Elsevier

Phytochemistry

Volume 69, Issue 14, October 2008, Pages 2539-2545
Phytochemistry

Initiation of rubber biosynthesis: In vitro comparisons of benzophenone-modified diphosphate analogues in three rubber-producing species

https://doi.org/10.1016/j.phytochem.2008.07.011Get rights and content

Abstract

Natural rubber, cis-1,4-polyisoprene, is a vital industrial material synthesized by plants via a side branch of the isoprenoid pathway by the enzyme rubber transferase. While the specific structure of this enzyme is not yet defined, based on activity it is probably a cis-prenyl transferase. Photoactive functionalized substrate analogues have been successfully used to identify isoprenoid-utilizing enzymes such as cis- and trans-prenyltransferases, and initiator binding of an allylic pyrophosphate molecule in rubber transferase has similar features to these systems. In this paper, a series of benzophenone-modified initiator analogues were shown to successfully initiate rubber biosynthesis in vitro in enzymatically-active washed rubber particles from Ficus elastica, Hevea brasiliensis and Parthenium argentatum.

Rubber transferases from all three species initiated rubber biosynthesis most efficiently with farnesyl pyrophosphate. However, rubber transferase had a higher affinity for benzophenone geranyl pyrophosphate (Bz-GPP) and dimethylallyl pyrophosphate (Bz–DMAPP) analogues with ether-linkages than the corresponding GPP or DMAPP. In contrast, ester-linked Bz–DMAPP analogues were less efficient initiators than DMAPP. Thus, rubber biosynthesis depends on both the size and the structure of Bz-initiator molecules. Kinetic studies thereby inform selection of specific probes for covalent photolabeling of the initiator binding site of rubber transferase.

Graphical abstract

Enzymatically-active washed rubber particles produce rubber in vitro when provided with appropriate substrates. Here, a series of benzophenone-modified initiator analogues successfully bound to the membrane-localized rubber transferase at the particle surface, and initiated rubber biosynthesis in Ficus elastica, Hevea brasiliensis and Parthenium argentatum (shown in micrograph, scale bar = 1 μm).

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Introduction

Natural rubber, cis-1,4-polyisoprene, is a strategically important plant-derived material used in thousands of industrial applications. Currently, Hevea brasiliensis (Brazilian rubber tree) is the sole source of natural rubber; most countries depend on imports of H. brasiliensis rubber to sustain demand. Further, decades of inbreeding have rendered commercial H. brasiliensis varieties susceptible to abiotic stress and pathogen attack.

Alternative natural rubber-producing plants capable of growing in temperate climates are actively sought. Guayule, Parthenium argentatum, is a natural rubber-producing woody shrub native to the southwestern United States and northern Mexico (Bonner, 1943, Backhaus, 1985, Madhavan et al., 1989, Whitworth and Whitehead, 1991). Recently, guayule rubber has been commercialized as an alternative source of rubber, but the need for natural rubber far outweighs the projected growth of the guayule supply. Genetic engineering holds significant potential for increased rubber yields, thereby enhancing the competitiveness of the US domestic rubber crop. Unfortunately, such efforts in crop improvement have been hampered by a lack of gene sequence knowledge, especially for gene(s) encoding the rubber transferase.

Rubber transferase is localized to the surface of cytosolic vesicles known as rubber particles, and biosynthesis is initiated through the binding of an allylic pyrophosphate (APP) primer. Progressive additions of isopentenyl pyrophosphate (IPP) molecules ultimately result in the formation of high molecular weight cis-1,4-polyisoprene (McMullin and McSweeney, 1966, Walsh, 1979, Tanaka, 2001). Enzymatically-active, partially-purified (washed) rubber particles can be isolated such that, when provided with an appropriate APP primer, magnesium ion cofactor, and IPP monomer, rubber is produced in vitro (Archer and Audley, 1967, Light and Dennis, 1989, Madhavan et al., 1989). Kinetic studies determined that rubber transferase is highly tolerant of APP primers of differing lengths and stereochemistries, including dimethyl allyl pyrophosphate (DMAPP), geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP), and others (Archer and Audley, 1987, Cornish et al., 1998). Structural analyses of natural rubber (Tanaka, 1989, Tanaka, 2001, Tanaka et al., 1996) and kinetic analyses of the rubber transferase (Cornish et al, 1998) suggest FPP functions as the actual APP primer in vivo.

