mM the IPP incorporation rate is higher at [IPP] ) 375 µM than at any other [IPP] tested (Figure 3). This suggests that the enzyme might have evolved to have higher activity at these specific [IPP] and [Mg2+], implying that the in vivo [IPP] and [Mg2+] for high rubber transferase activity are similar to the in vitro ones. In vitro, metal ion cofactor concentration affects the initiation rate, elongation rate, and molecular weight of rubber. The H. brasiliensis rubber transferase has a marked sensitivity to metal ion concentration at KIPP m (Figure 3). than other prenylRubber transferase has a higher KIPP m transferases,16 which ensures that IPP is only used for rubber

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production when it is nonlimiting to other enzymes. The metal ion species has been shown to not affect the affinity of FPP synthase for the IPP‚metal complex.24 Thus, variation of metal ion concentration likely affects rubber transferase activity due to direct interaction of free metal ion with the enzyme. Our results indicate that the interaction of the H. brasiliensis rubber transferase is stronger with manganese than magnesium ions. Magnesium is the primary metal ion cofactor of rubber transferase in vivo, as its concentration in latex is much higher than the manganese concentration 11 and is close to AMg whereas the endogenous [Mn2+] is max, 265 times smaller than AMn max and so is physiologically irrelevant. We have shown that the affinity of the rubber transferase for IPP‚Mg varies by 2 orders of magnitude depending on [Mg2+] (Table 1). Therefore, if H. brasiliensis is able to regulate the cytosolic metal ion concentration in vivo, it could use this mechanism to control the rate of rubber production, in addition to regulating the concentration of FPP and IPP. There is a significant difference in the endogenous magnesium concentration between the rubber producing species H. brasilensis (12 mM) and F. elastica (53 mM),11 and which approximately match their determined Amax. This might be one of the reasons that H. brasiliensis produces a high molecular weight rubber in vivo, whereas F. elastica produces a low molecular weight rubber, and in vitro both can produce high and low molecular weight rubber.14 Although magnesium is an essential cofactor for a number of cell functions, and it is known that [Mg2+] regulates the activity of photosynthetic enzymes in the chloroplast, little is understood about its uptake and transport in plants.34 Currently, the effect of [metal ion] on the conformation of the rubber transferase complex is unknown. However, magnesium was shown to affect the structure of porphorbilinogen synthase (PBGS).35 In the absence of magnesium, PBGS exist as a hexamer, has a low affinity for its substrate (5-aminolevulinic acid, ALA), and the reaction is slow. In the presence of magnesium, PBGS exist as an octamer and has a high affinity for ALA.35 The remarkable increase in affinity of the rubber transferase for the IPP‚Mg complex, when the total magnesium in solution is increased from 4 to 8 mM (AMg max), also may be due to a major conformational change. The presence of magnesium increases the PBGS affinity for ALA by 2 orders of magnitude,35 which is the same order of magnitude change in substrate affinity observed for the rubber transferase. Conclusions Rubber transferase requires a metal ion cofactor for activity,4,5,7,8,10-12 and it has been shown that [metal ion] in vitro can affect the rubber biosynthesis of Ficus elastica, HeVea brasiliensis, and Parthenium argentatum.11 In this work, we propose a regulatory mechanism for rubber biosynthesis in H. brasiliensis involving [metal ion]. At a low level of metal ion, only a small amount of rubber is synthesized, as the [FPP‚metal] and [IPP‚metal] are low. Increasing the [metal ion] leads to a higher amount of FPP‚metal and IPP‚metal in solution, resulting in higher FPP and IPP incorporation rates. However, at yet higher [metal

da Costa et al.

