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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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Phylogenetic distribution of Solanaceous TEAS and HPS-like terpene synthases(a) An un-rooted phylogenetic tree of 5-EA and PSD terpene synthases was created form available sequences (Supplementary Table 1 online) where branches are colored according to the established or putative functions as TEAS-like (blue) or HPS-like (red). (b) Sequence alignment of the M9 residue positions of the sequences (panel a) with HPS like residues shaded in grey.
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Figure 3: Phylogenetic distribution of Solanaceous TEAS and HPS-like terpene synthases(a) An un-rooted phylogenetic tree of 5-EA and PSD terpene synthases was created form available sequences (Supplementary Table 1 online) where branches are colored according to the established or putative functions as TEAS-like (blue) or HPS-like (red). (b) Sequence alignment of the M9 residue positions of the sequences (panel a) with HPS like residues shaded in grey.

Mentions: To investigate the natural distribution of these activities in the current work, we constructed a phylogenetic tree from available TEAS-like and HPS-like sequences from related solanaceous plants (Fig. 3a and Supplementary Table 1 online). While the product spectra of terpene synthases cannot be readily predicted from traditional phylogenetic analyses13,14, a clear functional division was apparent between the tobacco and pepper synthases as compared to their orthologues in tomato, potato and henbane. This division was also apparent at the level of our nine-residue subset with the exception of the Capsicum annuum synthase (Fig. 3b). This TEAS-like enzyme differs from both TEAS and HPS-like groups at 3 positions, and most notably, contains a threonine at position 438 like HPS, suggesting the first mutational steps in the TEAS-HPS divergence likely occurred at these positions. Evaluating the functional divergence of TEAS and HPS within the context of these nine amino acid substitutions provides a simplified experimental system to ask the broader question: how prevalent and hence evolvable are these parental and alternative biosynthetic activities throughout the intervening lineages connecting these extant enzymes? Measuring the distribution of biosynthetic activities over this sequence space defines a functionally relevant portion of the overall catalytic landscape, and provides a window into the complex functional terrain underlying the evolution of these enzymes. While variation at other positions may have contributed to the functional divergence of TEAS and HPS in meaningful ways, this focused set of functionally important residues makes it experimentally tractable to quantitatively characterize a catalytic landscape of secondary metabolism to biochemical resolution.


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)

Phylogenetic distribution of Solanaceous TEAS and HPS-like terpene synthases(a) An un-rooted phylogenetic tree of 5-EA and PSD terpene synthases was created form available sequences (Supplementary Table 1 online) where branches are colored according to the established or putative functions as TEAS-like (blue) or HPS-like (red). (b) Sequence alignment of the M9 residue positions of the sequences (panel a) with HPS like residues shaded in grey.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2664519&req=5

Figure 3: Phylogenetic distribution of Solanaceous TEAS and HPS-like terpene synthases(a) An un-rooted phylogenetic tree of 5-EA and PSD terpene synthases was created form available sequences (Supplementary Table 1 online) where branches are colored according to the established or putative functions as TEAS-like (blue) or HPS-like (red). (b) Sequence alignment of the M9 residue positions of the sequences (panel a) with HPS like residues shaded in grey.
Mentions: To investigate the natural distribution of these activities in the current work, we constructed a phylogenetic tree from available TEAS-like and HPS-like sequences from related solanaceous plants (Fig. 3a and Supplementary Table 1 online). While the product spectra of terpene synthases cannot be readily predicted from traditional phylogenetic analyses13,14, a clear functional division was apparent between the tobacco and pepper synthases as compared to their orthologues in tomato, potato and henbane. This division was also apparent at the level of our nine-residue subset with the exception of the Capsicum annuum synthase (Fig. 3b). This TEAS-like enzyme differs from both TEAS and HPS-like groups at 3 positions, and most notably, contains a threonine at position 438 like HPS, suggesting the first mutational steps in the TEAS-HPS divergence likely occurred at these positions. Evaluating the functional divergence of TEAS and HPS within the context of these nine amino acid substitutions provides a simplified experimental system to ask the broader question: how prevalent and hence evolvable are these parental and alternative biosynthetic activities throughout the intervening lineages connecting these extant enzymes? Measuring the distribution of biosynthetic activities over this sequence space defines a functionally relevant portion of the overall catalytic landscape, and provides a window into the complex functional terrain underlying the evolution of these enzymes. While variation at other positions may have contributed to the functional divergence of TEAS and HPS in meaningful ways, this focused set of functionally important residues makes it experimentally tractable to quantitatively characterize a catalytic landscape of secondary metabolism to biochemical resolution.

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