Submitted to: Journal of Organic Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/21/2007
Publication Date: 6/8/2007
Citation: Degraw, A.J., Zhao, Z., Strickland, C.L., Taban, A., Hsieh, J., Jeffries, M., Xie, W., Shintani, D., Mcmahan, C.M., Cornish, K., Distefano, M. 2007. A photoactive isoprenoid diphosphate analogue containing a stable phosphonate linkage: synthesis and biochemical studies with prenyltransferases. Journal of Organic Chemistry. 72: 4587-4595. Interpretive Summary: Our CRIS project aims to develop domestic sources of natural rubber to improve supply security in the Unites States, and provide a solution for people with Type I latex allergy while enhancing rural development with new industrial rubber-producing crops. The biosynthesis of rubber is part of the isoprenoid pathway in rubber-producing plants. While many steps are well understood, complete pathway metabolic engineering depends on identification of rubber transferase, the key enzyme producing the macromolecule. This study describes the use of crosslinkable initiator analogs which may be of assistance in isolating, purifying, and identifying the protein sequence of the rubber transferase enzyme.
Technical Abstract: A number of biochemical processes rely on isoprenoids, including the post-translational modification of signaling proteins and the biosynthesis of a wide array of compounds. Photoactivatable analogues have been developed to study isoprenoid utilizing enzymes such as the isoprenoid synthases and prenyltransferases. While these initial analogues proved to be excellent structural analogues with good cross linking capability, they lack the stability needed when the goals include isolation of cross-linked species, tryptic digestion, and subsequent peptide sequencing. Here, the synthesis of a benzophenone-based farnesyl pyrophosphate analogue containing a stable phosphonophosphate group is described. Inhibition kinetics, photolabeling experiments, as well as x-ray crystallographic analysis with a protein prenyltransferase are described, verifying this compound as a good isoprenoid mimetic. In addition, the utility of this new analogue was explored by using it to photoaffinity label crude protein extracts obtained from Hevea brasiliensis latex. Those experiments suggest that a small protein, Rubber Elongation Factor, interacts directly with farnesyl diphosphate during rubber biosynthesis. These results indicate that this benzophenone-based isoprenoid analogue will be useful for identifying enzymes that utilize farnesyl diphosphate as a substrate.