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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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Activities of the M9 lineage(a) A 3-D scatter plot of the product output (chemical space) was constructed where the x, y, and z-axes correspond to percentages of the major products 5-EA (2), 4-EE (4), and PSD (3), respectively (Supplementary Table 3 online). Each sphere represents one of the 418 active mutant proteins from the M9 library with wild type TEAS, M9 and EES highlighted as enlarged spheres. The tetrahedron encompassing the scatter plot was partitioned to represent each of the major reaction products by choosing the midpoint of each axis for subdividing into geometrically equivalent tetrahedrons. Each shaded volume, blue (5-EA, 2), purple (4-EE, 4), or red (PSD, 3) indicates product specificity of 50% or greater. Mutants in the remaining central volume (cyan) are defined as promiscuous. (b) Schematic of the scatter plot (panel a) summarizing the distribution of activities where the number of mutants in each quadrant is expressed as a percentage of the total number characterized.
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Figure 4: Activities of the M9 lineage(a) A 3-D scatter plot of the product output (chemical space) was constructed where the x, y, and z-axes correspond to percentages of the major products 5-EA (2), 4-EE (4), and PSD (3), respectively (Supplementary Table 3 online). Each sphere represents one of the 418 active mutant proteins from the M9 library with wild type TEAS, M9 and EES highlighted as enlarged spheres. The tetrahedron encompassing the scatter plot was partitioned to represent each of the major reaction products by choosing the midpoint of each axis for subdividing into geometrically equivalent tetrahedrons. Each shaded volume, blue (5-EA, 2), purple (4-EE, 4), or red (PSD, 3) indicates product specificity of 50% or greater. Mutants in the remaining central volume (cyan) are defined as promiscuous. (b) Schematic of the scatter plot (panel a) summarizing the distribution of activities where the number of mutants in each quadrant is expressed as a percentage of the total number characterized.

Mentions: A three-dimensional scatter plot illustrates how the product specificities of mutants distribute throughout chemical space (Fig. 4a). The 3 dominant products 5-EA (2), 4-EE (4), and PSD (3) define a two dimensional triangular plane, whereas the collective minor products contribute the third dimension of the tetrahedron. Mutants with varying degrees of catalytic promiscuity radiate uniformly from a cluster of TEAS-like activities, together forming a continuum with more HPS-like mutants. By contrast, EES-like enzymes are rare, appearing as a sparsely populated subgroup. Subdividing the scatter plot into three smaller tetrahedrons of equal volume geometrically defines product specificity as >50% 5-EA (2), 4-EE (4) or PSD (3), whereas the central volume represents promiscuous activities (Fig. 4b). The majority of mutants are promiscuous (51%) showing expanded product distributions and up-regulation of other TEAS minor products, predominantly germacrene A (5)16, a neutral intermediate along the TEAS and HPS cyclization pathways as well as the major product of a closely-related family of plant synthases. Kinetic analyses of select members of the library with diverse product specificities reveals that most mutants possess catalytic activities (Kcat) within 10-fold of wild type TEAS, indicating that most combinations of mutations alter product specificity without significantly compromising the overall catalytic rate (Supplementary Table 4 online).


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)

Activities of the M9 lineage(a) A 3-D scatter plot of the product output (chemical space) was constructed where the x, y, and z-axes correspond to percentages of the major products 5-EA (2), 4-EE (4), and PSD (3), respectively (Supplementary Table 3 online). Each sphere represents one of the 418 active mutant proteins from the M9 library with wild type TEAS, M9 and EES highlighted as enlarged spheres. The tetrahedron encompassing the scatter plot was partitioned to represent each of the major reaction products by choosing the midpoint of each axis for subdividing into geometrically equivalent tetrahedrons. Each shaded volume, blue (5-EA, 2), purple (4-EE, 4), or red (PSD, 3) indicates product specificity of 50% or greater. Mutants in the remaining central volume (cyan) are defined as promiscuous. (b) Schematic of the scatter plot (panel a) summarizing the distribution of activities where the number of mutants in each quadrant is expressed as a percentage of the total number characterized.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2664519&req=5

Figure 4: Activities of the M9 lineage(a) A 3-D scatter plot of the product output (chemical space) was constructed where the x, y, and z-axes correspond to percentages of the major products 5-EA (2), 4-EE (4), and PSD (3), respectively (Supplementary Table 3 online). Each sphere represents one of the 418 active mutant proteins from the M9 library with wild type TEAS, M9 and EES highlighted as enlarged spheres. The tetrahedron encompassing the scatter plot was partitioned to represent each of the major reaction products by choosing the midpoint of each axis for subdividing into geometrically equivalent tetrahedrons. Each shaded volume, blue (5-EA, 2), purple (4-EE, 4), or red (PSD, 3) indicates product specificity of 50% or greater. Mutants in the remaining central volume (cyan) are defined as promiscuous. (b) Schematic of the scatter plot (panel a) summarizing the distribution of activities where the number of mutants in each quadrant is expressed as a percentage of the total number characterized.
Mentions: A three-dimensional scatter plot illustrates how the product specificities of mutants distribute throughout chemical space (Fig. 4a). The 3 dominant products 5-EA (2), 4-EE (4), and PSD (3) define a two dimensional triangular plane, whereas the collective minor products contribute the third dimension of the tetrahedron. Mutants with varying degrees of catalytic promiscuity radiate uniformly from a cluster of TEAS-like activities, together forming a continuum with more HPS-like mutants. By contrast, EES-like enzymes are rare, appearing as a sparsely populated subgroup. Subdividing the scatter plot into three smaller tetrahedrons of equal volume geometrically defines product specificity as >50% 5-EA (2), 4-EE (4) or PSD (3), whereas the central volume represents promiscuous activities (Fig. 4b). The majority of mutants are promiscuous (51%) showing expanded product distributions and up-regulation of other TEAS minor products, predominantly germacrene A (5)16, a neutral intermediate along the TEAS and HPS cyclization pathways as well as the major product of a closely-related family of plant synthases. Kinetic analyses of select members of the library with diverse product specificities reveals that most mutants possess catalytic activities (Kcat) within 10-fold of wild type TEAS, indicating that most combinations of mutations alter product specificity without significantly compromising the overall catalytic rate (Supplementary Table 4 online).

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