Crystal Structure of Pentalenene Synthase: Mechanistic Insights on Terpenoid Cyclization Reactions in Biology

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Science  19 Sep 1997:
Vol. 277, Issue 5333, pp. 1820-1824
DOI: 10.1126/science.277.5333.1820


The crystal structure of pentalenene synthase at 2.6 angstrom resolution reveals critical active site features responsible for the cyclization of farnesyl diphosphate into the tricyclic hydrocarbon pentalenene. Metal-triggered substrate ionization initiates catalysis, and the α-barrel active site serves as a template to channel and stabilize the conformations of reactive carbocation intermediates through a complex cyclization cascade. The core active site structure of the enzyme may be preserved among the greater family of terpenoid synthases, possibly implying divergence from a common ancestral synthase to satisfy biological requirements for increasingly diverse natural products.

Sesquiterpenes comprise a group of natural products secreted by marine and terrestrial plants, fungi, and certain microorganisms. The structural diversity and stereochemical complexity of the C15-isoprenoid skeletons of these metabolites are remarkable. Indeed, of the more than 300 cyclic sesquiterpenes that have been characterized to date, each is derived from a common acyclic precursor, farnesyl diphosphate (1), in a reaction catalyzed by a sesquiterpene cyclase (2). Many cyclic sesquiterpenes exhibit useful medicinal properties and have been essential components of the pharmacopoeia since times of antiquity. For example, the sesquiterpenes furanoeudesma-1,3-diene and curzarene are responsible for the analgesic effects of myrrh by interacting with brain opioid receptors, thereby explaining the prescription of myrrh for use as a pain killer by Pliny the Elder, Hippocrates, and their predecessors (3).

In addition to the 15-carbon sesquiterpenes, the greater family of terpenoids includes the 10-carbon monoterpenes derived from geranyl diphosphate, the 20-carbon diterpenes derived from geranylgeranyl diphosphate, the 25-carbon sesterterpenes derived from geranylfarnesyl diphosphate, and the 30-carbon triterpenes (sterols) derived from two farnesyl diphosphate molecules. Terpenoid cyclases (also known as terpenoid synthases) perform critical biosynthetic tasks in metabolic pathways as diverse as cholesterol biosynthesis in mammals (4) and paclitaxel (Taxol™) synthesis in the Pacific yew (5). These complex intramolecular cyclizations are unified by the biogenetic isoprene rule (1) and require precise conformational and stereochemical control (2,6).

The biogenetic isoprene rule postulates that most sesquiterpene cyclization reactions occur through variations of a common mechanism involving (i) ionization of farnesyl diphosphate and electrophilic attack of the resultant allylic cation on one of the remaining π bonds of the substrate, (ii) subsequent cationic transformations (additional electrophilic cyclizations, hydride transfers, and Wagner-Meerwein rearrangements), culminated by (iii) quenching of the positive charge by deprotonation or capture of an exogenous nucleophile such as water. In the absence of a crystal structure it is not clear how a terpenoid cyclase mediates such complex reactions. For instance, how does the enzyme select and enforce the correct precatalytic substrate conformation? The starting conformation of farnesyl diphosphate is known to be a critical determinant of the ultimate cyclization product (2). In addition, how does the enzyme trigger substrate ionization and stabilize positive charge in the resulting cascade of highly reactive, electrophilic intermediates? Finally, how does the enzyme manage proper charge quenching to terminate the cyclization cascade?

We now report the x-ray crystal structure of recombinant pentalenene synthase (E.C. This terpenoid cyclase has been isolated from Streptomyces UC5319, cloned, expressed inEscherichia coli, and crystallized (7). Pentalenene synthase catalyzes the cyclization of farnesyl diphosphate into pentalenene, a tricyclic sesquiterpene that is the hydrocarbon precursor of the pentalenolactone family of antibiotics. The three-dimensional structure of pentalenene synthase reveals active site features responsible for the conformational and stereochemical control of farnesyl diphosphate cyclization and clarifies the structural basis of the catalytic metal requirement.

