Aristolochene synthase from Aspergillus terreus catalyzes the cyclization of the universal sesquiterpene precursor, farnesyl diphosphate, to form the bicyclic hydrocarbon aristolochene. The 2.2 Å resolution X-ray crystal structure of aristolochene synthase reveals a tetrameric quaternary structure in which each subunit adopts the α-helical class I terpene synthase fold with the active site in the "open", solvent-exposed conformation. Intriguingly, the 2.15 Å resolution crystal structure of the complex with Mg 2+ 3 -pyrophosphate reveals ligand binding only to tetramer subunit D, which is stabilized in the "closed" conformation required for catalysis. Tetramer assembly may hinder conformational changes required for the transition from the inactive open conformation to the active closed conformation, thereby accounting for the attenuation of catalytic activity with increasing enzyme concentration. In both conformations, but especially so in the closed conformation, the active site contour is highly complementary in shape to that of aristolochene, and a catalytic function is proposed for the pyrophosphate anion based on its orientation with regard to the presumed binding mode of aristolochene. A similar active site contour is conserved in aristolochene synthase from P. roqueforti despite the substantial divergent evolution of these two enzymes, while strikingly different active site contours are found in the sesquiterpene cyclases 5-epi aristolochene synthase and trichodiene synthase. Thus, the terpenoid cyclase active site plays a critical role as a template to bind the flexible polyisoprenoid substrate in the proper conformation for catalysis. Across the greater family of terpenoid cyclases, this template is highly evolvable within a conserved α-helical fold for the synthesis of terpene natural products of diverse structure and stereochemistry.The terpenoid family consists of tens of thousands of structurally and stereochemically complex natural products that ultimately derive from a mere handful of linear isoprenoid precursors. For example, cyclic sesquiterpenes comprise a broad family of C 15 -isoprenoids that serve myriad biological functions in plants, bacteria, and fungi (1,2), yet each sesquiterpene derives from the universal sesquiterpene precursor, farnesyl diphosphate, through a reaction catalyzed by a sesquiterpene cyclase (2-9). In general, a terpene cyclase governs a specific cyclization cascade with exquisite precision to generate a single, unique product. However, † This work was supported by National Institutes of Health Grants GM 56838 (D.W.C.) and GM 30301 (D.E.C.) ‡ Atomic coordinates of aristolochene synthase from A. terreus and its complex with Mg 2+ 3 -pyrophosphate have been deposited in the Protein Data Bank with accession codes 2E4O and 2OA6, respectively. *To whom correspondence should be addressed at the Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania, 231 South 34 th St., Philadelphia, Pennsylvania 19104-6323. Phone: 215-898-5714. Fax: 215...

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...

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