Biomacromolecules 2000, 1, 632-641
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Rubber Molecular Weight Regulation, in Vitro, in Plant Species that Produce High and Low Molecular Weights in Vivo Katrina Cornish,*,† Javier Castillo´ n,‡ and Deborah J. Scott† Western Regional Research Center, USDA-ARS, 800 Buchanan Street, Albany, California 94710; and Floral and Nursery Plants Research Unit, USDA-ARS, BARC-West, Building 010A, Room 238, 10300 Baltimore Avenue, Beltsville, Maryland 20705 Received April 17, 2000; Revised Manuscript Received August 10, 2000
In three rubber-producing species, in vitro, the rates of initiation and polymerization and the biopolymer molecular weight produced were affected by the concentration of farnesyl diphosphate (FPP) initiator and isopentenyl diphosphate (IPP) elongation substrate (monomer). Ficus elastica, a low molecular weightproducer in vivo, synthesized rubber polymers approximately twice the molecular weight of those made by HeVea brasiliensis or Parthenium argentatum (which produce high molecular weights in vivo), possibly due to its lower IPP Km. In all species, increasing FPP concentrations increased rubber biosynthetic rate and new molecules initiated but decreased molecular weight by competition with the allylic diphosphate (APP) end of elongating rubber molecules for the APP binding site. Increasing IPP concentrations increased rubber biosynthetic rate and rubber molecular weight, but only when FPP concentrations were below the FPP Km’s or where negative cooperativity operated. In conclusion, rubber transferase is not the prime regulator of rubber molecular weight in vivo. Introduction Natural rubber, arguably the most important biomacromolecule in the industrialized world, is produced by over 2500 plant species distributed among four of the six superorders of the Dicotyledonae.1-3 The rubber is synthesized by rubber transferase enzymes (cis-prenyl transferase, EC 2.5.1.20) integral to the monolayer membrane that surrounds microscopic, cytosolic rubber particles.4-7 This enzyme produces the rubber polymer (cis-1,4-polyisoprene) from isoprene monomers derived from isopentenyl diphosphate (IPP, strictly speaking, a pyrophosphate) (Figure 1). The rubber transferase also requires an allylic diphosphate cosubstrate (APP, also a pyrophosphate, produced by soluble trans prenyl transferases) to initiate polymer formation, and a divalent cation, such as Mg2+ or Mn2+, as cofactor8-10 (Figure 1). Rubber transferases exhibit similar kinetic constants and pH optima, and are able to accept a similar range of APP’s as initiating substrate.8,10-12 Also, studies have shown that the longer the APP, up to C15 or C20, the faster the rate of rubber biosynthesis in vitro.6,8,12 Structural studies have led to the suggestion that the C15 farnesyl diphosphate (FPP) may be the most common initiator in vivo, at least in HeVea brasiliensis.13 The molecular weight of the rubber biopolymer produced by different species varies widely, and most do not produce the high molecular weights required for commercial applications (molecular weight is strongly correlated with rubber * Corresponding author. Telephone: 510 559-5950. Fax: 510 5595663. E-mail:
[email protected]. † Western Regional Research Center, USDA-ARS. ‡ Floral and Nursery Plants Research Unit, USDA-ARS.
quality14). In vitro biochemical studies may lead to a better understanding of the regulation of rubber molecular weight, in vitro and in vivo, as well as provide information on use in the synthesis of synthetic polyisoprene and related polymers. In Parthenium argentatum, a high rubber molecular weight producing species,15 we have shown that molecular weight, in vitro, is greatly affected by substrate concentration and identity and that high concentrations of APP initiator can compete with the APP end of the elongating rubber molecule and displace the rubber molecule.16 Presumably this enzyme would be similarly affected in vivo. However, P. argentatum is one of the most generalized rubber-producing species known, making rubber in ordinary bark parenchyma cells instead of complex laticifers,17 and biochemical studies restricted to this one species cannot provide the complete story. In this paper, we investigate the effect of the concentration of IPP and the APP cosubstrate FPP on the initiation, IPPincorporation rate, and molecular weight of rubber polymers synthesized in vitro by enzymatically active rubber particles of H. brasiliensis and Ficus elastica. Both of these species produce their rubber as latex in a highly complex anastamosized cell system of laticifers, but the molecular weight of their rubber, in vivo, is very different: H. brasiliensis, the primary commercial source of natural rubber today, produces high quality, high molecular weight rubber (>1 000 000), whereas F. elastica produces low quality, low molecular weight rubber (106
115 0.6 192 13 232 103-104
385 0.01 38 500 2 618 176 >106
a Reactions were performed for isopentenyl diphosphate (IPP) in the presence of 20 m trans,trans-farnesyl diphosphate (FPP) and for FPP in 1 mM IPP. Apparent Km’s were calculated from log plots of v/(Vmax - v) against [S]. The projected rubber molecular weight (Mr) from in vitro experiments was calculated, assuming that the ratio of IPP Km/FPP Km reflects the number of isoprene units incorporated, as (Km(IPP)/Km(FPP)) 68 + 176. (Molecular weight of isoprene subunit ) 68, diphosphate with one negative charge ) 176.)