The pentalenene synthase crystal structure was determined by multiple isomorphous replacement and refined at 2.6 Å resolution (R = 0.198, Rfree = 0.273; Table1). The enzyme has a globular structure (Fig. 1) of approximate dimensions 60 Å by 50 Å by 40 Å defined by an aggregation of 11 α helices (designated A to K). Helices B, C, G, H, and K surround an active site cavity approximately 15 Å deep and 9 Å wide. Polypeptide loops connecting these helices are relatively short (average three residues) on the side of the protein distal to the active site, and relatively long (average ten residues) on the side of the protein proximal (that is, surrounding) to the active site cavity. This feature is a consequence of the helix packing arrangement necessary to enclose a central, well-defined cavity. A disulfide linkage is found between Cys128 and Cys136.

Table 1

Summary of x-ray crystal structure determination. Crystals of pentalenene synthase form as short hexagonal rods and belong to space groupP6 3 with hexagonal unit cell dimensions of a = b = 179.8 Å, c = 56.6 Å; two 38-kD monomers reside in the asymmetric unit and are related by two-fold noncrystallographic symmetry (NCS) (7). Diffraction data from pentalenene synthase crystals were collected at room temperature on an R-AXIS IIc image plate area detector, and intensity data integration and reduction were performed with MOSFLM (19) and CCP4 (20), respectively. For phase determination by multiple isomorphous replacement (MIR), initial heavy atom positions were determined in difference Patterson maps and refined with the program MLPHARE (20, 21). The model was fit into an electron density map calculated with solvent-flipped and NCS-averaged MIR phases extended to 3.3 Å resolution with SOLOMON (20, 22). Subsequent refinement and rebuilding of the native model was performed with X-PLOR (23) and O (24), respectively. Group B factors (one main chain and one side chain B factor per residue) were refined and a bulk solvent correction was applied. Strict NCS constraints were maintained as judged by R free; refinement at 3.3 Å resolution converged to a crystallographic R factor of 0.215 (R free = 0.277). A crystal derivatized with 1.0 mM trimethyllead acetate diffracted to 2.6 Å resolution when flash-frozen at the Cornell High Energy Synchrotron Source (CHESS, beamline A-1, λ = 0.91 Å). Although this crystal was nonisomorphous with native crystals (at room temperature), the 3.3 Å resolution model of pentalenene synthase served as the starting point for rigid-body refinement, followed by iterative rounds of simulated annealing refinement and rebuilding against the 2.6 Å resolution data with X-PLOR (23) and O (24), respectively. Restrained individual B factors were refined and a bulk solvent correction was applied. The quality of the model was improved in the final stages of refinement by releasing the NCS constraints into appropriately weighted restraints as judged byR free. Refinement converged smoothly to a final crystallographic R factor of 0.198 (R free = 0.273). Disordered segments in the final model include Pro2-Gln3, Phe158-Asp164, and Arg314-His337 at the COOH-terminus. The final model has excellent stereochemistry with no residues adopting unfavorable backbone conformations. Since there are no significant structural differences between the 3.3 Å resolution and the 2.6 Å resolution models, and since the lead binding site is removed from the active site (lead makes an interlattice contact between two protein molecules), active site features of the 2.6 Å resolution structure represent those of the native enzyme.

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Figure 1

Ribbon plot (25) of pentalenene synthase. The mouth of the active site cavity opens toward the top of the figure, and the aspartate-rich segment beginning with Asp80 is red. The dotted line is the disordered Phe158-Asp164 loop, and the Cys128-Cys136 disulfide linkage is yellow.