Genetic sequences of rubber transferase remain unknown because it is a membrane-associated enzyme present in relatively low abundance (Cornish, 1993, Cornish, 2001a, Cornish, 2001b). Classical biochemical approaches depend upon their ability to follow enzymatic activity throughout protein purification, but activity in rubber particles is rapidly lost upon disruption of their structural integrity. As an alternative, we have chosen an approach of covalent photoaffinity tagging of rubber transferase using benzophenone (Bz)-containing analogs of the rubber biosynthetic initiator, FPP. This approach allows rubber transferase to be followed throughout purification even after enzymatic activity is lost.

Benzophenone-containing photoaffinity labeling probes undergo C–H bond insertion reactions upon excitation with long wavelength (350 nm) light. Stable adducts between a variety of functional groups present in biomolecules and the carbonyl carbon of the benzophenone group form in these reactions, making them highly useful for identifying active site residues and ligand binding sites in proteins (Dorman and Prestwich, 1994, Turek-Etienne et al., 2003) as exemplified in Fig. 1. Photoaffinity labeling studies have been used to identify binding regions in specific enzymes and to isolate a number of previously unidentified proteins, (Yokoyama et al., 1995, Gaon et al., 1996a, Turek et al., 1997, Turek et al., 2001, Zhang et al., 1988, Zhang et al., 2004, Webb et al., 1999) including protein prenyltransferases (Omer et al., 1993, Bukhtiyarov et al., 1995, Edelstein and Distefano, 1997) with remarkable specificity (Dorman and Prestwich, 1994). FPP binding to the rubber transferase active site occurs in a similar manner to the FPP-requiring enzymes mentioned above (Mau et al., 2003). Indeed, we have previously employed a Bz-containing inhibitor of rubber synthesis to label proteins found in enzymatically active rubber particles suggesting that this could be a valuable approach (DeGraw et al., 2007). However, in that case the molecule used was an inhibitor. A better approach would be to use isoprenoid diphosphate analogues that could be bona fide initiators of rubber synthesis. Thus, to identify an appropriate Bz-containing initiator, we have tested a series of Bz-modified FPP analogues for their ability to initiate biosynthesis in rubber particles purified from three different rubber-producing species, Ficus elastica, H. brasiliensis and P. argentatum. Initiator analogues varied by alkyl chain length, by linkage between the alkyl chain and the Bz group (ether vs. ester), and by the position of the Bz relative to the alkyl chain (meta vs. para) (Fig. 2). In studies with farnesyltransferase, all of these analogues (Fig. 2) could inactivate and covalently label the enzyme upon photolysis (Gaon et al., 1996b, Turek et al., 1996, Yokoyama et al., 1995, Turek et al., 2001) suggesting they would be good probes for studying the rubber transferase.

Section snippets

In vitro rubber synthesis by F. elastica, H. brasiliensis and P. argentatum rubber transferases with endogenous initiators

Initial kinetic studies were performed to determine the binding affinities for the naturally-occurring allylic pyrophosphate initiators (i.e. FPP, GPP and DMAPP) using washed rubber particles purified from three different rubber-producing plant species (F. elastica, H. brasiliensis and P. argentatum). In all cases, rubber transferase activity was measured as incorporation of [1-14C] IPP into higher molecular weight rubber produced in vitro and normalized to the amount of rubber present in WRP

Concluding remarks

Benzophenone-modified initiator analogues successfully initiated rubber biosynthesis in the three species studied, reinforcing the observation that the active site of the rubber transferase enzyme is able to accept a wide range of allylic pyrophosphate initiators. The analogs studied, all fairly flexible molecules which can adopt multiple conformations, range in Vmax only by a factor of 10. Despite a range of structural differences these enzyme–substrate systems all successfully initiated

General experimental procedures

[1-14C]IPP (55 mCi/mmol) was obtained from American Radiolabeled Chemicals, Inc. (St. Louis, MO, USA). MultiScreen HTS DV opaque filter plates and vacuum manifolds were from Millipore Co. (Bedford, MA, USA). ScintiVerse BD Cocktail was from Fisher Scientific (Santa Clara, CA, USA). All other chemicals were obtained from Sigma–Aldrich Chemical Company (St. Louis, MO, USA).

Preparation of enzymatically-active rubber particles

Mature, whole P. argentatum shrubs were freshly harvested, shipped overnight, stored at 4 °C, and processed within 96 h. Bark

Acknowledgements

We thank Drs. James Thompson and Christopher Mau for their critical review of this manuscript. We acknowledge Dr. Deborah Scott for helpful suggestions. Ms. De Wood of USDA-ARS provided the beautiful electron micrograph of the washed rubber particle. Dr. Terry Coffelt of USDA-ARS-USALARC kindly provided P. argentatum materials for the washed rubber particle isolation, and Dr. R. Krishnakumar the H. brasiliensis washed rubber particles. This work was supported by the National Science Foundation

References (42)

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