Figure 9. Mean molecular weight ranges (×103) of rubber molecules synthesized by H. brasiliensis, F. elastica, and P. argentatum at various IPP and FPP concentrations (black, thick bars), compared with the ranges predicted from the substrate ratios (grey, thin bars). Values were calculated as given in the Experimental Section.
fold change over the IPP concentration range (Figure 9f)). Also, P. argentatum produced molecular weights higher than the substrate range prediction at low IPP concentrations (Figure 9f). However, the IPP had greater effects on the rubber molecular weight produced by the other two rubber transferases, a 2.6-8.7-fold change in H. brasiliensis (Figure 9d) and a 5.0-16.7-fold change in F. elastica (Figure 9e). Also, although the H. brasiliensis rubber transferase was affected by FPP in a similar way to P. argentatum (a 6.814.6-fold change in molecular weight over the FPP concentration range (Figure 9a)), the F. elastica rubber molecular weight was more effected by IPP (8.2-27.7-fold (Figure 9e)) than by FPP (5-16.7-fold (Figure 9b)). These results suggest that the molecular weight of rubber produced by the P. argentatum rubber transferase is more tightly regulated than the rubber transferases from the other two species. At first glance, our data suggest that polymer molecular weight in vivo may be regulated, at least in part, by different endogenous substrate pool sizes, rather than by radically different rubber transferase enzymes in different species. At this time, we do not know the endogenous substrate concentrations in vivo, and direct quantification is problematical because rapid catabolism occurs during substrate purification. Nonetheless, a “low molecular weight” species such as F. elastica might have much higher endogenous levels of APP than “high molecular weight” species such as P. argentatum or H. brasiliensis, and perhaps different levels of the prenyl transferases that catalyze the synthesis of these molecules or of catabolic enzymes that convert or degrade them. It has also been noted that the rubber produced by greenhouse-grown F. elastica plants includes a small amount (ca. 2%) of high molecular weight rubber.15 It seems possible that these high molecular weight rubber polymers may have been synthesized during a period of increased demand for APP by other enzymes of the isoprenoid pathway, such as might be created by a spurt of rapid growth or development. Our in vitro results suggest that the inevitable, but transient, lowering in endogenous APP concentration would result in
much higher molecular weight polymer being synthesized while those conditions prevailed. However, Km’s reflect the substrate concentration around which the particular enzyme operates most efficiently and are often thought to reflect the endogenous, or physiological, substrate concentration in vivo. If we assume this to be true, the rubber transferase IPP and APP Km’s predict the ideal conditions for the enzyme (high IPP levels, low APP levels). The high rubber transferase IPP Km ensures that rubber is made only at times when IPP is nonlimiting for other prenyl transferases, all of which have low IPP Km’s much closer to the APP Km’s (reviewed in ref 22). Under optimal conditions, and if no other factors are involved, the rubber molecular weight synthesized would presumably reflect the substrate equilibrium, and can be calculated from the IPP Km to APP Km ratio. In the case of P. argentatum and F. elastica, the ratio of IPP Km to FPP Km does translate to match the high and low molecular weights produced in vivo, respectively (Table 3), by these species. However, the IPP Km to FPP Km ratio of the H. brasiliensis rubber transferase predicts that this species would produce low molecular weight rubber in vivo (Table 3), which is not the case. Thus, it seems likely that specific polymer termination events occur in vivo, in addition to species-specific substrate effects. Very little is known about rubber polymer termination, as yet, apart from some rubber structural studies indicating that the compartmentalized rubber polymers are dephosphorylated and that different polymer end groups have been noted in different species.13 Additional research should be carried out to specifically address these issues. Conclusions In conclusion, we have shown that rubber molecule initiation, biosynthetic rate and molecular weight, in vitro, are dependent upon substrate concentration and the ratio of IPP and FPP, but are affected by intrinsic properties of the rubber transferases as well. Thus, the higher the IPP concentration, the higher the biosynthetic rate, and when FPP levels are limiting, or when negative cooperativity inhibits access of FPP to the FPP binding site, the higher the polymer molecular weight produced. The higher the FPP concentration, the greater the number of rubber molecules, the lower the rubber molecular weight, and, at limiting IPP concentrations, the higher the overall biosynthetic rate. The three
Rubber Transferases
rubber transferases investigated are all capable of producing a wide range of molecular weights, depending upon substrate concentration, clearly demonstrating that the transferases are not the prime determinates of different product size in vivo. However, despite these commonalities, considerable differences exist between the species with respect to cosubstrate effects, binding constants, effective concentration ranges of the initiation and elongation (monomer) substrates, and the role of negative cooperativity in molecular weight regulation in vitro. The P. argentatum rubber transferase appears to exert more control over the molecular weight it produces than those from the other two species, and may, therefore, provide the best prospect for genetic transformation of candidate annual crop species. However, our in vitro experiments have not provided a unifying biochemical mechanism that could explain the different rubber molecular weights found in vivo in different plant species, such as the high molecular weight rubber in H. brasiliensis and P. argentatum and the low molecular weight rubber in F. elastica. It is possible these evolutionarily divergent species employ different mechanisms to regulate molecular weight. Alternatively, and perhaps more likely, given the considerable similarities in rubber biosynthesis enjoyed by the species, similar endogenous regulatory factors may operate in vivo. These factors, which may include, but are unlikely to be limited to, endogenous substrate concentrations, must be understood in order for us to achieve our goal of engineering temperate zone annual crops to produce high quality rubber. Acknowledgment. We thank Drs. R. W. Lenz, C. J. D. Mau, and G. A. R. Nobes for their critical reviews of this manuscript, and Ms. M. H. Chapman for technical assistance.
