Abstract(+)-δ-Cadinene synthase (DCS) from Gossypium arboreum (tree cotton) is a sesquiterpene cyclase that catalyzes the cyclization of farnesyl diphosphate in the first committed step of the biosynthesis of gossypol, a phytoalexin that defends the plant from bacterial and fungal pathogens. Here, we report the X-ray crystal structure of unliganded DCS at 2.4 Å resolution and the structure of its complex with three putative Mg 2+ ions and the substrate analogue inhibitor 2-fluorofarnesyl diphosphate (2F-FPP) at 2.75 Å resolution. These structures illuminate unusual features that accommodate the trinuclear metal cluster required for substrate binding and catalysis. Like other terpenoid cyclases, DCS contains a characteristic aspartate-rich motif D 307 DTYD 311 on helix D that interacts with Mg 2+A and Mg 2+ C . However, DCS appears to be unique among terpenoid cyclases in that it does not contain the "NSE/DTE" motif on helix H that specifically chelates Mg 2+ B , which is usually found as the signature sequence (N, D)D(L, I, V)X(S, T)XXXE (boldface indicates Mg 2+ B ligands). Instead, DCS contains a second aspartate-rich motif, D 451 DVAE 455 , that interacts with Mg 2+ B . In this regard, DCS is more similar to the isoprenoid chain elongation enzyme farnesyl diphosphate synthase, which also contains two aspartate-rich motifs, rather than the greater family of terpenoid cyclases. Nevertheless, the structure of the DCS-2F-FPP complex shows that the structure of the trinuclear magnesium cluster is generally similar to that of other terpenoid cyclases despite the alternative Mg 2+ B -binding motif. Analyses of DCS mutants with alanine substitutions in the D 307 DTYD 311 and D 451 DVAE 455 segments reveal the contributions of these segments to catalysis.Antimicrobial natural terpenoid products known as phytoalexins are generated by tree cotton (Gossypium arboreum, indigenous to India and Pakistan) in response to threats from bacterial or fungal pathogens (1-2). These products are classified as secondary metabolites and are † This work was supported by National Institutes of Health grant GM56838 (D.W.C.), BBSRC grants 6/B17177 (R.K.A.) and BB/ G003572/1 (R.K.A), EPSRC grant EP/D06958/1 (R.K.A), Royal Society grant 2007R2 (R.K.A.), and Cardiff University. ‡ The atomic coordinates of unliganded (+)-δ-cadinene synthase and its complex with 2-fluorofarnesyl diphosphate have been deposited in the Protein Data Bank (www.rcsb.org) with accession codes 3G4D and 3G4F, respectively. Δ These authors made equal contributions to this study. * To whom correspondence should be addressed: DWC: Tel: 215-898-5714. Fax: 215-573-2201. Email: chris@sas.upenn NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript therefore non-essential for plant viability; however, they mediate important interactions between plants and their environments (3). The 64 kDa sesquiterpenoid cyclase (+)-δ-cadinene synthase (DCS, isozyme XC1; SWISS-PROT accession code Q39761) catalyzes the formation of (+)-δ-cadinene from farnesyl diphosphat...

Farnesyl diphosphate (FPP),2 a flexible 15-carbon isoprenoid, is the universal precursor of Ͼ300 different cyclic sesquiterpenes found in numerous plants, bacteria, and fungi (1, 2). The cyclization of FPP is catalyzed by a sesquiterpene cyclase that utilizes a trinuclear magnesium cluster to trigger the departure of the pyrophosphate (PP i ) leaving group, thereby forming an allylic carbocation that typically reacts with one of the remaining bonds of the substrate (3-7). The remarkable diversity of sesquiterpene structure and stereochemistry is the consequence of precise control exerted by the cyclase over the conformations of the flexible substrate and carbocation intermediates in the cyclization cascade.Aristolochene synthase from Aspergillus terreus is a sesquiterpene cyclase that catalyzes the cyclization of FPP to form aristolochene (Fig. 1a), the parent hydrocarbon of a large group of fungal toxins such as gigantenone, PR-toxin, and bipolaroxin (8). In contrast to aristolochene synthase from Penicillium roqueforti, which generates aristolochene predominantly (Ͼ90%) but also small amounts of germacrene A and valencene (9, 10), aristolochene synthase from A. terreus generates aristolochene exclusively (9). Each cyclase adopts the common ␣-helical fold of a class I terpenoid cyclase and contains two conserved metal binding motifs: the "aspartate-rich" motif D 90 DLLE that coordinates to Mg 3 ⅐PP i stabilizes the active site in a closed conformation that is completely sequestered from bulk solvent (Fig. 1b) (11). In addition to multiple metal coordination interactions, the PP i anion accepts hydrogen bonds from conserved residues Arg , and Tyr 315 when bound to the closed conformation (Fig. 1c). It is likely that the diphosphate group of FPP makes identical metal coordination and hydrogen bond interactions in the Michaelis complex, i.e. the complex between the enzyme and the productively bound substrate that immediately precedes the initiation of the cyclization cascade.Substrate conformation is a crucial determinant of the biosynthetic outcome of the terpenoid cyclase reaction. The active site of aristolochene synthase from A. terreus serves as a high fidelity template that fixes FPP in a single, productive conformation in the Michaelis complex; otherwise, aberrant cyclization products would result. To study the conformational control of FPP in the active site of aristolochene synthase from A. terreus, we now report the structures of crystalline complexes * This work was supported, in whole or in part, by National Institutes of Health Grants GM 56838 (to D. W. C.), GM 30301 (to D. E. C.), and GM 13956 (to R. M. C.). This work was also supported by the Engineering and Physical Sciences Research Council (Grant EP/D069580 to R. K. A.) and by Cardiff University (Studentship to F. Y.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Th...

