Taxadiene synthase structure and evolution of modular architecture in terpene biosynthesis

Abstract

The first step in the biosynthesis of the anticancer compound Taxol (paclitaxel) and many other natural C20 diterpenes is the cyclization of an isoprenoid, catalysed by taxadiene synthase. The X-ray crystal structure of this enzyme from the Pacific yew has now been determined. Its C-terminal catalytic domain binds and activates the substrate in a manner seen in class I terpenoid cyclases, but the N-terminal domain and a third 'insertion' domain adopt the fold of a class II terpenoid cyclase. This suggests that this enzyme could be the ancestral progenitor of all terpenoid cyclases. The first X-ray crystal structure of a diterpene cyclase is reported — this enzyme, taxadiene synthase, catalyses the cyclization of an isoprenoid in the first committed step of the biosynthesis of the cancer chemotherapeutic drug Taxol. The C-terminal catalytic domain binds and activates the substrate in a manner seen in class I terpenoid cyclases, but the N-terminal domain and a third 'insertion' domain together adopt the fold of a class II terpenoid cyclase. It is proposed that this enzyme could be the ancestral progenitor of all terpenoid cyclases. With more than 55,000 members identified so far in all forms of life, the family of terpene or terpenoid natural products represents the epitome of molecular biodiversity. A well-known and important member of this family is the polycyclic diterpenoid Taxol (paclitaxel), which promotes tubulin polymerization1 and shows 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 catalysed by taxadiene synthase5. The full-length form of this diterpene cyclase contains 862 residues, but a roughly 80-residue amino-terminal transit sequence is cleaved on 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, hereafter designated TXS. Specifically, we have determined structures of TXS complexed with 13-aza-13,14-dihydrocopalyl diphosphate (1.82 Å resolution) and 2-fluorogeranylgeranyl diphosphate (2.25 Å resolution). The TXS structure reveals a modular assembly of three α-helical domains. The carboxy-terminal catalytic domain is a class I terpenoid cyclase, which binds and activates substrate GGPP with a three-metal ion cluster. 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.