Taxadiene synthase structure and evolution of modular architecture in terpene biosynthesis

Abstract

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.

Structure, function and inhibition of the two- and three-domain 4Fe-4S IspG proteins

“…Finally, we propose a model for the three-dimensional structures of the 3-domain IspGs using computational methods. This approach has been successful with other prenyl synthases, in particular with the diterpene cyclases, where the use (24) of a variety of computational tools enabled successful prediction of the three-helical αβγ domain structures, recently confirmed by two crystallographic investigations (25,26).…”

Modular Pathway Engineering of Diterpenoid Synthases and the Mevalonic Acid Pathway for Miltiradiene Production

“…22,23 Protein modeling 24 using AtCPS as the template showed SmCPS belongs to the class II synthase and the active site DXDD motif for H + -initiated cyclization is located between the βγ domains in the N-terminus (Supporting Information Figure S7). Protein modeling using taxadiene synthase as the template showed the active site of SmKSL is located at the α domain in the Cterminus (Supporting Information Figure S7).…”

The Primary Diterpene Synthase Products of Picea abies Levopimaradiene/Abietadiene Synthase (PaLAS) Are Epimers of a Thermally Unstable Diterpenol

“…Computational Structure Analysis-We modeled the structures of PaLAS and PaIso based on the structure of taxadiene synthase from T. brevifolia (22) to draw insight into the origin of product differences. The most energetically favorable binding located 13-hydroxy-8 (14)-abietene in the center of the …”

“…Computational Structure Analysis-Homology models of PaIso and PaLAS were built using the CPHmodels 3.0 server (21) based on the tertiary structure of taxadiene synthase from Taxus brevifolia (Protein Data Bank code 3P5R, chain A) (22) and further subjected to a final energy minimization using the YASARA server (23). Modeled structures were certified as high quality with Ramachandran plot statistics exceeding 90% using PROCHECK (24).…”