Trichodiene synthase

A descriptor for an enzyme active site that permits binding of a family of related compounds (e.g., mimics of the reaction intermediate) that can be derived from the initial binding and conformational changes in the substrate. This concept arose from the observation that a number of monoterpene cyclases were incapable of discriminating between enantiomers of the reaction intermediate, even though the enzyme catalyzes the synthesis of an enantiomerically pure product from an achiral substrate. An example is trichodiene synthase which catalyzes the cyclization of farnesyl diphosphate to trichodiene. 

A term used to describe inhibition induced by a second inhibitor. A particular inhibitor (F) may be a weak inhibitor for a certain enzyme however, in the presence of a second inhibitor (F), inhibition is greatly enhanced. An example of such a system is the inhibitory effect of inorganic pyrophosphate on trichodiene synthase by certain aza analogues Both enantiomers of serine are weak inhibitors of y-glutamyl transpeptidase (L-serine has a Ki value of 10.7 mM), but in the presence of borate ion, they are potent inhibitors ... 

Gallo, A. (2004). Isolation and characterisation of a trichodiene synthase homologous gene in Trichoderma harzianum. Physiol. Mol. Plant Pathol. 65,11-29. 

Straus, N. A., and Wong, B. (1998). Stachybotrys chartarum trichodiene synthase (TR15) gene, complete cds. GenBank accession no. AF053926, direct submission, 17. March 1998.. ... 

Zook, M., K. Johnson, T. Hohn, and R. Hammerschmidt. Structural characterization of 15-hydroxytrychodiene, a ses-quiterpenoid produced by tansformed tobacco cell suspension cultures expressing a trichodiene synthase gene from Fusarium sporotrichoid.es. Phytochemistry 1996 43(6) 1235-1237. Eklund, A.M., I. Eorsblom, ]. E. Berg, C. Damberg, and I. Wahlberg. Tobacco chemistry. Four new cyclized cembranoids from tobacco. Acta Chem Scand Ser A 1998 52(10) 1254-1262. 

Crystal structure determinations of trichodiene synthases were reported, including structures of ternary complexes incorporating inorganic pyrophosphate and an aza analogue of the bisabolyl carbocation intermediate.220,221 The conformation and orientation of the carbocation analogue lead to the conclusion that carbocation intermediates... 

CANE, D. E., XUE, Q., FITZSIMONS, B. C., Trichodiene synthase, probing the role of the highly conserved aspartate-rich region by site-directed mutagenesis, Biochem., 1996 35,12369-12376. 

RYNKIEWICZ, M. J., CANE, D. E CHRISTIANSON, D. W., Structure of trichodiene synthase from Fusarium sporotrichioides provides mechanistic inferences on the terpene cyclization cascade, Proc. Natl. Acad. Sci. USA, 2001, 98, 13543-13548. 

Cane, D. E. and Xue, Q. (1996) Trichodiene synthase. Enzymatic formation of multiple sesquiterpenes by alteration of the cyclase active site. J Am Chem. Soc. 118,1563,1564. 

Cell suspension culture of N. tabaccum transformed with a gene encoding trichodiene synthase from F. spomtrichioides. 

Studies in which [2--mevalonic acid lactone ( mevalonate ) (105) was used as a precursor showed that three molecules were incorporated into trichothecolone (101), the label appearing at positions 4, 8, and 14 (249), The involvement of 2-rran5-6-rra -famesyldiphosphate (106) had been shown previously using " C-labelled material 250), The biosynthesis thus follows a standard sesquiterpene pathway from mevalonate through famesyldiphosphate to nerolidyldiphosphate (107) (257, 252), which is then cyclised by the enzyme trichodiene synthase to the bicy-clic hydrocarbon trichodiene (49) (253). The evidence for the participation of nerolidyldiphosphate has been reviewed (254, 255). None of the possible enzyme bound intermediates has been isolated from what is shown (107, 108) as a concerted process involving a 1,4-hydride shift and two 1,2-methyl shifts (256),... 

Trichodiene synthase has been obtained from T, roseum (259) and subsequently from F, sporotrichioides (260, 261) and F, sambucinum (261), the F, sporotrichioides enzyme has been characterised. Isomerisation of famesyldiphosphate to nerolidyldiphosphate is the rate-determining step in the reaction pathway (262). Factors affecting substrate recognition by the active site have been studied (263), and a number of amino-acid... 

Another important application arising from the work with trichodiene synthase has been the development of Tri5 specific polymerase chain reaction-based assays for the detection of potential trichothecene-producing Fusarium species in pure culture and in contaminated grain (269,275,276). 

Miller JD, MacKenzie S (2000) Secondary Metabolites of Fusarium venenatum Strains with Deletions in the Tri5 Gene Encoding Trichodiene Synthase. Mycologia... 