Biomacromolecules, Vol. 1, No. 4, 2000 641 (2) Flowering plants of the world; Heywood, V. H., Ed.; Oxford University Press: London, 1978; pp 1-335. (3) Bonner, J. In Guayule Natural Rubber: A Technical Publication with Emphasis on Recent Findings; Whitworth, J. W., Whitehead, E. E., Eds.; Guayule Administrative Management Committee and USDA Cooperative State Research Service, Office of Arid Lands Studies, The University of Arizona: Tucson, AZ, 1991; pp 1-16. (4) Cornish, K. Eur. J. Biochem. 1993, 218, 267-271. (5) Cornish, K.; Backhaus, R. A. Phytochemistry 1990, 29, 3809-3813. (6) Cornish, K.; Siler, D. J. J. Plant Physiol. 1995, 147, 301-305. (7) Cornish, K.; Wood, D. F.; Windle, J. J. Planta 1999, 210, 85-96. (8) Archer, B. L.; Audley, B. G. Bot. J. Linn. Soc. 1987, 94, 181-196. (9) Madhavan, S.; Greenblatt, G. A.; Foster, M. A.; Benedict, C. R. 1989. Plant Physiol. 1989, 89, 506-511. (10) Cornish, K.; Siler, D. J. Plant Physiol. Biochem. 1996, 34, 377384. (11) Lynen, F. J. Rubber Res. Inst. Malaya 1969, 21, 389-406. (12) Cornish, K.; Castillo´n, J.; Chapman, M. H. In Biochemical Principles and Mechanisms of Biosynthesis and Biodegradation of Polymers; Steinbu¨chel, A., Ed.; Wiley-VCH-Verlag: New York, 1998; pp 316323. (13) Tanaka, Y.; Aik-Hwee, E.; Ohya, N.; Nishiyama, N.; Tangpakdee, J.; Kawahara, S.; Wititsuwannakul, R. Phytochemistry 1996, 41, 1501-1505. (14) Swanson, C. L.; Buchanan, R. A.; Otey, F. H. J. Appl. Polym. Sci. 1979, 23, 743-748. (15) Cornish, K.; Siler, D. J.; Grosjean, O. K.; Goodman, N. J. Nat. Rubber Res. 1993, 8, 275-285. (16) Castillo´n, J.; Cornish, K. Phytochemistry 1999, 51, 43-51. (17) Guayule Natural Rubber: A Technical Publication with Emphasis on Recent Findings; Whitworth, J. W., Whitehead, E. E., Eds.; Guayule Administrative Management Committee and USDA Cooperative State Research Service, Office of Arid Lands Studies, The University of Arizona: Tucson, AZ, 1991; pp 1-445. (18) Siler, D. J.; Cornish, K. Phytochemistry 1993, 32, 1097-1102. (19) Cornish, K.; Bartlett, D. L. Phytochem. Anal. 1997, 8, 130-134. (20) Cornish, K.; Siler, D. J. J. Plant Physiol. 1995, 147, 301-305. (21) Segel, I. H. Enzyme Kinetics; John Wiley and Sons: New York, 1993; pp 1-957. (22) Ogura, K.; Koyama, T. Chem. ReV. 1998, 98, 1263-1276. (23) Wang, K.; Ohnuma, S. TIBS 1999, 24, 445-451.
References and Notes (1) Bealing, F. J. J. Rubber Res. Inst. Malaya 1969, 21, 445-455.
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