Location: Crop Improvement & Utilization Research
Title: Initiation of rubber biosynthesis: in vitro comparisons of benzophenone-modified diphosphate analogue structure in three natural rubber-producing species Authors
|Distefano, Mark - UNIV OF MINNESOTA|
|Degraw, Amanda - UNIV OF MINNESOTA|
|Cornish, Katrina - YULEX CORPORATION|
|Shintani, David - UNIV OF NV, RENO|
Submitted to: Phytochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 25, 2008
Publication Date: September 15, 2008
Citation: Xie, W., Mcmahan, C.M., Whalen, M.C., Distefano, M., Degraw, A., Cornish, K., Shintani, D. 2008. Initiation of rubber biosynthesis: in vitro comparisons of benzophenone-modified diphosphate analogue structure in three natural rubber-producing species. Phytochemistry. 69:2539-2545. Interpretive Summary: The USDA/ARS Domestic Natural Rubber project seeks to develop rubber-producing crops suitable for cultivation in the temperature climate of the United States. One candidate crop, guayule (Parthenium argentatum Gray), produces high quality natural rubber and is under cultivation on the southwestern US. The rubber is isolated in the form of latex safe for people with Type I Latex allergy, and commercial products are under development. Sustainability of this new industrial crop depends on continued improvement in the efficiency of rubber production. Metabolic engineering of guayule has been successfully accomplished; however to date no increase in rubber production has been confirmed. One hurdle is the lack of knowledge of the structure of rubber transferase. Rubber transferase, the key enzyme in rubber biosynthesis pathway, is probably a cis-prenyl transferase, but the structure of this enzyme, and the corresponding associated genes, is yet to be elucidated. In collaboration with the University of Nevada at Reno and the University of Minnesota, analogs of the natural rubber biosynthesis initiators have been synthesized. The analogs contain a photoreactive (benzophenone) functional group that allows them to be crosslinked to the enzyme with which they are bound. Similar molecules have been successfully used to isolate other isoprenyl pathway enzymes. In this work, we show that the structurally-modified analogs do indeed serve as initiators of rubber biosynthesis, paving the way for their use as probe molecules for rubber transferase structural studies.
Technical Abstract: Natural rubber is synthesized by initiation with one allylic pyrophosphate (APPs) molecule followed by elongation with thousands of isopentenyl pyrophosphate (IPP) molecules by the enzyme rubber transferase (a cis-prenyl transferase). To better understand how rubber transferase initiates and binds APPs, two benzophenone-linked geranyl pyrophosphate (Bz-GPP) analogs and four benzophenone-linked dimethylallyl pyrophosphate (Bz-DMAPP) analogs were synthesized and used for rubber synthesis studies in vitro using washed rubber particles from Ficus elastica, Hevea brasiliensis and Parthenium argentatum. Rubber transferases from all three species initiate rubber biosynthesis most efficiently with farnysyl pyrophosphate (FPP) as expected. However, Bz-GPP ether analogs have a higher affinity to rubber transferase than GPP. Similarly, Bz-DMAPP ether analogs are more efficient initiators when compared to that of DMAPP. In contrast Bz-DMAPP ester analogs are less efficient initiators than DMAPP. Thus rubber synthesis was affected by the size and structure of initiators and the resulting information will allow further study of the enzyme binding sites by photolabeling the active sites of rubber transferase.