Isoprenoids are a diverse class of metabolically-synthesized organic chemicals with function related to polymer length. Isoprenoids are relevant most-visibly as the main component of natural rubber. Natural rubber is, specifically, a cis-1,4-polyisoprene composed of an elongated chain of isoprene subunits that are initiated with a trans-conformation farnesyl pyrophosphate and terminated with a pyrophosphate. Cis-prenyltransferases (CPT) are the class of enzymes that catalyze formation of single-chain isoprenoids of different lengths (figure 1) and corresponding functions. Bacteria use CPTs to produce undecaprenyl pyrophosphate to build cell walls, making this CPT a potential target for antibacterials. Eukaryotes use a CPT to produce dehydrodolichyl pyrophosphate -a precursor to dolichol- which is necessary for protein decoration. Plants have novel CPTs that ultimately make natural rubber, a vital material for our civilization. CPTs are typically associated with lipid bodies and other hydrophobic compartments, yet their function is usually tested in aqueous reactions. One clue that aqueous reactions are inappropriate for studying CPT activity was the discovery that detergents were needed to stimulate the reaction. We therefore propose that in vitro study of CPTs require simulation of in vivo hydrophobic compartments, and propose aqueous-organic two-phase systems (AOTPS) as a model reaction platform. This work uses undecaprenyl pyrophosphate synthase from Microccocus luteus as a model CPT as it was the first cloned CPT with a crystal structure. Our goals, beyond gaining basic knowledge into the specific kinetics of CPTs, are to (1) learn how to manipulate CPT-product molecular weight and polydispersity with AOTPS models, and to (2) develop a useful tool for the scientific communities to study CPTs (such as the putative rubber transferase) in vitro. Other benefits to an AOTPS models for CPT activity is that we can potentially sequester product away from the enzyme and substrates, reducing product inhibition and simplifying separation of targets.
Figure 1. Simplified schematic of cis-prenyltransferase chain elongation whereby isoprenoid subunits condense on the end of a growing polyisoprene chain and release a pyrophosphate from the existing end.
Figure 2. Simplified Schematic of a cis-prenyltransferase reaction in an aqueous-organic two-phase system.
- Y. Kharel, and T. Koyama (2003). "Molecular analysis of cis-prenyl chain elongating enzymes", The Royal Society of Chemistry. 20, 111-118.
- B. Ku, J.-C. Jeong, B. Mijts, C. Schmidt-Dannert, and J.S. Dordick (2005), “Preparation, Characterization, and Optimization of an In Vitro C30 Carotenoid Pathway", Appl. Environ. Microbiol. 71, 6578-6583.
- Y. Kharel, S. Takahashi, S. Yamashita, and T. Koyama (2006), “Manipulation of prenyl chain length determination mechanism of cis-prenyltransferases”, Febs Journal. 273, 647-657.