Quantitative exploration of the catalytic landscape separating divergent plant sesquiterpene synthases

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. We report the first systematic quantitative characterization of a catalytic landscape underlying the evolution of sesquiterpene chemical diversity. Based on our previous discovery of a set of 9 naturally occurring amino acid substitutions that functionally inter-converted orthologous sesquiterpene synthases from Nicotiana tabaccum and Hyoscyamus muticus, we created a library of all possible residue combinations (29 = 512) in the N. tabaccum parent. The product spectra of 418 active enzymes to reveal a rugged landscape where several minimal combinations of the 9 mutations encode convergent solutions to the inter-conversions of parental activities. Quantitative comparisons indicate context dependence for mutational effects - epistasis - in product specificity and promiscuity. These results provide a measure of the mutational accessibility of phenotypic variability among a diverging lineage of terpene synthases.

[1]  D. Herschlag,et al.  Catalytic promiscuity and the evolution of new enzymatic activities. , 1999, Chemistry & biology.

[2]  D. Shin,et al.  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. , 1998, Plant & cell physiology.

[3]  T. Creighton.  Methods in Enzymology , 1968, The Yale Journal of Biology and Medicine.

[4]  M. Hilker,et al.  The significance of background odour for an egg parasitoid to detect plants with host eggs. , 2005, Chemical senses.

[5]  J. Gershenzon,et al.  The function of terpene natural products in the natural world. , 2007, Nature chemical biology.

[6]  Nigel F. Delaney,et al.  Darwinian Evolution Can Follow Only Very Few Mutational Paths to Fitter Proteins , 2006, Science.

[7]  P. Agarwal,et al.  Network of coupled promoting motions in enzyme catalysis , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[8]  Dan S. Tawfik,et al.  Robustness–epistasis link shapes the fitness landscape of a randomly drifting protein , 2006, Nature.

[9]  J. Noel,et al.  Biosynthetic potential of sesquiterpene synthases: alternative products of tobacco 5-epi-aristolochene synthase. , 2006, Archives of biochemistry and biophysics.

[10]  M. Nei,et al.  MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. , 2007, Molecular biology and evolution.

[11]  T. Mabry,et al.  Biochemical Systematics and Ecology , 1974 .

[12]  R. Ranganathan,et al.  Evolutionarily conserved pathways of energetic connectivity in protein families. , 1999, Science.

[13]  S. Benkovic,et al.  Coupling interactions of distal residues enhance dihydrofolate reductase catalysis: mutational effects on hydride transfer rates. , 2002, Biochemistry.

[14]  Rudy M. Baum,et al.  The Chemistry Of Biology , 2008 .

[15]  T. Kokubun,et al.  Plant--fungal interactions: the search for phytoalexins and other antifungal compounds from higher plants. , 2001, Phytochemistry.

[16]  R. Rosenfeld.  Nature , 2009, Otolaryngology--head and neck surgery : official journal of American Academy of Otolaryngology-Head and Neck Surgery.

[17]  Thomas E. Ferrin,et al.  Designed divergent evolution of enzyme function , 2006, Nature.

[18]  P. Facchini,et al.  Gene family for an elicitor-induced sesquiterpene cyclase in tobacco. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[19]  S. Copley.  Enzymes with extra talents: moonlighting functions and catalytic promiscuity. , 2003, Current opinion in chemical biology.

[20]  M. Austin,et al.  Evolving biosynthetic tangos negotiate mechanistic landscapes. , 2008, Nature chemical biology.

[21]  J. Noel,et al.  A single-vial analytical and quantitative gas chromatography-mass spectrometry assay for terpene synthases. , 2004, Analytical biochemistry.

[22]  D. Janzen,et al.  Herbivores: Their Interaction With Secondary Plant Metabolites , 1982 .

[23]  C. Poulter,et al.  Chimeras of Two Isoprenoid Synthases Catalyze All Four Coupling Reactions in Isoprenoid Biosynthesis , 2007, Science.

[24]  Victor Guallar,et al.  Archives of Biochemistry and Biophysics , 1951, Nature.

[25]  J. Gershenzon,et al.  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 , 2004, The Plant Cell Online.

[26]  Journal of Chemical Physics , 1932, Nature.

[27]  R. Jensen.  Enzyme recruitment in evolution of new function. , 1976, Annual review of microbiology.

[28]  R. Croteau,et al.  Mutational analysis of a monoterpene synthase reaction: altered catalysis through directed mutagenesis of (-)-pinene synthase from Abies grandis. , 2005, Archives of biochemistry and biophysics.

[29]  Paul E O'Maille,et al.  Gene library synthesis by structure-based combinatorial protein engineering. , 2004, Methods in enzymology.

[30]  A. Makris,et al.  Rational Conversion of Substrate and Product Specificity in a Salvia Monoterpene Synthase: Structural Insights into the Evolution of Terpene Synthase Function[W] , 2007, The Plant Cell Online.

[31]  K. Akiyama,et al.  Plant sesquiterpenes induce hyphal branching in arbuscular mycorrhizal fungi , 2005, Nature.

[32]  Paul E O'Maille,et al.  Structure-based combinatorial protein engineering (SCOPE). , 2002, Journal of Molecular Biology.

[33]  J. Harborne.  The comparative biochemistry of phytoalexin induction in plants , 1999 .

[34]  J. Bohlmann,et al.  Plant terpenoid synthases: molecular biology and phylogenetic analysis. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[35]  M. Kelsey.  “Annual Review of Microbiology” , 1946, Nature.

[36]  Stephen P. Miller,et al.  Direct Demonstration of an Adaptive Constraint , 2006, Science.

[37]  R. Peters,et al.  A single residue switch converts abietadiene synthase into a pimaradiene specific cyclase. , 2007, Journal of the American Chemical Society.

[38]  J. Noel,et al.  Identifying and manipulating structural determinates linking catalytic specificities in terpene synthases. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[39]  T. Kokubun,et al.  Plant—Fungal Interactions: The Search for Phytoalexins and Other Antifungal Compounds from Higher Plants , 2010 .

[40]  M. Pedras,et al.  Phytoalexins from crucifers: synthesis, biosynthesis, and biotransformation. , 2000, Phytochemistry.

[41]  E. Ortlund,et al.  Crystal Structure of an Ancient Protein: Evolution by Conformational Epistasis , 2007, Science.

[42]  Diane M. Martin,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. , 2003, The Plant cell.

[43]  G. M. Dyson.  The Chemical Senses , 1946, Nature.