Abstract
Throughout molecular evolution, organisms create assorted chemicals in response to varying ecological niches. Catalytic landscapes underlie metabolic evolution, wherein mutational steps alter the biosynthetic properties of enzymes. Here we report the first systematic quantitative characterization of the catalytic landscape underlying the evolution of sesquiterpene chemical diversity. On the basis of our previous discovery of a set of nine naturally occurring amino acid substitutions that functionally interconverted orthologous sesquiterpene synthases from Nicotiana tabacum and Hyoscyamus muticus, we created a library of all possible residue combinations (29 = 512) in the N. tabacum enzyme. The product spectra of 418 active enzymes revealed a rugged landscape where several minimal combinations of the nine mutations encode convergent solutions to the interconversions of parental activities. Quantitative comparisons indicated context dependence for mutational effects—epistasis—in product specificity and promiscuity. These results provide a measure of the mutational accessibility of phenotypic variability in a diverging lineage of terpene synthases.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Grayer, R.J. & Kokubun, T. Plant-fungal interactions: the search for phytoalexins and other antifungal compounds from higher plants. Phytochemistry 56, 253–263 (2001).
Pedras, M.S., Okanga, F.I., Zaharia, I.L. & Khan, A.Q. Phytoalexins from crucifers: synthesis, biosynthesis, and biotransformation. Phytochemistry 53, 161–176 (2000).
Harborne, J.B. The comparative biochemistry of phytoalexin induction in plants. Biochem. Syst. Ecol. 27, 335–367 (1999).
Akiyama, K., Matsuzaki, K. & Hayashi, H. Plant sesquiterpenes induce hyphal branching in arbuscular mycorrhizal fungi. Nature 435, 824–827 (2005).
Mumm, R. & Hilker, M. The significance of background odour for an egg parasitoid to detect plants with host eggs. Chem. Senses 30, 337–343 (2005).
Feeny, P. Herbivores: Their Interactions with Secondary Plant Metabolites 2nd edn. (Academic Press, San Diego, 1992).
Gershenzon, J. & Dudareva, N. The function of terpene natural products in the natural world. Nat. Chem. Biol. 3, 408–414 (2007).
O'Maille, P.E., Bakhtina, M. & Tsai, M.D. Structure-based combinatorial protein engineering (SCOPE). J. Mol. Biol. 321, 677–691 (2002).
O'Maille, P.E., Chappell, J. & Noel, J.P. A single-vial analytical and quantitative gas chromatography-mass spectrometry assay for terpene synthases. Anal. Biochem. 335, 210–217 (2004).
Back, K., He, S., Kim, K.U. & Shin, D.H. Cloning and bacterial expression of sesquiterpene cyclase, a key branch point enzyme for the synthesis of sesquiterpenoid phytoalexin capsidiol in UV-challenged leaves of Capsicum annuum. Plant Cell Physiol. 39, 899–904 (1998).
Facchini, P.J. & Chappell, J. Gene family for an elicitor-induced sesquiterpene cyclase in tobacco. Proc. Natl. Acad. Sci. USA 89, 11088–11092 (1992).
Greenhagen, B.T., O'Maille, P.E., Noel, J.P. & Chappell, J. Identifying and manipulating structural determinates linking catalytic specificities in terpene synthases. Proc. Natl. Acad. Sci. USA 103, 9826–9831 (2006).
Dudareva, N. et al. (E)-beta-ocimene and myrcene synthase genes of floral scent biosynthesis in snapdragon: function and expression of three terpene synthase genes of a new terpene synthase subfamily. Plant Cell 15, 1227–1241 (2003).
Bohlmann, J., Meyer-Gauen, G. & Croteau, R. Plant terpenoid synthases: molecular biology and phylogenetic analysis. Proc. Natl. Acad. Sci. USA 95, 4126–4133 (1998).
O'Maille, P.E., Tsai, M.D., Greenhagen, B.T., Chappell, J. & Noel, J.P. Gene library synthesis by structure-based combinatorial protein engineering. Methods Enzymol. 388, 75–91 (2004).
O'Maille, P.E., Chappell, J. & Noel, J.P. Biosynthetic potential of sesquiterpene synthases: alternative products of tobacco 5-epi-aristolochene synthase. Arch. Biochem. Biophys. 448, 73–82 (2006).
Copley, S.D. Enzymes with extra talents: moonlighting functions and catalytic promiscuity. Curr. Opin. Chem. Biol. 7, 265–272 (2003).
Jensen, R.A. Enzyme recruitment in evolution of new function. Annu. Rev. Microbiol. 30, 409–425 (1976).
O'Brien, P.J. & Herschlag, D. Catalytic promiscuity and the evolution of new enzymatic activities. Chem. Biol. 6, R91–R105 (1999).
Wilderman, P.R. & Peters, R.J. A single residue switch converts abietadiene synthase into a pimaradiene specific cyclase. J. Am. Chem. Soc. 129, 15736–15737 (2007).
Yoshikuni, Y., Ferrin, T.E. & Keasling, J.D. Designed divergent evolution of enzyme function. Nature 440, 1078–1082 (2006).
