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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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Overall structure of TEAS, location and identity of M9 residues(a) Nucleotide and amino acid identity of substitutions between TEAS and HPS. Shading is used to indicate nucleotide substitutions in HPS relative to the TEAS reference sequence. (b) The primary structure is composed of N-terminal (blue) and C-terminal (gold) terpenoid synthase domains. Amino acid positions of the M9 library are indicated using TEAS numbering. (c) Tertiary structure of TEAS (pdb id 5eat) shown as ribbons with domains colored (as in a) and Mg2+ and FPP (1) modeled into the active site. (d) The spatial distribution of M9 library residues is depicted, where the active site is rendered as a continuous van der Waal’s surface (positions 402 and 516 highlighted in red) and second tier residues (colored side chains) are arrayed behind the active site proper.
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Figure 2: Overall structure of TEAS, location and identity of M9 residues(a) Nucleotide and amino acid identity of substitutions between TEAS and HPS. Shading is used to indicate nucleotide substitutions in HPS relative to the TEAS reference sequence. (b) The primary structure is composed of N-terminal (blue) and C-terminal (gold) terpenoid synthase domains. Amino acid positions of the M9 library are indicated using TEAS numbering. (c) Tertiary structure of TEAS (pdb id 5eat) shown as ribbons with domains colored (as in a) and Mg2+ and FPP (1) modeled into the active site. (d) The spatial distribution of M9 library residues is depicted, where the active site is rendered as a continuous van der Waal’s surface (positions 402 and 516 highlighted in red) and second tier residues (colored side chains) are arrayed behind the active site proper.

Mentions: We previously used a structure-guided approach to identify a functionally linked subset of nine amino acid residues out of the 135 naturally occurring amino acid differences between TEAS and HPS (Fig. 2). Mutational swaps of this nine-residue subset are sufficient to interconvert the product specificities of the encoded mutant proteins in the background of each parent enzyme12. Eight of these nine amino acid substitutions are achievable by single non-synonymous nucleotide changes per codon (Fig. 2a). However, position 406 (TEAS numbering) requires a two base change to interconvert between Tyr and Leu in TEAS and HPS, respectively, suggesting the possible intermediacy of Phe at this position in a common ancestor. Interestingly, only two of the nine amino acid differences are localized on the active site surface, while the remainder are scattered throughout the second tier (Fig. 2d). Replacing these two active site residues of TEAS with their HPS counterparts redirects the final deprotonation-neutralization step to produce 4-epi-eremophilene (4-EE, 4), a terpene not previously identified in nature12. Thus, the resulting 4-epi-eremophilene synthase (EES) represents an intermediate enzyme with hybrid parental activities (Fig. 1a).


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)

Overall structure of TEAS, location and identity of M9 residues(a) Nucleotide and amino acid identity of substitutions between TEAS and HPS. Shading is used to indicate nucleotide substitutions in HPS relative to the TEAS reference sequence. (b) The primary structure is composed of N-terminal (blue) and C-terminal (gold) terpenoid synthase domains. Amino acid positions of the M9 library are indicated using TEAS numbering. (c) Tertiary structure of TEAS (pdb id 5eat) shown as ribbons with domains colored (as in a) and Mg2+ and FPP (1) modeled into the active site. (d) The spatial distribution of M9 library residues is depicted, where the active site is rendered as a continuous van der Waal’s surface (positions 402 and 516 highlighted in red) and second tier residues (colored side chains) are arrayed behind the active site proper.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2664519&req=5

Figure 2: Overall structure of TEAS, location and identity of M9 residues(a) Nucleotide and amino acid identity of substitutions between TEAS and HPS. Shading is used to indicate nucleotide substitutions in HPS relative to the TEAS reference sequence. (b) The primary structure is composed of N-terminal (blue) and C-terminal (gold) terpenoid synthase domains. Amino acid positions of the M9 library are indicated using TEAS numbering. (c) Tertiary structure of TEAS (pdb id 5eat) shown as ribbons with domains colored (as in a) and Mg2+ and FPP (1) modeled into the active site. (d) The spatial distribution of M9 library residues is depicted, where the active site is rendered as a continuous van der Waal’s surface (positions 402 and 516 highlighted in red) and second tier residues (colored side chains) are arrayed behind the active site proper.
Mentions: We previously used a structure-guided approach to identify a functionally linked subset of nine amino acid residues out of the 135 naturally occurring amino acid differences between TEAS and HPS (Fig. 2). Mutational swaps of this nine-residue subset are sufficient to interconvert the product specificities of the encoded mutant proteins in the background of each parent enzyme12. Eight of these nine amino acid substitutions are achievable by single non-synonymous nucleotide changes per codon (Fig. 2a). However, position 406 (TEAS numbering) requires a two base change to interconvert between Tyr and Leu in TEAS and HPS, respectively, suggesting the possible intermediacy of Phe at this position in a common ancestor. Interestingly, only two of the nine amino acid differences are localized on the active site surface, while the remainder are scattered throughout the second tier (Fig. 2d). Replacing these two active site residues of TEAS with their HPS counterparts redirects the final deprotonation-neutralization step to produce 4-epi-eremophilene (4-EE, 4), a terpene not previously identified in nature12. Thus, the resulting 4-epi-eremophilene synthase (EES) represents an intermediate enzyme with hybrid parental activities (Fig. 1a).

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