Zook M, Johnson K, Hohn T, Hammerschmidt R (1996) Structural Characterization of 15-Hydroxytrichodiene, a Sesquiterpenoid Produced by Transformed Tobacco Cell Suspension Cultures Expressing a Trichodiene Synthase Gene from Fusarium sporotrichioides. Phytochemistry 43 1235... 

Hohn TM, Beremand MN (1989) Regulation of Trichodiene Synthase in Fusarium sporotrichioides and Gibberella pulicaris (Fusarium sambucinum). Appl Environ Microbiol 55 1500... 

Cane DE, Yang G, Xue Q, Shim JH (1995) Trichodiene Synthase. Substrate Specificity and Inhibition. Biochemistry 34 2471... 

Cane DE, Shim JH, Xue Q, Fitzsimons BC, Hohn TM (1995) Trichodiene Synthase. Identification of Active Site Residues by Site-Directed Mutagenesis. Biochemistry 34 2480 (1997) 36 9636... 

Cane DE, Xue Q, Fitzsimons BC (1996) Trichodiene Synthase. Probing the Role of the Highly Conserved Aspaitate-Rich Region by Site-Direeted Mutagenesis. Biochemistry 35 12369... 

Hohn TM, Desjardins AE (1992) Isolation and Gene Disruption of the Tox5 Gene Encoding Trichodiene Synthase in Gibberella pulicaris. Mol Plant-Microbe Interact 5 249... 

Hohn TM, Plattner RD (1989) Expression of the Trichodiene Synthase Gene of Fusarium sporotrichioides in Escherichia coli Results in Sesquiterpene Production. Arch Biochem Biophys 275 92... 

Cane DE, Wu Z, Oliver IS, Hohn TM (1993) Overproduction of Soluble Trichodiene Synthase from Fusarium sporotrichioides in Escherichia coli. Arch Biochem Biophys 300 416... 

Desjardins AE, Hohn TM, McCormick SP (1992) Effect of Gene Dismption of Trichodiene Synthase on the Virulence of Gibberella pulicaris. Mol Plant-Microbe Interact 5 214... 

The nearly complete catalytic intolerance for glutamyl and alanyl substitutions in the DDxxD motif of limonene synthase is novel and unlike the much less pronounced effects of comparable substitutions in the sesquiterpene cyclase trichodiene synthase [97, 98]. However, pre-steady state kinetic analysis of trichodiene synthase [101] and several other sesquiterpene synthases [102] has recently shown that product release is rate limiting in these cases, and thus can mask the kinetic influence of the aspartate mutations on earlier steps in the catalytic cycle. In the instance of monoterpene cyclase catalysis, product release is not the slow step since comparison of k at values with GPP and LPP as substrate clearly reveals the initial ionization-isomerization to be rate limiting. Thus, perturbations that influence the first ionization step will be fully reflected in overall rate suppression for limonene synthase. This kinetic sensitivity at the initial steps of the reaction cycle does not, however, explain the near complete intolerance of limonene synthase to aspartate substitution in the DDxxD motif and it is thus tempting to speculate a more specific, but presently unidentified, influence on the requisite isomerization of GPP. 

Although the primary structures of the microbial sesquiterpene cyclases share little with their plant counterparts, similar tertiary structures, common electrophilic reaction mechanisms, and conservation of the DDxxD substrate binding motif make it instructive to describe trichodiene synthase from Fusarium sporotrichioidesy about which considerable information is available. 

Consistent with the participation of arginine residues in diphosphate orientation and ionization by monoterpene and sesquiterpene synthases [91,117] is the substitution of a highly conserved RxR motif by RxC in y-humulene synthase (residues 306-308). Perhaps this substitution in y-humulene synthase perturbs diphosphate binding in much the same way as was suggested for the central arginine mutation in the DRRYR motif of trichodiene synthase which resulted in multiproduct formation [117]. 

This a-domain is common to all class I terpene synthases and is the only domain in bacterial and fungal class I enzymes such as the pentalenene synthase from Streptomyces ey oliatus or the trichodiene synthase from Fusarium sporotri-chioides (Fig. 87.14a) [191,194]. Plant hemi-, mono-, and sesquiterpene synthases exhibit a second helical p-domain that resembles a barrel structure. An example of this class is given by the (+)-5-cadinene synthase from G. arboreum (Fig. 87.14b) [195]. In plant diterpene cyclases and in the exceptional case of the ( )-a-bisabolene synthase from A. grandis (Fig. 87.14c), a third helical y-domain with a barrel-like structure is present [197, 200, 202]. The p- and y-domains in the plant enzymes are nonfunctional but are required for correct enzyme folding. 

Tri5 (encoding trichodiene synthase). TRI5 catalyzes cyclization of all-tran -FPP via nerolidyl pyrophosphate to TDN [36,45]. 

In another example, a gene, isolated from the fungus Fusarium spmrotrichioides, which encodes for trichodiene synthase (the enzyme responsible for the production of the toxic sesquiterpenoid trichodiene) has been introduced into tobacco (23) and trichodiene was accumulated at low... 

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