ion], the metal ion can interact directly with the rubber transferase inhibiting enzyme activity causing a decrease in IPP incorporation rate. The strength of interaction depends on the metal ion identity, with free manganese ions having a stronger interaction than free magnesium ions. Metal ion concentration has a stronger effect on IPP incorporation rate than on FPP incorporation rate and, as the [metal ion] affects both the initiation rate and the elongation rate, it also affects the rubber molecular weight. The affinity of the enzyme for IPP‚Mg is greatly affected by the magnesium concentration. The change in affinity might be due to a conformational change of the rubber transferase, similar to that which occurs in porphorbilinogen synthase (PBGS). The affinity of PBGS for aminolevulinic acid, its substrate, increases by 2 orders of magnitude in the presence of magnesium, where PBGS exists as an octamer.35 Rubber transferase undergoes a similar change in affinity when the exogenous magnesium concentration was increased from 4 to 8 mM. Although metal ion identity affects the rubber molecular weight in vitro, magnesium is the primary cofactor in vivo.11 Our results suggest that rubber biosynthesis in vivo in H. brasiliensis may be regulated by cytosolic magnesiusm concentration in addition to the rates of FPP and IPP synthesis. Acknowledgment. We thank Drs. Charles Lee, JiannTsyh Lin, and Deborah J. Scott for their critical reviews of this manuscript. We thank Dr. R. Krishnakumar for H. brasiliensis latex. This work was supported, in part, by the USDA-CSREES Initiative for Future Agriculture and Food Systems grant number 2001-04125. References and Notes (1) Bealing, F. J. Carbohydrate metabolism in HeVea latexsavailability and utilization of substrates. J. Rubber Res. Inst. Malaysia 1969, 21, 445-455. (2) Bonner, J. Guayule Natural Rubber: A Technical Publication with Emphasis on Recent Findings. In Guayule AdministratiVe Management Committee and USDA CooperatiVe State Research SerVice; Whitworth, E. E., Ed.; The University of Arizona: Tucson, AZ, 1991; p 1-16. (3) Swanson, C. L.; Buchanan, R. A.; Otey, F. H. Molecular-Weights of Natural Rubbers from Selected Temperate Zone Plants. J. Appl. Polym. Sci. 1979, 23 (3), 743-748. (4) Archer, B. L.; Audley, B. G. New Aspects of Rubber Biosynthesis. Bot. J. Linnean Soc. 1987, 94(1-2), 181-196. (5) Madhavan, S.; Greenblatt, G. A.; Foster, M. A.; Benedict, C. R. Stimulation of Isopentenyl Pyrophosphate Incorporation into Polyisoprene in Extracts from Guayule Plants (Parthenium-Argentatum Gray) by Low-Temperature and 2-(3,4-Dichlorophenoxy)Triethylamine. Plant Physiol. 1989, 89 (2), 506-511. (6) Cornish, K.; Wood, D. F.; Windle, J. J. Rubber particles from four different species, examined by transmission electron microscopy and electron-paramagnetic-resonance spin labeling, are found to consist of a homogeneous rubber core enclosed by a contiguous, monolayer biomembrane. Planta 1999, 210 (1), 85-96. (7) Cornish, K.; Backhaus, R. A. Rubber Transferase-Activity in Rubber Particles of Guayule. Phytochemistry 1990, 29 (12), 3809-3813. (8) Cornish, K. The Separate Roles of Plant Cis and Trans Prenyl Transferases in Cis-1,4-Polyisoprene Biosynthesis. Eur. J. Biochem. 1993, 218 (1), 267-271. (9) Cornish, K.; Siler, D. J. Effect of different allylic diphosphates on the initiation of new rubber molecules and on cis-1,4-polyisoprene biosynthesis in guayule (Parthenium argentatum Gray). J. Plant Physiol. 1995, 147 (3-4), 301-305. (10) Benedict, C. R.; Madhavan, S.; Greenblatt, G. A.; Venkatachalam, K. V.; Foster, M. A. The Enzymatic-Synthesis of Rubber Polymer in Parthenium-Argentatum Gray. Plant Physiol. 1990, 92 (3), 816821.

Metal Effects in Rubber Biosynthesis (11) Scott, D. J.; da Costa, B. M. T.; Espy, S. C.; Keasling, J. D.; Cornish, K. Activation and inhibition of rubber transferases by metal cofactors and pyrophosphate substrates. Phytochemistry 2003, 64 (1), 123134. (12) Tanaka, Y.; Aik-Hwee, E.; Ohya, N.; Nishiyama, N.; Tangpakdee, J.; Kawahara, S.; Wititsuwannakul, R. Initiation of rubber biosynthesis in Hevea brasiliensis: Characterization of initiating species by structural analysis. Phytochemistry 1996, 41 (6), 1501-1505. (13) Cornish, K. Similarities and differences in rubber biochemistry among plant species. Phytochemistry 2001, 57 (7), 1123-1134. (14) Cornish, K.; Castillon, J.; Scott, D. J. Rubber molecular weight regulation, in vitro, in plant species that produce high and low molecular weights in vivo. Biomacromolecules 2000, 1 (4), 632641. (15) Castillon, J.; Cornish, K. Regulation of initiation and polymer molecular weight of cis-1,4-polyisoprene synthesized in vitro by particles isolated from Parthenium argentatum (Gray). Phytochemistry 1999, 51 (1), 43-51. (16) Ogura, K.; Koyama, T. Enzymatic aspects of isoprenoid chain elongation. Chem. ReV. 1998, 98 (4), 1263-1276. (17) Wang, K.; Ohnuma, S. Chain-length determination mechanism of isoprenyl diphosphate synthases and implications for molecular evolution. Trends Biochem. Sci. 1999, 24 (11), 445-451. (18) Kang, H. S.; Kim, Y. S.; Chung, G. C. Characterization of natural rubber biosynthesis in Ficus benghalensis. Plant Physiol. Biochem. 2000, 38 (12), 979-987. (19) Kang, H.; Kang, M. Y.; Han, K. H. Identification of natural rubber and characterization of rubber biosynthetic activity in fig tree. Plant Physiol. 2000, 123 (3), 1133-1142. (20) Scott, D. J.; Cornish, K. Activation and inhibition of rubber transferase activity in relationship to the concentrations of metal cosubstrate and diphosphate substrates. Abstr. Papers Am. Chem. Soc. 2000, 219, U215-U216. (21) Cornish, K.; Bartlett, D. L. Stabilisation of particle integrity and particle bound cis-prenyl transferase activity in stored, purified rubber particles. Phytochem. Anal. 1997, 8 (3), 130-134. (22) Siler, D. J.; Cornish, K. A Protein from Ficus-Elastica Rubber Particles Is Related to Proteins from Hevea-Brasiliensis and Parthenium-Argentatum. Phytochemistry 1993, 32 (5), 1097-1102. (23) Mau, C. J. D.; Scott, D. J.; Cornish, K. Multiwell filtration system results in rapid, high-throughput rubber transferase microassay. Phytochem. Anal. 2000, 11 (6), 356-361.

