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Taxadiene synthase structure and evolution of modular architecture in terpene biosynthesis

Nature volume 469, pages 116–120 (2011)Cite this article

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Abstract

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.

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Figure 1: Proposed catalytic mechanism of taxadiene synthase.
Figure 2: Structural relationships among terpenoid cyclases.
Figure 3: Binding of substrate analogue to TXS.
Figure 4: Active-site cavities of terpenoid synthases.

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Accession codes

Primary accessions

Protein Data Bank

Data deposits

The atomic coordinates and structure factors of the TXS–ACP and TXS–FGP complexes are deposited in the Protein Data Bank with accession codes 3P5P and 3P5R, respectively.

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Acknowledgements

We thank C. MacDermaid and J. Saven for advice and assistance with molecular modelling calculations, and E. Oldfield for helpful comments on the manuscript. We thank the National Synchrotron Light Source at Brookhaven National Laboratory for beamline access. The US National Institutes of Health provided grants GM56838 (D.W.C.), GM13956 (R.M.C.) and CA55254 (R.C.) in support of this research. Y.J. thanks the University of Illinois for support through the John C. Bailar and R. C. Fuson Fellowships.

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Authors and Affiliations

  1. Department of Chemistry, Roy and Diana Vagelos Laboratories, University of Pennsylvania, 231 South 34th Street, Philadelphia, Pennsylvania 19104-6323, USA,

    Mustafa Köksal & David W. Christianson

  2. Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, 61801, Illinois, USA

    Yinghua Jin & Robert M. Coates

  3. Department of Chemistry and Biochemistry, University of Colorado, Boulder, 80309, Colorado, USA

    Yinghua Jin

  4. Institute of Biological Chemistry, Washington State University, Pullman, 99164-6340, Washington, USA

    Rodney Croteau

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  1. Mustafa Köksal
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  4. Rodney Croteau
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Contributions

M.K. and D.W.C. performed the X-ray crystallographic studies. R.C. supplied the M79-TXS construct from which M107-TXS-CHT was ultimately prepared. Y.J. and R.M.C. synthesized 2-fluorogeranylgeranyl diphosphate. All authors contributed to the interpretation of the results and preparation of the manuscript.

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Correspondence to David W. Christianson.

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The authors declare no competing financial interests.

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Köksal, M., Jin, Y., Coates, R. et al. Taxadiene synthase structure and evolution of modular architecture in terpene biosynthesis. Nature 469, 116–120 (2011). https://doi.org/10.1038/nature09628

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