Prenyl diphosphate substrates

This enzyme [EC 2.5.1.1] (also referred to as prenyl-transferase and geranyl-diphosphate synthase) catalyzes the reaction of dimethylallyl diphosphate and isopen-tenyl diphosphate to produce geranyl diphosphate and pyrophosphate (or, diphosphate). The enzyme will not accept larger prenyl diphosphates as substrates. 

Synthetic derivatives and analogs of prenyl diphosphates have historically played a key role in defining key featnres of the mechanism of enzymes that ntilize these key intermediates in the isoprenoid pathway. This has also been the case with the investigation of the protein prenyl-transferases. A brief introduction to the protein prenyltransferase enzymes is given along with outlines on the previous use of prenyl diphosphate tools and key aspects of their synthesis. The development of prenyl diphosphate-based FTase inhibitors is described. The use of prenyl diphosphate derivatives as mechanistic and structural probes is next discussed. In particular, the use of fluorinated, isotopically labeled, and photoaffinity derivatives is presented. An overview of the extensive work on the determination of FTase isoprenoid substrate specificity is then given, and the chapter concludes with a section on the development of prenyl diphosphate tools for proteomic studies. 

Hemiterpenoids are produced from the isoprenyl diphosphate DMAPP. All other terpenoids are produced from DMAPP and IPP via longer-chain prenyl diphosphate intermediates formed by prenyl transferases. Prenyl transferases (20) catalyze the formation of geranyl diphosphate (GPP), famesyl diphosphate (FPP), and geranylgeranyl diphosphate (GGPP) from one molecule of DMAPP and one, two, or three molecules of IPP, respectively (Fig. 1). Isoprenyl diphosphates are the substrates for all TPS, which lead to the hemiterpenoids, monoterpenoids, sesquiterpenoids, and diterpenoids, which will be highlighted with selected examples in the following sections. 

S-prenylation is the most recent of the four major types of lipid modifications to be described. As with -acylation, S-prenylation is posttranslational. The lipid substrates for these modifications are farnesyl diphosphate and geranylgeranyl diphosphate. The mechanism... 

Prenylation, the key step in terpene biosynthesis, is catalyzed by prenyltransferases. These enzymes are responsible for the condensation of isopentenyl pyrophosphate (IPP) with an allyl pyrophosphate, thus yielding isoprenoids. Numerous studies have been performed with fluorinated substrates in order to determine the mechanism of the reactions that involve these enzymes prenyltransferases, farnesyl diphosphate synthase (FDPSase), famesyltransferase (PFTase), and IPP isomerase. These studies are based on the potential ability of fluorine atoms to destabilize cationic intermediates, and then slow down S l type processes in these reactions. 

A further element shared by all sesquiterpene synthases is the need for a divalent metal ion as cofactor. The metal ion is essential for substrate binding but also for product specificity. The metal ions stabihze the negatively charged pyrophosphate group of farnesyl diphosphate as illustrated by the crystal structure of 5-epz-aristolochene synthase [9]. The highly conserved sequence (I, L, V)DDxxD(E) serves to bind the metal ions in all known terpene and prenyl synthases (Fig. 5) [18-22]. A further interesting property among terpene synthases is that the active sites are enriched in relatively inert amino acids, thus it is the shape and dynamic of the active site that determines catalytic specificity [23]. 

Prenyltransferase activities have been studied in C. roseus both at the enzyme level and at the product level. Biosynthetic capabilities were investigated by incubating [1- C]IPP with aliquots of cell-free homogenates prepared from P. aphanidermatum treated and untreated suspension-cultured cells of C. roseus. After elicitation, the total incorporation of IPP into prenyl lipids was decreased, in particular into squalene. But the incorporation of IPP into some (as yet unidentified) compounds was increased (99). The prenyltransferases and subsequent enzyme activities are relatively easily extracted and remain complexed so that the product of one enzyme can be used as a substrate for the next enzyme. With an assay for these enzymes as described in detail in Threlfall and Whitehead (101), about a dozen enzyme activities could be detected in series using cell-free preparations of elicited Tabemaemontana divaricata cells (27). In the elicited C. roseus cells, the activities of IPP isomerase, famesyl diphosphate synthase, squalene synthase, squalene-2,3-epoxidase (and probably also a squalene-2,3-epoxide cyclase) were thus detected. Compared with the control nontreated cells, squalene production seemed to be reduced particularly (99). 

The first reaction involves a prenyl transfer step in which C(10 of one of the allylic substrates (the donor) is bonded to the C(2)-C(3) double bonds of the other (the receptor) to produce a cyclopropylcarbinyl diphosphate with a CV-2-3 structure (Poulter, 1990). In this manner, famesyl pyrophosphate (8) yields presqualene pyrophosphate (9). Only (/ )-presqualene pyrophosphate has been found in nature. 

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