A similar, but not identical, fold to that of pentalenene synthase is found in farnesyl diphosphate synthase (Fig.2), which catalyzes the synthesis of farnesyl diphosphate (the substrate of a sesquiterpene cyclase) from isopentenyl diphosphate and dimethylallyl diphosphate (8). Despite only 15% sequence identity between the two enzymes, the different substrates utilized by each enzyme, and the different outcomes of the electrophilic condensation reactions catalyzed, general structural features of their active sites are conserved. This structural homology is reminiscent of suggestions that enzymes catalyzing successive steps in a metabolic pathway may evolve with similar structures through divergence, regardless of the degree of amino acid sequence identity; function evolves more rapidly than sequence and sequence evolves more rapidly than tertiary structure (9). This expectation is certainly consistent with the unexpected structural homology observed between pentalenene synthase and the catalytic core of epi-aristolochene synthase despite only 16% sequence identity (10). In anticipation of broad structural relationships among this family of biosynthetic enzymes, we designate the α-helical topology observed for pentalenene synthase as a minimal “terpenoid synthase fold.”

Figure 2

Ribbon plot (25) of farnesyl diphosphate synthase (8) (PDB accession code 1FPS). The core terpenoid synthase structure shared with pentalenene synthase is blue; the two aspartate-rich segments [beginning with Asp257 (left) and Asp117 (right)] are red. The Asp117 segment of farnesyl diphosphate synthase aligns with the Asp80 segment of pentalenene synthase (Fig. 1). Both substrates of this enzyme, isopentenyl diphosphate and dimethylallyl diphosphate, are believed to bind to the two aspartate-rich segments through bridging magnesium ions.

The active site cavity of pentalenene synthase (Fig.3) is identified by structural homology with farnesyl diphosphate synthase (8) as well as the binding of a nonreactive substrate analogue (11). The bottom of this cavity is predominantly hydrophobic in nature and contains aromatic residues Phe57, Phe76, Phe77, and Trp308, and aliphatic residues Leu53, Val177, Val179, Thr182, and Val301. These residues confer an overall shape to the cavity, which serves as a template for the binding of farnesyl diphosphate in the correct conformation for cyclization to pentalenene. The upper region of the active site cavity is somewhat more hydrophilic in nature and includes the polar or charged side chains of His309, Asn219, Arg44, Arg157, Arg173, Lys226, and Arg230. In addition, the carboxylate side chains of Asp80 and Asp84 (and to a lesser degree, Asp81, which salt links with Arg44) protrude into the upper active site cavity (Fig.4). These three aspartate residues comprise a signature “aspartate-rich segment” (12), which indicates a postulated metal-binding site (7); Mg2+ is required to facilitate pyrophosphate departure in the first step of the cyclization reaction (13). Basic residues Arg157, Arg173, Lys226, and Arg230 may also stabilize the pyrophosphate leaving group.

Figure 3

Close-up view of active site residues in pentalenene synthase; selected residues are indicated by sequence number and discussed in the text. The aspartate-rich segments are red and the proposed catalytic base His309 is green.

Figure 4

Averaged omit map of the aspartate-rich segment calculated with Fourier coefficients |F o| − |F c| and phases derived from the final model less the atoms of Phe77-Asp84 (contoured at 4.5σ). Given that the carboxylate side chains of Asp80and Asp84 are oriented toward the active site cavity, these residues most likely bind Mg2+ in the enzyme-substrate complex.

Initiation of a cyclization cascade is triggered by the ionization of a substrate molecule bound in the enzyme active site with precisely controlled folding, shape, and size (2, 4, 6). Mechanistic studies of pentalenene synthase by stereospecifically labeled substrates are consistent with the mechanism and stereochemistry (Fig.5) wherein farnesyl diphosphate undergoes ionization and electrophilic attack of the incipient allylic cation-pyrophosphate pair on the distal π bond (7, 14). The carboxylate side chains of Asp80 and Asp84are oriented most favorably for Mg2+ complexation; the coordination of pyrophosphate to Mg2+, as well as hydrogen bond interactions with nearby basic residues, trigger leaving group departure. Deprotonation of the resultant humulyl cation is thought to result in the formation of the intermediate humulene. Stereospecific reprotonation of humulene yields the protoilludyl cation, which in turn undergoes rearrangement and cyclization to the product pentalenene. Inspection of the pentalenene synthase active site reveals that the likely catalytic base is His309. The Nδ-H group of this residue donates a hydrogen bond to the backbone carbonyl of Ser305, leaving the lone electron pair at Nε oriented toward the active site cavity and poised for catalysis at the pH optimum of 8.2 to 8.4. Notably, a catalytically essential histidine residue has been identified in the humulene synthase from sage leaf (15).

