It is generally believed that proteins with promiscuous functions divergently evolved to acquire higher specificity and activity1,2,3,4,5, and that this process was highly dependent on the ability of proteins to alter their functions with a small number of amino acid substitutions (plasticity)6. The application of this theory of divergent molecular evolution to promiscuous enzymes may allow us to design enzymes with more specificity and higher activity. Many structural and biochemical analyses have identified the active or binding site residues important for functional plasticity (plasticity residues)6,7,8,9,10. To understand how these residues contribute to molecular evolution, and thereby formulate a design methodology, plasticity residues were probed in the active site of the promiscuous sesquiterpene synthase γ-humulene synthase11,12. Identified plasticity residues were systematically recombined based on a mathematical model in order to construct novel terpene synthases, each catalysing the synthesis of one or a few very different sesquiterpenes. Here we present the construction of seven specific and active synthases that use different reaction pathways to produce the specific and very different products. Creation of these enzymes demonstrates the feasibility of exploiting the underlying evolvability of this scaffold, and provides evidence that rational approaches based on these ideas are useful for enzyme design.
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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)
Copley, S. D. Enzymes with extra talents: moonlighting functions and catalytic promiscuity. Curr. Opin. Chem. Biol. 7, 265–272 (2003)
James, L. C. & Tawfik, D. S. Conformational diversity and protein evolution—a 60-year-old hypothesis revisited. Trends Biochem. Sci. 28, 361–368 (2003)
Gerlt, J. A., Babbitt, P. C. & Rayment, I. Divergent evolution in the enolase superfamily: the interplay of mechanism and specificity. Arch. Biochem. Biophys. 433, 59–70 (2005)
Aharoni, A. et al. The ‘evolvability’ of promiscuous protein functions. Nature Genet. 37, 73–76 (2005)
Bone, R., Silen, J. L. & Agard, D. A. Structural plasticity broadens the specificity of an engineered protease. Nature 339, 191–195 (1989)
van Den Heuvel, R. H., Fraaije, M. W., Ferrer, M., Mattevi, A. & van Berkel, W. J. Inversion of stereospecificity of vanillyl-alcohol oxidase. Proc. Natl Acad. Sci. USA 97, 9455–9460 (2000)
Matsumura, I. & Ellington, A. D. In vitro evolution of β-glucuronidase into a β-galactosidase proceeds through non-specific intermediates. J. Mol. Biol. 305, 331–339 (2001)
Aharoni, A. et al. Directed evolution of mammalian paraoxonases PON1 and PON3 for bacterial expression and catalytic specialization. Proc. Natl Acad. Sci. USA 101, 482–487 (2004)
Steele, C. L., Crock, J., Bohlmann, J. & Croteau, R. Sesquiterpene synthases from grand fir (Abies grandis). Comparison of constitutive and wound-induced activities, and cDNA isolation, characterization, and bacterial expression of δ-selinene synthase and γ-humulene synthase. J. Biol. Chem. 273, 2078–2089 (1998)
Little, D. B. & Croteau, R. B. Alteration of product formation by directed mutagenesis and truncation of the multiple-product sesquiterpene synthases δ-selinene synthase and γ-humulene synthase. Arch. Biochem. Biophys. 402, 120–135 (2002)
Lesburg, C. A., Zhai, G., Cane, D. E. & Christianson, D. W. Crystal structure of pentalenene synthase: mechanistic insights on terpenoid cyclization reactions in biology. Science 277, 1820–1824 (1997)
Starks, C. M., Back, K., Chappell, J. & Noel, J. P. Structural basis for cyclic terpene biosynthesis by tobacco 5-epi-aristolochene synthase. Science 277, 1815–1820 (1997)
Caruthers, J. M., Kang, I., Rynkiewicz, M. J., Cane, D. E. & Christianson, D. W. Crystal structure determination of aristolochene synthase from the blue cheese mold, Penicillium roqueforti. J. Biol. Chem. 275, 25533–25539 (2000)
