Sesquiterpenes synthases

Schnee C, Koellner TG, Held M, Turlings TCJ, Gershenzon J, Degenhardt J (2006) The products of a single maize sesquiterpene synthase form a volatile defense signal that attracts natural enemies of maize herbivores. Proc Natl Acad Sci USA 103 1129-1134 Pichersky E, Gershenzon J (2002) The formation and function of plant volatUes perfumes for pollinator attraction and defense. Curr Opin Plant Biol 5 237-243... 

ThoU D, Chen E, Petri J, Gershenzon J, Pichersky E (2005) Two sesquiterpene synthases are responsible for the complex mixture of sesquiterpenes emitted from Arabidopsis flowers. Plant J 42 757-771... 

Schnee C, Kollner TG, Gershenzon J, Degenhardt J (2002) The maize gene terpene synthase 1 encodes a sesquiterpene synthase catalyzing the formation of ( )-P-farnesene, ( )-nerolidol, and ( , )-famesol iter herbivore damage. Plant Physiol 130 2049-2060... 

Xu YH, Wang JW, Wang S, Wang JY, Chen XY (2004) Characterization of GaWRKYl, a cotton transcription factor that regulates the sesquiterpene synthase gene (-l-)-delta-cadinene synthase-A. Plant Physiol 135 507-515... 

Zook M, Hohn T, Bonnen A, Tsuji J, Hammerschmidt R (1996) Characterization of novel sesquiterpenoid biosynthesis in tobacco expressing a fungal sesquiterpene synthase. Plant Physiol 112 311-318... 

Steele CL, Crock J, Bohlmann J, Croteau R. (1998). Sesquiterpene synthases from grand fir Abies grandis). Comparison of constitutive and wound-induced activities, and cDNA isolation, characterization, and bacterial expression of delta-selinene synthase and gamma-humulene synthase./owrna/ of Biological Chemistry, 273,2078. 

In the last few years, sesquiterpene synthase from different plants has raised attention. In 2004, Schalk and Clark [88] described a process (patented by Fir-menich, Switzerland) that makes it possible to obtain sesquiterpene synthase and to produce various aliphatic and oxygenated sesquiterpenes from farnesyl diphosphate. For instance, valencene can be obtained in this way. 

One year later, Schalk [89] described a process for cloning sesquiterpene synthases from patchouli plants Pogostemon cablin) and the enzyme-catalysed terpenoid production. Various sesquiterpenes can be obtained by this method, for instance patchoulol and other germacrene-type sesquiterpenes. 

Most of the compounds shown in Figure 22-4 are derived from the C15 famesyl diphosphate. There are more than 300 known cyclic structures among these sesquiterpenes, and many sesquiterpene synthases have been characterized.91/91a Aristolochene is formed by the action of a 38-kDa cyclase that has been isolated from species of Penicillium and Aspergillus,92"4 Notice that the synthesis must involve two cyclization steps and migration of a methyl group. Three-dimensional structures are known for at least two terpene synthases,95 96 and comparison of gene sequences suggests that many others have similar structures. 

Another sesquiterpene synthase forms trichodi-ene, the parent compound for a family of mycotoxins and antibiotics.97/97a b A different sesquiterpene synthase, present in peppermint and also in a wide range of other plants and animals, forms the acyclic (E)-(J-... 

Figure 10.8 Famesyl diphosphate (FPP) can be cyclized in many different ways by sesquiterpene synthases giving rise to different classes of sesquiterpenes. The eremophilane class of sesquiterpenes has received considerable attention because the genes coding for the corresponding synthase enzymes were the first to be isolated. 25...

Figure 10.9 Each class of sesquiterpenes (see Fig. 10.8) consists of many structural variants that arise from stereochemical positioning of methyl groups and sites of double bond formation. Depicted are the possible variants within the eremophilane class of sesquiterpenes, which are hypothesized to arise from an equally related class of sesquiterpene synthases. Only those structural derivatives that have been found in nature or synthesized are denoted by name. The remaining structures should, therefore, be considered novel.

