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Quantitative exploration of the catalytic landscape separating divergent plant sesquiterpene synthases.

O'Maille PE, Malone A, Dellas N, Andes Hess B, Smentek L, Sheehan I, Greenhagen BT, Chappell J, Manning G, Noel JP - Nat. Chem. Biol. (2008)

Bottom Line: 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 (2(9) = 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.These results provide a measure of the mutational accessibility of phenotypic variability in a diverging lineage of terpene synthases.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, The Salk Institute for Biological Studies, Jack H. Skirball Center for Chemical Biology & Proteomics, 10010 North Torrey Pines Road, La Jolla, California 92037, USA.

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 (2(9) = 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.

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Average inter-neighbor distances (AID) in chemical and sequence spaceA representative mutant (unlabeled red sphere) is shown in chemical space along with all nine possible single mutant neighbors (numbered green spheres) to illustrate a, short, b, medium, and c, high average inter-neighbor mutational distances (AID). Sequences of each representative mutant are referenced across the top of the three alignment tables with mutational neighbors and distances listed below. Each mutated position is boxed and residues of HPS origin are indicated with shading. (d) The average inter-neighbor distance (AID) for a subset of 236 mutants was plotted as a simple histogram, where the shoulders and apex of the distribution are labeled a, b and c to correspond to representative mutants above. (e) The distribution of AID as a function of the number of accumulated HPS substitutions was plotted, where M1 refers to all single mutants, M2 to all double mutants and so on.
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Figure 6: Average inter-neighbor distances (AID) in chemical and sequence spaceA representative mutant (unlabeled red sphere) is shown in chemical space along with all nine possible single mutant neighbors (numbered green spheres) to illustrate a, short, b, medium, and c, high average inter-neighbor mutational distances (AID). Sequences of each representative mutant are referenced across the top of the three alignment tables with mutational neighbors and distances listed below. Each mutated position is boxed and residues of HPS origin are indicated with shading. (d) The average inter-neighbor distance (AID) for a subset of 236 mutants was plotted as a simple histogram, where the shoulders and apex of the distribution are labeled a, b and c to correspond to representative mutants above. (e) The distribution of AID as a function of the number of accumulated HPS substitutions was plotted, where M1 refers to all single mutants, M2 to all double mutants and so on.

Mentions: The average inter-neighbor distance (AID) was calculated for each mutant and specific examples illustrate how this index relates to chemical space (scatter plot) and sequence space (alignment with mutational neighbors) (Fig. 6a–c). For a mutant with a low AID, most mutations registered negligible to modest effects on the product output as evident from the clustering of most mutational neighbors in a small region of chemical space (Fig. 6a). By contrast, mutants with a high AID show a broad scattering of mutational neighbors throughout chemical space (Fig. 6c) with demonstrable long jumps between highly specific TEAS-like to EES- or HPS-like activities by single mutational steps. Hence, the activities of some mutants are highly sensitive to mutational perturbations. For a promiscuous mutant with a moderate AID (Fig. 6b), nearly half of the mutational steps tighten product specificity. Considering the larger trends throughout the M9 library, the AID for the subset of 236 mutants was plotted as a simple histogram (Fig. 6d). Plotting the AID as a function of the number of mutations reveals that the distribution of averages is similar across the library (Fig. 6e).


Quantitative exploration of the catalytic landscape separating divergent plant sesquiterpene synthases.

O'Maille PE, Malone A, Dellas N, Andes Hess B, Smentek L, Sheehan I, Greenhagen BT, Chappell J, Manning G, Noel JP - Nat. Chem. Biol. (2008)

Average inter-neighbor distances (AID) in chemical and sequence spaceA representative mutant (unlabeled red sphere) is shown in chemical space along with all nine possible single mutant neighbors (numbered green spheres) to illustrate a, short, b, medium, and c, high average inter-neighbor mutational distances (AID). Sequences of each representative mutant are referenced across the top of the three alignment tables with mutational neighbors and distances listed below. Each mutated position is boxed and residues of HPS origin are indicated with shading. (d) The average inter-neighbor distance (AID) for a subset of 236 mutants was plotted as a simple histogram, where the shoulders and apex of the distribution are labeled a, b and c to correspond to representative mutants above. (e) The distribution of AID as a function of the number of accumulated HPS substitutions was plotted, where M1 refers to all single mutants, M2 to all double mutants and so on.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2664519&req=5

Figure 6: Average inter-neighbor distances (AID) in chemical and sequence spaceA representative mutant (unlabeled red sphere) is shown in chemical space along with all nine possible single mutant neighbors (numbered green spheres) to illustrate a, short, b, medium, and c, high average inter-neighbor mutational distances (AID). Sequences of each representative mutant are referenced across the top of the three alignment tables with mutational neighbors and distances listed below. Each mutated position is boxed and residues of HPS origin are indicated with shading. (d) The average inter-neighbor distance (AID) for a subset of 236 mutants was plotted as a simple histogram, where the shoulders and apex of the distribution are labeled a, b and c to correspond to representative mutants above. (e) The distribution of AID as a function of the number of accumulated HPS substitutions was plotted, where M1 refers to all single mutants, M2 to all double mutants and so on.
Mentions: The average inter-neighbor distance (AID) was calculated for each mutant and specific examples illustrate how this index relates to chemical space (scatter plot) and sequence space (alignment with mutational neighbors) (Fig. 6a–c). For a mutant with a low AID, most mutations registered negligible to modest effects on the product output as evident from the clustering of most mutational neighbors in a small region of chemical space (Fig. 6a). By contrast, mutants with a high AID show a broad scattering of mutational neighbors throughout chemical space (Fig. 6c) with demonstrable long jumps between highly specific TEAS-like to EES- or HPS-like activities by single mutational steps. Hence, the activities of some mutants are highly sensitive to mutational perturbations. For a promiscuous mutant with a moderate AID (Fig. 6b), nearly half of the mutational steps tighten product specificity. Considering the larger trends throughout the M9 library, the AID for the subset of 236 mutants was plotted as a simple histogram (Fig. 6d). Plotting the AID as a function of the number of mutations reveals that the distribution of averages is similar across the library (Fig. 6e).

Bottom Line: 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 (2(9) = 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.These results provide a measure of the mutational accessibility of phenotypic variability in a diverging lineage of terpene synthases.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, The Salk Institute for Biological Studies, Jack H. Skirball Center for Chemical Biology & Proteomics, 10010 North Torrey Pines Road, La Jolla, California 92037, USA.

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 (2(9) = 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.

Show MeSH
Related in: MedlinePlus