Figure 5

Proposed mechanism for cyclization of farnesyl pyrophosphate to intermediates humulene and protoilludyl cation, with subsequent rearrangement into pentalenene (7, 14). The pyrophosphate leaving group is omitted for clarity; however, it may remain bound in the enzyme active site during the cyclization cascade and contribute to the electrostatic stabilization of carbocation intermediates.

The precatalytic binding conformation of farnesyl diphosphate in the pentalenene synthase active site can be modeled unambiguously on the basis of the well-determined stereochemical details of the cyclization reaction (16). A properly folded substrate molecule will contact the catalytic base His309, and the diphosphate moiety will contact the aspartate-rich sequence of the enzyme through a bridging magnesium ion (or ions) (Fig.6). Approximately 80% of the substrate surface area is buried in the enzyme-substrate complex, helping to prevent premature quenching of carbocation intermediates by solvent (16).

Figure 6

(A) Farnesyl diphosphate must bind in the active site cleft with the correct conformation required for cyclization to pentalenene, with the C9-H oriented toward catalytic base His309 and the pyrophosphate moiety oriented toward the aspartate-rich segment through bridging Mg2+ (metal ions not shown for the sake of clarity). The trajectory of C-C bond formation leading to humulene synthesis in the initial cyclization step is indicated by a dashed line. (B) Pentalenene (as well as the preceding intermediates) is complementary in shape to the active site cavity. Comparison of substrate and product binding suggests that the active site is a template for correct conformation and stereochemistry in the cyclization cascade.

The U-shaped conformation of substrate farnesyl diphosphate is centered about an axis defined by Phe77 and Asn219 (Fig.6). We propose that these two residues in particular, and the remaining active site residues in general, provide a template that channels reactive conformations along the exclusive reaction coordinate leading to pentalenene formation. As such, these residues destabilize incorrect substrate and intermediate conformations that would otherwise lead to spurious cyclization products; alternative substrate starting conformations do not fit well in the enzyme active site. We therefore propose that Phe77 and Asn219 are optimally located to stabilize highly reactive carbocation intermediates through favorable quadrupole-charge (17) and dipole-charge interactions, respectively:

Further stabilization may result from a quadrupole-charge interaction with Phe76, which is well positioned to stabilize the protoilludyl cation and the derived rearrangement product, which is generated by the 1,2-hydride shift. Similarly, Trp308 may also be well positioned to stabilize positive charge at sites corresponding to the original C10, C9, and C8 atoms of farnesyl diphosphate.

It is interesting to consider that at least two-thirds of the carbon atoms of the farnesyl diphosphate backbone undergo substantial changes in hybridization, configuration, and bonding during the cyclization cascade catalyzed by a typical sesquiterpene cyclase (2). The pentalenene synthase structure shows that the enzyme serves as a template to channel conformation and stereochemistry in the chemistry of cyclization, and the enzyme elegantly protects and stabilizes reactive carbocation intermediates. These features are likely to be common to all terpenoid cyclases. Alteration of active site residues in a terpenoid cyclase can result in the formation of aberrant cyclization products, so that the architecture and chemical nature of the substrate binding pocket are critical for channeling the correct cyclization chemistry (18). Now that the three-dimensional structure of a terpenoid synthase is available at atomic resolution, we are in a position to test the specific catalytic function of individual amino acids, as well as entire helical subdomains, in structure-based redesign experiments.

  • * To whom correspondence should be addressed. E-mail: chris{at}


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