Another possibihty to gain the product encoded by a silent gene cluster can be achieved by a method called the in vitro reconstitution approach. A necessity is that the substrates of the pathway are predictable. The in vitro reconstitution of an entire biosynthetic pathway usually involves the separate overexpression of each gene, the purification of the resulting protein and the performance of enzymatic studies. Thus, the discovery of a fully elaborated metabolic product by this approach is likely to be very laborious. For example, after discovering a cryptic sesquiterpene synthase in the genome of S. coelicolor (64), the new epi-isozaene (8) could be produced successfully. This metabolite was shown to be an intermediate in the assembly line of the known Streptomyces sesquiterpene albaflavenone (9) (135). 

Lin X, Hopson R, Cane DE (2006) Genome Mining in Streptomyces coelicolor Molecular Cloning and Characterization of a New Sesquiterpene Synthase. J Am Chem Soc 128 6022... 

The recently published crystal structure of tobacco epi-aristolochene synthase (a sesquiterpene synthase) has provided the first look at the three-dimensional configuration of a plant terpene s)mthase (Starks et al, 1997). 

Crock, J., Wildung, M. and Croteau, R. (1997) Isolation and bacterial expression of a sesquiterpene synthase cDNA clone from peppermint (Mentha x piperita, L.) that produces the aphid alarm pheromone (E)-beta-famesene. Proc. Natl. Acad. Sci. USA, 94, 12833-8. 

Lucker, J., Bowen, P. and Bohlmann, J. (2004) Vitis vinifera terpenoid cyclases functional identification of two sesquiterpene synthase cDNAs encoding... 

The first step taken in the biosynthetic pathway of artemisinin was the cyclization of the general mevalonate pathway originated sesquiterpenoid precursor farnesyl diphosphate (FPP) into IS, 6R, 7R, i0it)-amorpha-4,ll-diene by amorpha-4,11-diene synthase (AMDS) (Fig. 4) [6-8]. The crystal structure of this sesquiterpene synthase is not known. From aU plant... 

The three-dimensional structures of three non-plant sesquiterpene synthases reveals a single domain composed entirely of a-hehces and loops despite the low homology on amino acid sequence level [14,16,17]. The secondary elements of 5-epi-aristolochene synthase, a plant sesquiterpene synthase, conform to this pattern with the exception of two domains solely composed of a-helices and loops. It is reasonable, but still a matter of debate, to extrapolate these data to the case of amorpha-4,11-diene synthase, which will probably only display a-hehces and loops once the crystal structme has been solved. 

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]. 

Picaud et al. purified recombinant AMDS and determined its pH optimum to 6.5 [24]. Several sesquiterpene synthases show maximum activity in this range examples are tobacco 5-epz-aristolochene synthase [25,26], germa-crene A synthase from chickory [26] and nerolidol synthase from maize [27]. Terpenoid synthases are, however, not restricted to a pH optimum in this range. Intriguing examples are the two (-l-)-(5-cadinene synthase variants from cotton, which exhibit maximum activity at pH 8.7 and 7-7.5, respectively [28] and 8-epi-cedrol synthase from A. annua [29] with the pH optimum around... 

X 10 p,M s with Mn " " as a cofactor. These very low efficiencies are common to several sesquiterpene synthases but substantial differences have been reported. The synthase reached a kcatK value of 9.7 x 10 p,M s for conversion of FPP at pH 9.5 using Mg + as a metal ion cofactor. This increase in efficiency is interesting and shows the broad window in which the enzyme... 

Modification of enzymes involved in the synthesis from universal precursors of other secondary metabolites has also been demonstrated. The sesquiterpene synthases are a large family of enzymes that act by a common mechanism and catalyze the cyclization of famesyl diphosphate, each to form a distinct sesquiterpene. The active site of the synthase that produces trichodiene, the... 

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