Enzymes prenyltransferase

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. 

Prenyltransferases are a class of enzymes that transfer allylic prenyl groups to acceptor molecules. Prenyl transferases commonly refer to prenyl diphosphate synthases (even though the class of prenyl transferases also includes enzymes that catalyze the transfer of prenyl groups to acceptors that include not only isopentenyl diphosphate (IPP) but also aromatic compounds and proteins etc.). 

The prenyl chain elongation catalyzed by prenyltransferases is quite unique because the reaction proceeds consecutively and terminates precisely at discrete chain lengths according to the specificities of the enzymes. The chain length of products varies so widely that it ranges from geraniol (CIO) to natural rubber (C > 5000). 

FIGURE 3.5 Biosynthetic route to isoflavonoids (and some derivatives) from the 5-deoxyflavanone liquiritigenin. A possible route to the retrochalcone echinatin is also shown. Unlabelled arrows indicate biosynthetic steps for which the enzyme(s) have not been characterized. Enzyme abbreviations are defined in the text and in Table 3.1, except for P2CP, pterocarpan 2-C-prenyltransferase P4CP, pterocarpan 4-C-prenyltransferase. 

However, there are also examples of prenyltransferase in which substrates are only poorly accepted with low rate constants (Kc.lt/Knl). especially when their structures differ significantly from the natural substrates of the enzymes. There are two ways to avoid such drawbacks in future (1) enzyme optimisation by site-directed mutation and (2) significantly increased catalyst overexpression, so that the amount of enzyme is not the limiting factor. 

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. 

Terpene synthases, also known as terpene cyclases because most of their products are cyclic, utilize a carbocationic reaction mechanism very similar to that employed by the prenyltransferases. Numerous experiments with inhibitors, substrate analogues and chemical model systems (Croteau, 1987 Cane, 1990, 1998) have revealed that the reaction usually begins with the divalent metal ion-assisted cleavage of the diphosphate moiety (Fig. 5.6). The resulting allylic carbocation may then cyclize by addition of the resonance-stabilized cationic centre to one of the other carbon-carbon double bonds in the substrate. The cyclization is followed by a series of rearrangements that may include hydride shifts, alkyl shifts, deprotonation, reprotonation and additional cyclizations, all mediated through enzyme-bound carbocationic intermed iates. The reaction cascade terminates by deprotonation of the cation to an olefin or capture by a nucleophile, such as water. Since the native substrates of terpene synthases are all configured with trans (E) double bonds, they are unable to cyclize directly to many of the carbon skeletons found in nature. In such cases, the cyclization process is preceded by isomerization of the initial carbocation to an intermediate capable of cyclization. 

Croteau, R. and Purkett, P.T. (1989) Geranyl pyrophosphate synthase characterization of the enzyme and evidence that this chain-length specific prenyltransferase is associated with monoterpene biosynthesis in sage (Salvia officinalis). Arch. Biochem. Biophys., 271,524—35. 

Although several previous reports claimed that the enzyme had been purified, Gebler and Poulter (1992) appear to have been the first to fully characterize the activity of the purified DMAT synthase. The enzyme was purified from Claviceps fusiformis ATCC 26245 [erroneously annotated in type specimen collections as a C. purpurea strain (Pazoutova and Tudzynski, 1999)]. The monomeric size was estimated at 53 kDa, and by gel filtration analysis the native enzyme was determined (at 105 kDa) to be a homodimer. Unlike other prenyltransferases, no metal ion requirement has been noted. However, when assayed in a buffer with 4 mM Ca2+, the purified protein gave a specific activity of 500 nmol/min/mg, essentially the same as with 4 mM Mg2+, but approximately twice that of the measured without added divalent cations and with the chelator EDTA included in the assay buffer. These divalent metal cations eliminated negative cooperativity of substrate binding observed both for dimethylallyl diphosphate and L-tryptophan, indicating that Ca2+ and Mg2+ probably had allosteric effects. In buffer with 4 mM MgCl2 the KM for dimethylallyl diphosphate was 8 jlM, and the KM for L-tryptophan was 12 xM. The enzyme product was authenticated by mass spectrometry, UV spectrometry, and -NMR. 

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