Terpene biosynthesis enzymes

The enzyme catalyzed reactions that lead to geraniol and farnesol (as their pyrophosphate esters) are mechanistically related to the acid catalyzed dimerization of alkenes discussed m Section 6 21 The reaction of an allylic pyrophosphate or a carbo cation with a source of rr electrons is a recurring theme m terpene biosynthesis and is invoked to explain the origin of more complicated structural types Consider for exam pie the formation of cyclic monoterpenes Neryl pyrophosphate formed by an enzyme catalyzed isomerization of the E double bond m geranyl pyrophosphate has the proper geometry to form a six membered ring via intramolecular attack of the double bond on the allylic pyrophosphate unit... 

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. 

Another example of successful engineering of terpene biosynthesis is the constitutive overexpression of the gene encoding the first-step enzyme 1-deoxy-D-xylulose-5-phosphate synthase (DXPS) in the DXP pathway in bacteria and Arabidopsis. In both cases, increased enzyme activity caused increased accumulation of downstream terpenoids, suggesting that DXPS is rate-limiting [8]. 

Since squalene can be produced from farnesyl pyrophosphate with NADPH and a suitable enzyme system, the general features of the above scheme for terpene biosynthesis are well supported by experiment. 

Table 5.2 Isolated genes encoding several major classes of enzymes In terpene biosynthesis...

Most successful attempts to isolate the enzymes involved in terpene biosynthesis have come from these early stages. Crude in vitro systems will frequently convert - [2- C]mevalonic acid (1) into its phosphate (2) and pyrophosphate (3), isopentenyl pyrophosphate (4), and dimethylallyl pyrophosphate (5). However, only traces of radioactivity are recovered from the prenol pyrophosphates (6). As well as phosphorylating mevalonic acid the same enzyme, or a related one, is... 

Several of the individual enzymes of the early steps of terpene biosynthesis have been isolated. Mevalonic kinase [EC 2.7.1.36, (1) — (2)] has been prepared from several sources, for example Phaseolus vulgaris and pig liver. A feed-back control mechanism was suggested for the animal enzyme. Kekwick determined detailed kinetic data for pyrophosphomevalonate decarboxylase [EC 4.1.1.33,... 

The Search for Genes Encoding Short-Chain Isoprenyl Diphosphate Synthases-Branch-point Enzymes of Terpene Biosynthesis... 

The stereochemistry is well established, and many questions concerning the overall mechanism of the condensation have now been resolved. Famesyl pyrophosphate synthetase (EC 2.5.1.1) is the key enzyme in the biosynthetic pathways for several classes of terpenes. This enzyme catalyzes l -4 condensation between IPP and DMAPP, or geranyl pyrophosphate, polymerizations that constitute the major building steps of terpenoid biosynthesis (Fig. 21.2) (Poulter and Rilling, 1978 Poulter et al., 1978, 1981). The condensation... 

As presented in this chapter, today, much is known about the process of terpene biosynthesis. The accumulated knowledge includes a detailed picture about the biosynthesis of the terpenoid monomers IPP and DMAPP either via the mevalonate or the DXP route and their interconversion by isomerases. Also, the stereochemical courses and enzyme mechanisms of all transformations have been largely elucidated. Especially the recently obtained structural data of prenyltransferases and various kinds of terpene synthases resulted in an evolutionary model that involves six domains (a, P, 7,8, e, and Q for the biosynthesis of linear polyisoprenoids from IPP and DMAPP and their subsequent transformation into (poly)cyclic terpenes. All these insights may open up new chances in controlling terpene biosynthesis, e.g., by directed evolution of terpene cyclases or domain swaps in multi-domain enzymes for the production of new terpenes, reconstitution of terpene biosynthetic pathways in heterologous hosts for production optimization, or targeted inhibitirm of pathways in pathogens for disease control. 

Other enzymes involved in terpene biosynthesis have also been harnessed for biocatalytic reactions of arenes. Prenyltransferase enzymes that can affect the addition of Cj isoprenyl units both at carbon and at heteroatoms have been used for biocatalytic arene alkylations. For example, L-tryptophan 49 undergoes prenylation at various positions on the indole core in a wholly regiose-lective fashion, depending on the enzyme used [28] (Scheme 32.6). 

Takahashi et al. 1998) in possessing an N-terminal amino acid sequence which is not part of the mature protein but functions as a transit peptide which is used by the plant cell to target the nascent enzyme to the organelle where terpene biosynthesis takes place. Once inside the plastid, the transit peptide is cleaved by proteolysis and the remainder folds into the active, soluble protein. Transit peptides are a ubiquitous feature of the plastidic terpene biosynthesis pathway in plants (for other examples see Williams et al. 1998 Bohlmann etal. 2000 Okada etal. 2000). 

The usual method of study is to suggest a possible precursor and to feed it to the biosynthesizing system. The precursor has to be labelled in some way to trace it through the sequence of reactions, and that is usually by some isotopic element. It may be a radio-active isotope, such as H, " 0, or that can be followed by its radiation or it can be a stable heavy isotope, such as H, C, N, or 0, that can be traced by mass spectrometry or nuclear magnetic resonance (NMR) spectroscopy (Table 5.1). Another possible way is to use mutant strains of an organism that lack the enzymes to complete a particular synthesis, or to add a specific enzyme inhibitor, so that intermediates accumulate and can be identified. A mutant strain of yeast was important in discovering mevalonic acid and its place in terpene biosynthesis (Chapter 6) and a number of mutants of the bacterium Escherichia coli helped to understand the shikimic acid pathway (Chapter 8). 

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