Hyatt, D.C. & Croteau, R. Mutational analysis of a monoterpene synthase reaction: altered catalysis through directed mutagenesis of (-)-pinene synthase from Abies grandis. Arch. Biochem. Biophys. 439, 222–233 (2005).
Kampranis, S.C. et al. Rational conversion of substrate and product specificity in a Salvia monoterpene synthase: structural insights into the evolution of terpene synthase function. Plant Cell 19, 1994–2005 (2007).
Kollner, T.G., Schnee, C., Gershenzon, J. & Degenhardt, J. The variability of sesquiterpenes emitted from two Zea mays cultivars is controlled by allelic variation of two terpene synthase genes encoding stereoselective multiple product enzymes. Plant Cell 16, 1115–1131 (2004).
Weinreich, D.M., Delaney, N.F., Depristo, M.A. & Hartl, D.L. Darwinian evolution can follow only very few mutational paths to fitter proteins. Science 312, 111–114 (2006).
Ortlund, E.A., Bridgham, J.T., Redinbo, M.R. & Thornton, J.W. Crystal structure of an ancient protein: evolution by conformational epistasis. Science 317, 1544–1548 (2007).
Bershtein, S., Segal, M., Bekerman, R., Tokuriki, N. & Tawfik, D.S. Robustness-epistasis link shapes the fitness landscape of a randomly drifting protein. Nature 444, 929–932 (2006).
Miller, S.P., Lunzer, M. & Dean, A.M. Direct demonstration of an adaptive constraint. Science 314, 458–461 (2006).
Thulasiram, H.V., Erickson, H.K. & Poulter, C.D. Chimeras of two isoprenoid synthases catalyze all four coupling reactions in isoprenoid biosynthesis. Science 316, 73–76 (2007).
Agarwal, P.K., Billeter, S.R., Rajagopalan, P.T., Benkovic, S.J. & Hammes-Schiffer, S. Network of coupled promoting motions in enzyme catalysis. Proc. Natl. Acad. Sci. USA 99, 2794–2799 (2002).
Rajagopalan, P.T., Lutz, S. & Benkovic, S.J. Coupling interactions of distal residues enhance dihydrofolate reductase catalysis: mutational effects on hydride transfer rates. Biochemistry 41, 12618–12628 (2002).
Lockless, S.W. & Ranganathan, R. Evolutionarily conserved pathways of energetic connectivity in protein families. Science 286, 295–299 (1999).
Austin, M.B., O'Maille, P.E. & Noel, J.P. Evolving biosynthetic tangos negotiate mechanistic landscapes. Nat. Chem. Biol. 4, 217–222 (2008).
Tamura, K., Dudley, J., Nei, M. & Kumar, S. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24, 1596–1599 (2007).
Acknowledgements
We thank M. Austin and J. Melnick for critical review of the manuscript, Y. Zhai for computational support and J. Gullberg and A. Nordstom for insightful discussions. This work was supported by National Institutes of Health grant GM54029 to J.C. and J.P.N. J.P.N. is supported by the Howard Hughes Medical Institute.
Author information
Authors and Affiliations
Contributions
P.E.O. designed the study, conducted experiments, analyzed data and wrote the manuscript; A.M. conducted experiments and developed small-scale protein purification; N.D. conducted experiments, analyzed data and contributed revisions to the manuscript; B.A.H. conducted quantum mechanics calculations and contributed revisions to the manuscript; L.S. conducted quantum mechanics calculations; I.S. conducted experiments; B.T.G. and J.C. designed the study and contributed revisions to the manuscript; G.M. analyzed data, developed the biosynthetic tree and chemical distance analysis, and contributed revisions to the manuscript; J.P.N. designed the study, analyzed the data and wrote the manuscript.
Corresponding author
Ethics declarations
Competing interests
J.P.N. and J.C. declare an interest in Allylix, Inc. as scientific co-founders and stockholders. Allylix, Inc. is engaged in the commercial production of high-value sesquiterpene products.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1 and 2, Supplementary Tables 1–7 and Supplementary Methods (PDF 6532 kb)
Rights and permissions
About this article
Cite this article
O'Maille, P., Malone, A., Dellas, N. et al. Quantitative exploration of the catalytic landscape separating divergent plant sesquiterpene synthases. Nat Chem Biol 4, 617–623 (2008). https://doi.org/10.1038/nchembio.113
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nchembio.113
This article is cited by
-
Carotenoid cleavage enzymes evolved convergently to generate the visual chromophore
Nature Chemical Biology (2024)
-
Engineered enzymes for the synthesis of pharmaceuticals and other high-value products
Nature Synthesis (2024)
-
Genetic complementation fosters evolvability in complex fitness landscapes
Scientific Reports (2023)
-
Mining methods and typical structural mechanisms of terpene cyclases
Bioresources and Bioprocessing (2021)
-
Pervasive cooperative mutational effects on multiple catalytic enzyme traits emerge via long-range conformational dynamics
Nature Communications (2021)