With more than 55,000 members identified to date in all forms of life, the family of terpene or terpenoid natural products represents the epitome of molecular biodiversity. A particularly eminent member of this family is the polycyclic diterpenoid Taxol (paclitaxel), which promotes tubulin polymerization1 and exhibits remarkable efficacy in cancer chemotherapy2. The first committed step of Taxol biosynthesis in the Pacific yew (Taxus brevifolia)3 is the cyclization of the linear isoprenoid substrate geranylgeranyl diphosphate (GGPP) to form taxa-4(5),11(12)diene4, which is catalyzed by taxadiene synthase5. The full-length form of this diterpene cyclase contains 862-residues, but an ~80-residue N-terminal transit sequence is cleaved upon maturation in plastids6. We now report the X-ray crystal structure of a truncation variant lacking the transit sequence and an additional 27 residues at the N-terminus, henceforth designated TXS. Specifically, we have determined structures of TXS complexed with 13-aza-13,14-dihydrocopalyl diphosphate (ACP, 1.82 Å resolution) and 2-fluorogeranylgeranyl diphosphate (FGP, 2.25 Å resolution). The TXS structure is the first of a diterpene cyclase and reveals a modular assembly of three α-helical domains. The C-terminal catalytic domain is a class I terpenoid cyclase, which binds and activates substrate GGPP with a three-metal ion cluster. Surprisingly, the N-terminal domain and a third "insertion" domain together adopt the fold of a vestigial class II terpenoid cyclase. A class II cyclase activates the isoprenoid substrate by protonation instead of ionization, and the TXS structure reveals a definitive connection between the two distinct cyclase classes in the evolution of terpenoid biosynthesis.

The structure of ent-copalyl diphosphate synthase (CPS) reveals three α-helical domains (α, β, γ), as also observed in the related diterpene cyclase taxadiene synthase. However, active sites are located at the interface of the βγ domains in CPS but exclusively in the α domain of taxadiene synthase. Modular domain architecture in plant diterpene cyclases enables the evolution of alternative active sites and chemical strategies for catalyzing isoprenoid cyclization reactions.

The X-ray crystal structure of recombinant isoprene synthase from grey poplar leaves (Populus x canescens) (PcISPS) has been determined at 2.7 Å resolution, and the structure of its complex with three Mg 2+ ions and the unreactive substrate analogue dimethylallyl-S-thiolodiphosphate has been determined at 2.8 Å resolution. Analysis of these structures suggests that the generation of isoprene from substrate dimethylallyl diphosphate occurs via a syn-periplanar elimination mechanism in which the diphosphate leaving group serves as a general base. This chemical mechanism is responsible for the annual atmospheric emission of 100 billion kilograms of isoprene by terrestrial plant life. Importantly, the PcISPS structure promises to guide future protein engineering studies potentially leading to hydrocarbon fuels and products that do not rely on traditional petrochemical sources.

Background The diterpene cyclase ent-copalyl diphosphate synthase (CPS) catalyzes the first committed step in the biosynthesis of gibberellins. The previously reported 2.25 Å resolution crystal structure of CPS complexed with (S)-15-aza-14,15-dihydrogeranylgeranyl thiolodiphosphate (1) established the αβγ domain architecture, but ambiguities regarding substrate analog binding remained. Method Use of crystallization additives yielded CPS crystals diffracting to 1.55 Å resolution. Additionally, active site residues that hydrogen bond with D379, either directly or through hydrogen bonded water molecules, were probed by mutagenesis. Results This work clarifies structure-function relationships that were ambiguous in the lower resolution structure. Well-defined positions for the diphosphate group and tertiary ammonium cation of 1 as well as extensive solvent structure, are observed. Conclusions Two channels involving hydrogen bonded solvent and protein residues lead to the active site, forming hydrogen bonded "proton wires" that link general acid D379 with bulk solvent. These proton wires may facilitate proton transfer with the general acid during catalysis. Activity measurements made with mutant enzymes indicate that N425, which donates a hydrogen bond directly to D379, and T421, which hydrogen bonds with D379 through an intervening solvent molecule, help orient D379 for catalysis. Residues involved in hydrogen bonds with the proton wire, R340 and D503, are also important. Finally, conserved residue E211, which is located near the diphosphate group of 1 is proposed to be a ligand to Mg2+ required for optimal catalytic activity. General Significance This work establishes structure-function relationships for class II terpenoid cyclases.

The crystal structure of 2-methylisoborneol synthase (MIBS) from Streptomyces coelicolor A3(2) has been determined in complex with substrate analogues geranyl-S-thiolodiphosphate and 2-fluorogeranyl diphosphate at 1.80 Å and 1.95 Å resolution, respectively. This terpenoid cyclase catalyzes the cyclization of the naturally-occuring, non-canonical C-methylated isoprenoid substrate, 2-methylgeranyl diphosphate, to form the bicyclic product 2-methylisoborneol, a volatile C11 homoterpene alcohol with an earthy, musty odor. While MIBS adopts the tertiary structure of a class I terpenoid cyclase, its dimeric quaternary structure differs from that previously observed in dimeric terpenoid cyclases from plants and fungi. The quaternary structure of MIBS is nonetheless similar in some respects to that of dimeric farnesyl diphosphate synthase, which is not a cyclase. The structures of MIBS complexed with substrate analogues provide insights regarding differences in the catalytic mechanism of MIBS and the mechanisms of (+)-bornyl diphosphate synthase and endo-fenchol synthase, plant cyclases that convert geranyl diphosphate into products with closely related bicyclic bornyl skeletons, but distinct structures and stereochemistries.