Rynkiewicz, M. J., Cane, D. E. & Christianson, D. W. Structure of trichodiene synthase from Fusarium sporotrichioides provides mechanistic inferences on the terpene cyclization cascade. Proc. Natl Acad. Sci. USA 98, 13543–13548 (2001)
Glasby, J. S. Encyclopaedia of the Terpenoids (Wiley, Chichester/New York, 1982)
Aubourg, S., Lecharny, A. & Bohlmann, J. Genomic analysis of the terpenoid synthase (AtTPS) gene family of Arabidopsis thaliana. Mol. Genet. Genomics 267, 730–745 (2002)
Martin, D. M., Faldt, J. & Bohlmann, J. Functional characterization of nine Norway spruce TPS genes and evolution of gymnosperm terpene synthases of the TPS-d subfamily. Plant Physiol. 135, 1908–1927 (2004)
Bohlmann, J., Meyer-Gauen, G. & Croteau, R. Plant terpenoid synthases: molecular biology and phylogenetic analysis. Proc. Natl Acad. Sci. USA 95, 4126–4133 (1998)
Lichtarge, O., Bourne, H. R. & Cohen, F. E. An evolutionary trace method defines binding surfaces common to protein families. J. Mol. Biol. 257, 342–358 (1996)
Martin, V. J., Pitera, D. J., Withers, S. T., Newman, J. D. & Keasling, J. D. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nature Biotechnol. 21, 796–802 (2003)
Stemmer, W. P. Rapid evolution of a protein in vitro by DNA shuffling. Nature 370, 389–391 (1994)
Sali, A. Comparative protein modeling by satisfaction of spatial restraints. Mol. Med. Today 1, 270–277 (1995)
Baker, D. & Sali, A. Protein structure prediction and structural genomics. Science 294, 93–96 (2001)
Rynkiewicz, M. J., Cane, D. E. & Christianson, D. W. X-ray crystal structures of D100E trichodiene synthase and its pyrophosphate complex reveal the basis for terpene product diversity. Biochemistry 41, 1732–1741 (2002)
Mildvan, A. S. Inverse thinking about double mutants of enzymes. Biochemistry 43, 14517–14520 (2004)
Joo, H., Lin, Z. & Arnold, F. H. Laboratory evolution of peroxide-mediated cytochrome P450 hydroxylation. Nature 399, 670–673 (1999)
James, L. C., Roversi, P. & Tawfik, D. S. Antibody multispecificity mediated by conformational diversity. Science 299, 1362–1367 (2003)
Adams, R. P. Identification of Essential Oil Components by Gas Chromatography/Mass Spectroscopy (Allured Publishing Corporation, Carol Stream, Illinois, 1995).
We would like to thank P. C. Babbitt, J. D. Newman, M. C. Chang and S. C.-H. Pegg for discussions and critical reading of the manuscript. We are also grateful for D. Herschlag for critical comments. This research was funded by the Bill & Melinda Gates Foundation, the US Department of Agriculture, and the National Science Foundation.Author Contributions Y.Y. and J.D.K. conceived the project; Y.Y., J.D.K. and T.E.F. designed the experiments; and Y.Y. and J.D.K. wrote the paper.
J.D.K. owns stock in, and is a founder of, Amyris Biotechnologies. Amyris may use this technology to produce terpenes. However, Amyris currently has no plans to use the technology.
This file contains Supplementary Tables 1–9. (PDF 175 kb)
This file contains Supplementary Figures 1–11. *In Figure 1 and Supplementary Figure 1, the structure of longifolene (4) has been corrected (it was missing a methyl group). In Supplementary Figure 1, the longipinene structure was also wrongly labelled 'longifolene' and vice versa. This correction was made on 27 February 2006. (DOC 1686 kb)
Three-dimensional coordinates for the homology structure of γ-humulene synthase (PDF 158 kb)
DNA sequence of designed γ-humulene synthase (PDF 59 kb)
Additional methods used in this study. (PDF 110 kb)
Detailed protocols for systematic remodeling of plasticity residues (PDF 103 kb)
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Yoshikuni, Y., Ferrin, T. & Keasling, J. Designed divergent evolution of enzyme function. Nature 440, 1078–1082 (2006). https://doi.org/10.1038/nature04607
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