Biomacromolecules, Vol. 6, No. 1, 2005 289 (24) King, H. L.; Rilling, H. C. Avian Liver Prenyltransferase - Role of Metal in Substrate Binding and Orientation of Substrates During Catalysis. Biochemistry 1977, 16 (17), 3815-3819. (25) Pickett, J. S.; Bowers, K. E.; Fierke, C. A. Mutagenesis studies of protein farnesyltransferase implicate aspartate beta 352 as a magnesium ligand. J. Biol. Chem. 2003, 278 (51), 51243-51250. (26) Chen, Y. H.; Chen, A. P. C.; Chen, C. T.; Wang, A. H. J.; Liang, P. H. Probing the conformational change of Escherichia coli undecaprenyl pyrophosphate synthase during catalysis using an inhibitor and tryptophan mutants. J. Biol. Chem. 2002, 277 (9), 7369-7376. (27) Ohnuma, S.; Koyama, T.; Ogura, K. Alteration of the Product Specificities of Prenyltransferases by Metal-Ions. Biochem. Biophys. Res. Commun. 1993, 192 (2), 407-412. (28) Poulter, C. D.; Rilling, H. C. In Biosynthesis of Isoprenoid Compounds; Porter, J. W., Spurgeon, S. L., Eds.; John Wiley & Sons: New York, 1982; pp 161-224. (29) Chayet, L.; Rojas, M. C.; Cori, O.; Bunton, C. A.; McKenzie, D. C. Complexes of Bivalent-Cations with Neryl and Geranyl Pyrophosphate - Their Role in Terpene Biosynthesis. Bioorg. Chem. 1984, 12 (4), 329-338. (30) Reuter, B.; Kno¨ss, W.; Hemmerlin, A.; Bach, T. J. Proteinindependent interaction of divalent metal ions with geranylgeranyl diphosphate and other prenyl diphosphates. Pharm. Pharm. Lett. 1997, 7, 141-144. (31) Shannon, R. D. Revised Effective Ionic-Radii and Systematic Studies of Interatomic Distances in Halides and Chalcogenides. Acta Crystallogr. A 1976, 32(SEP1), 751-767. (32) Liang, P. H.; Ko, T. P.; Wang, A. H. J. Structure, mechanism and function of prenyltransferases. Eur. J. Biochem. 2002, 269 (14), 3339-3354. (33) Segel, I. H. Enzyme Activation. In Enzyme kinetics: behaVior and analysis of rapid equilibrium and steady-state enzyme systems; Segel, I. H., Ed.; Wiley: New York, 1993; Chapter 5, pp 227-272. (34) Shaul, O. Magnesium transport and function in plants: the tip of the iceberg. Biometals 2002, 15 (3), 309-323. (35) Breinig, S.; Kervinen, J.; Stith, L.; Wasson, A. S.; Fairman, R.; Wlodawer, A.; Zdanov, A.; Jaffe, E. K. Control of tetrapyrrole biosynthesis by alternate quaternary forms of porphobilinogen synthase. Nat. Struct. Biol. 2003, 10 (9), 757-763.

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Regulation of Rubber Biosynthetic Rate and Molecular Weight in

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