The mechanism of the conversion of (E,E)-farnesyl diphosphate (FPP, 1a) to aristolochene (6) catalyzed by aristolochene synthase from Penicillium roqueforti has been proposed to proceed through the neutral intermediate germacrene A (4a). However, much of the experimental evidence is also in agreement with a mechanism in which germacrene A is not an intermediate in the predominant mechanism that leads to the formation of aristolochene, but rather an off-pathway product that is formed in a side reaction. Hence, to elucidate the mechanism of FPP cyclisation the substrate analogue 2-fluoroFPP (1b) was synthesized, and upon incubation with aristolochene synthase was converted to a single pentane extractable product according to GC-MS analysis. On the basis of NMR analyses this product was identified as 2-fluorogermacrene A (4b). Variable temperature (1)H NMR spectroscopy indicated the existence of two conformers of 4b that were in slow exchange at -60 degrees C, while at 90 degrees C the two isomers gave rise to averaged NMR signals. In the major isomer (approximately 75%) the methyl groups on C3 and C7 were most likely in the down-down orientation as had been observed for other (E,E)-germacranes. This work suggests that after an initial concerted cyclisation of FPP to germacryl cation deprotonation leads to the formation of germacrene A, and provides compelling evidence that germacrene A is indeed an on-pathway product of catalysis by aristolochene synthase.

The structure of hydantoin (imidazolidine‐2,4‐dione), C3H4N2O2, has been determined from a twinned crystal. The two carbonyl bond lengths are nearly equal, even though one of them adjoins electron‐donating NH groups to either side while the other is adjacent to only one. Ab initio molecular‐orbital calculations yield more negative Löwdin charge on the former than the latter. Hydantoin mol­ecules form two chains linked by N—H⋯O hydrogen bonds, from which inversion centres create a `chain of rings'. Out of 50 hydantoin moieties in 49 independent mol­ecules of hydantoin derivatives in the Cambridge Structural Database [Version 5.25; Allen (2002). Acta Cryst. B58, 380–388], five show this arrangement, six are a variant using the same O atom twice, five form a chain of edge‐fused 12‐membered hydrogen‐bonded rings, and all but three of the remainder have one eight‐membered ring and/or one chain connecting their hydantoin rings.

The catalytic mechanism of the enzyme aristolochene synthase from Penicillium roqueforti (PR-AS) has been probed with the farnesyl diphosphate analogues 6- and 14-fluoro farnesyl diphosphate (1a and 1c). Incubation of these analogues with PR-AS followed by analysis of the reaction products by GC-MS and NMR spectroscopy indicated that these synthetic FPP analogues were converted to the fluorinated germacrene A analogues 3b and 3c, respectively. In both cases the position of the fluorine atom prevented the formation of the eudesmane cation analogues 4b and 4c. These results highlight that germacrene A is an on-path reaction intermediate during PR-AS catalysis and shed light on the mechanism by which germacrene A is converted to eudesmane cation. They support the proposal that the role of PR-AS in the cyclisation is essentially passive in that it harnesses the inherent chemical reactivity present in the substrate by promoting the initial ionisation of farnesyl diphosphate and by acting as a productive template to steer the reaction through an effective series of cyclisations and rearrangements to (+)-aristolochene (7a).